Karl-Dieter Müller

Neochlamydia hartmannellae gen. nov., sp. nov. (Parachlamydiaceae), an endoparasite of the amoeba Hartmannella vermiformis.

Free-living amoebae are increasingly being recognized to serve as vehicles of dispersal for various bacterial human pathogens and as hosts for a variety of obligate bacterial endocytobionts. Several Chlamydia-like Acanthamoeba endocytobionts constituting the recently proposed family Parachlamydiaceae are of special interest as potential human pathogens. In this study coccoid bacterial endocytobionts of a Hartmannella vermiformis isolate were analysed. Infection of H. vermiformis with these bacteria resulted in prevention of cyst formation and subsequent host-cell lysis. Transfection experiments demonstrated that the parasites were not capable of propagating within other closely related free-living amoebae but were able to infect the distantly related species Dictyostelium discoideum. Electron microscopy of the parasites revealed typical morphological characteristics of the Chlamydiales, including the existence of a Chlamydia-like life-cycle, but indicated that these endocytobionts, in contrast to Chlamydia species, do not reside within a vacuole. Comparative 16S rRNA sequence analysis showed that the endocytobiont of H. vermiformis, classified as Neochlamydia hartmannellae gen. nov., sp. nov., is affiliated to the family Parachlamydiaceae. Confocal laser scanning microscopy in combination with fluorescence in situ hybridization using rRNA-targeted oligonucleotide probes confirmed the intracellular localization of the parasites and demonstrated the absence of other bacterial species within the Hartmannella host. These findings extend our knowledge of the phylogenetic diversity of the Parachlamydiaceae and demonstrate for the first time that these endocytobionts can naturally develop within amoebae of the genus Hartmannella.

Direct identification of bacteria in urine samples by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and relevance of defensins as interfering factors

Standard methods for the identification of uropathogens that are based on the determination of metabolic activity require cultivation on agar plates, which often takes more than 1 day. If microbial growth on agar plates is slow, or if metabolic activity is impaired by adverse interactions resulting from the patients condition or from medical treatment, the application of standard methods may lead to delayed or erroneous identification of bacteria. In recent studies, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has proven to be able to rapidly identify bacteria obtained from cultures. We tested the applicability of this analytical technique for the rapid identification of bacteria collected directly from urine samples and compared the results with those of conventional identification methods, such as the Vitek system, the MicroScan WalkAway system and the API system, and in some cases with the gas chromatographic determination of the bacterial long-chain fatty acid pattern. We analysed a total of 107 urine samples with bacterial counts ranging from 10(2) to ≥10(5) c.f.u. ml(-1). Mass spectrometric identification of bacteria was accomplished for 62 of these samples. In the mass spectra obtained from 40 of the 45 urine samples for which no identification result was achieved, a triplet of very intense peaks corresponding to the human α-defensins 1, 2 and 3 occurred at m/z values of around 3440 Da. This signal suppressed the intensity of the bacterial protein peaks and thus impaired database matching. Our results show that MALDI-TOF MS allows the reliable direct identification of bacteria in urine samples at concentrations as low as 10(3) c.f.u. ml(-1). In a subset of samples, human defensins may occur and impair the mass spectrometric identification of bacteria.

Acanthamoeba from human nasal mucosa infected with an obligate intracellular parasite

Summary An aggregation of numerous round particles could be observed inside the cytoplasm of trophozoites of an Acanthamoeba strain (Bn 9 ) isolated from human nasal mucosa [8]. Transmission and scanning electron microscopy revealed small coccoidal bacteria (0.4–0.6 μm in diameter) with a prominent trilamellar cell wall. Initially the bacteria multiplied inside food vacuoles of infected trophozoites, later on they could also be observed free in the cytoplasm. The affected host cells were finally ruptured liberating the bacteria into the environment. The procaryotes were ingested by further trophozoites that were not able to digest them but became lethally infected, too. The parasitic bacteria were successfully transfered to a pathogenic A. castellanii strain (C 3 ). Infected amoebae could be cured from parasites by rifampicin. The taxonomic position of the gram-positive cocci could not be determined. So far it was not possible to subcultivate the organisms in vitro. Therefore we assume that they are obligate intracellular parasites of amoebae, comparable to those rod-shaped bacteria described by Drozanski [3] as obligate pathogens of Acanthamoebae.

Microsporidia-like parasites of amoebae belong to the early fungal lineage Rozellomycota

Molecular phylogenies based on the small subunit ribosomal RNA gene (SSU or 18S ribosomal DNA (rDNA)) revealed recently the existence of a relatively large and widespread group of eukaryotes, branching at the base of the fungal tree. This group, comprising almost exclusively environmental clones, includes the endoparasitic chytrid Rozella as the unique known representative. Rozella emerged as the first fungal lineage in molecular phylogenies and as the sister group of the Microsporidia. Here we report rDNA molecular phylogenetic analyses of two endonuclear parasites of free-living naked amoebae having microsporidia-like ultrastructural features but belonging to the rozellids. Similar to microsporidia, these endoparasites form unflagellated walled spores and grow inside the host cells as unwalled nonphagotrophic meronts. Our endonuclear parasites are microsporidia-like rozellids, for which we propose the name Paramicrosporidium, appearing to be the until now lacking morphological missing link between Fungi and Microsporidia. These features contrast with the recent description of the rozellids as an intermediate wall-less lineage of organisms between protists and true Fungi. We thus reconsider the rozellid clade as the most basal fungal lineage, naming it Rozellomycota.

Protochlamydia naegleriophila as Etiologic Agent of Pneumonia

Using ameba coculture, we grew a Naegleria endosymbiont. Phenotypic, genetic, and phylogenetic analyses supported its affiliation as Protochlamydia naegleriophila sp. nov. We then developed a specific diagnostic PCR for Protochlamydia spp. When applied to bronchoalveolar lavages, results of this PCR were positive for 1 patient with pneumonia. Further studies are needed to assess the role of Protochlamydia spp. in pneumonia.

Vannella sp. harboring Microsporidia-like organisms isolated from the contact lens and inflamed eye of a female keratitis patient

Abstract Viable Hartmannella sp. and two strains of Vannella sp. – but no Acanthamoebae– multiplied on NN-agar inoculated with pieces of the contact lens from a female keratitis patient. Within the cytoplasm of one Vannella isolate, intracellular parasites could be observed whose earliest stages were developing within the nucleus, resembling those Microsporidia-like parasites seen within Vannella isolated recently from a warm tapwater system. This assumption was also confirmed by electron microscopy. In swabs taken directly from the cornea, Pseudomonas aeruginosa were identified, but they did not yield any growth of amebas in culture. However, cocultivation of parasite-free Vannella strains with the above-mentioned swab matter resulted in infected amebas harboring the same intracellular parasites seen before. This infection could be established only if the corresponding spores were present as infective agents in the swab matter. The successful treatment of the patient with antibiotics supports the assumption that P. aeruginosa was the main cause of the corneal ulceration. The extent to which the Microsporidia-like organisms may have been involved in the development of keratitis remains a matter of discussion.

Rediscovery of Nucleophaga amoebae, a novel member of the Rozellomycota

Recent studies showed that the huge diversity branching at or near the phylogenetic root of the fungal kingdom, mostly constituted by uncultured environmental clones, is actually characterized by intracellular predators/parasites of various eukaryotes. These form three related lineages: the Aphelidea, endoparasites of algae; the Rozellomycota, with Rozella species mainly endoparasites of water moulds, and Paramicrosporidium species endonuclear parasites of amoebae; and the Microsporidia, mainly endoparasites of animals. Increasing evidence suggests the emergence of Microsporidia from within Rozellomycota; however, their fungal or protistan nature is still unclear. Here, we report the molecular phylogeny based on the small subunit ribosomal RNA (SSU rDNA) gene, of an additional endoparasite of amoebae, corresponding to the old enigmatic chytrid Nucleophaga amoebae described in the nineteenth century. Our results show that Nucleophaga, possessing a morphotype intermediate between Rozella and Paramicrosporidium, emerges as a unique lineage within the Rozellomycota. The recovery and characterization of new members of Rozellomycota are of high value for the understanding of the early evolutionary history of the Fungi and related lineages.

Molecular identification of Nucleophaga terricolae sp. nov. (Rozellomycota), and new insights on the origin of the Microsporidia

Microsporidia are widespread endoparasites of animals, including humans. They are characterized by highly modified morphological and genetic features that cause difficulties in elucidating their enigmatic origin and evolution. Recent advances, however, indicate that the Microsporidia have emerged from the Rozellomycota, forming together either the most basal lineage of the Fungi or its closer relative. The Rozellomycota comprise a huge diversity of uncultured environmental clones, with a very few known species endoparasitic of algae and water moulds, like the chytrid-like Rozella, and of free-living amoebae, like Nucleophaga and the microsporidia-like Paramicrosporidium. A possible ancestral microsporidium, Mitosporidium, has recently been described from the water flea Daphnia, since the phylogenomic reconstruction showed that it branches to the root of the microsporidian tree, while the genome analysis revealed a fungal-like nuclear genome and the persistence of a mitochondrial genome. Here we report the 18S rDNA molecular phylogeny of an additional microsporidium-like endoparasite of amoebae, which has a developmental cycle almost identical to that of Nucleophaga amoebae. Our results show that the endoparasite is closely related to N. amoebae, forming a distinct species, for which we propose the name Nucleophaga terricolae. Furthermore, the Nucleophaga lineage is recovered as sister to the Microsporidia while Mitosporidium turns out to be member of a well-supported group of environmental clones. These results raise the question about the actual ancestry of the Microsporidia within the Rozellomycota. A precise and robust phylogeny will require further comparative genomic studies of these various strains, and should also consider the primitive microsporidia, for which genetic data are still lacking, because all these organisms are essentially morphologically similar.

Molecular characterization and ultrastructure of a new amoeba endoparasite belonging to the Stenotrophomonas maltophilia complex

Naegleria and Acanthamoeba spp. were recovered from biofilm of a flushing cistern in a lavatory and both were found to be infected by rod-shaped bacteria enclosed within a vacuole. These intracellular bacteria behave like parasites, causing lysis of host amoebae. The bacteria proved unculturable on bacteriological media, and but could be maintained as endocytobionts within Acanthamoeba on agar plates. A marked differential host preference was observed in co-culture assays with various strains of amoebae. Molecular phylogenetic analyses performed on almost complete 16S rDNA sequences showed that the bacteria emerged as an atypical rapidly-evolving strain within the Stenotrophomonas maltophilia complex (Gamma-Proteobacteria, Xanthomonadales).

“Candidatus Mesochlamydia elodeae” (Chlamydiae: Parachlamydiaceae), a novel chlamydia parasite of free-living amoebae

Vannella sp. isolated from waterweed Elodea sp. was found infected by a chlamydia-like organism. This organism behaves like a parasite, causing the death through burst of its host. Once the vannellae degenerated, the parasite was successfully kept in laboratory within a Saccamoeba sp. isolated from the same waterweed sample, which revealed in fine through electron microscopy to harbor two bacterial endosymbionts: the chlamydial parasite we introduce and another endosymbiont initially and naturally present in the host. Herein, we provide molecular-based identification of both the amoeba host and its two endosymbionts, with special focus on the chlamydia parasite. High sequence similarity values of the 18S rDNA permitted to assign the amoeba to the species Saccamoeba lacustris (Amoebozoa, Tubulinea). The bacterial endosymbiont naturally harbored by the host belonged to Sphingomonas koreensis (Alpha-Proteobacteria). The chlamydial parasite showed a strict specificity for Saccamoeba spp., being unable to infect a variety of other amoebae, including Acanthamoeba, and it was itself infected by a bacteriophage. Sequence similarity values of the 16S rDNA and phylogenetic analysis indicated that this strain is a new member of the family Parachlamydiaceae, for which we propose the name “Candidatus Mesochlamydia elodeae.”