In Situ Detection of Novel Bacterial Endosymbionts of Acanthamoeba spp. Phylogenetically Related to Members of the Order Rickettsiales

Abstract

Acanthamoebae are ubiquitous soil and water bactivores which may serve as amplification vehicles for a variety of pathogenic facultative bacteria and as hosts to other, presently uncultured bacterial endosymbionts. The spectrum of uncultured endosymbionts includes gram-negative rods and gram-variable cocci, the latter recently shown to be members of the Chlamydiales. We report here the isolation from corneal scrapings of two Acanthamoebastrains that harbor gram-negative rod endosymbionts that could not be cultured by standard techniques. These bacteria were phylogenetically characterized following amplification and sequencing of the near-full-length 16S rRNA gene. We used two fluorescently labelled oligonucleotide probes targeting signature regions within the retrieved sequences to detect these organisms in situ. Phylogenetic analyses demonstrated that they displayed 99.6% sequence similarity and formed an independent and well-separated lineage within theRickettsiales branch of the alpha subdivision of theProteobacteria. Nearest relatives included members of the genus Rickettsia, with sequence similarities of approximately 85 to 86%, suggesting that these symbionts are representatives of a new genus and, perhaps, family. Distance matrix, parsimony, and maximum-likelihood tree-generating methods all consistently supported deep branching of the 16S rDNA sequences within the Rickettsiales. The oligonucleotide probes displayed at least three mismatches to all other available 16S rDNA sequences, and they both readily permitted the unambiguous detection of rod-shaped bacteria within intact acanthamoebae by confocal laser-scanning microscopy. Considering the long-standing relationship of mostRickettsiales with arthropods, the finding of a related lineage of endosymbionts in protozoan hosts was unexpected and may have implications for the preadaptation and/or recruitment of rickettsia-like bacteria to metazoan hosts.

Ultrastructural and biomolecular detection of Rickettsiales‐like organisms in tissues of rainbow trout with Red Mark Syndrome

“…In 10 of 17 trout investigated by TEM, ultrastructural observations revealed the presence of intracytoplasmic oval to short rod-shaped electron-dense microorganisms (400 to 800 nm in length and 100 to 200 nm in width) that often showed a typical cell wall organization of Gramnegative bacteria comparable to other ultramicroscopical descriptions of intracellular bacteria belonging to the order Rickettsiales Silverman 1991;Vannini et al 2010;Vellaiswamy, Campagna & Raoult 2011). These microorganisms were mainly localized free within host cell cytoplasm as observed by other authors for many Rickettsiales, including various Midichloria species or various Rickettsia species (Fritsche et al 1999;Dumler et al 2001;Duh Vannini et al 2010;Szokoli et al 2016). Silverman (1991) demonstrated by ultrastructural analysis that members of the genus Rickettsia exist free within the cytoplasm whereas Ehrlichia and Coxiella are bound by a phagosomal or phagolysosomal membrane.…”

“…Recent phylogenetic studies described a new clade-family within Rickettsiales (Alphaproteobacteria), named Midichloriaceae, comprising several bacterial symbionts and the bacteria Midichloria mitochondri, a symbiont of the hard tick Ixodes ricinus (Sassera et al 2006;Vannini et al 2010). To date, the only ultrastructural study performed in RMS trout failed to reveal the presence of intralesional microorganisms (Verner-Jeffreys et al 2008), whereas in the present investigation intracellular bacteria displaying ultrastructural features similar to bacteria belonging to Rickettsiaceae and Midichloriaceae families, order Rickettsiales, regarding shape, dimension, cell wall organization and subcellular localization, have been observed (Fritsche et al 1999;Sacchi et al 2004;Sassera et al 2006;Vannini et al 2010;Szokoli et al 2016).…”

Novel bacterial endosymbionts of Acanthamoeba spp. related to the Paramecium caudatum symbiont Caedibacter caryophilus

“…Acanthamoebae are well known as hosts for a variety of bacterial endocytobionts. Parasitic and actual endosymbiotic host±endocytobiont relationships have been observed (Birtles et al, 1996;Fritsche et al, 1999). Naturally occurring endocytobionts of acanthamoebae that have previously been characterized phylogenetically are af®liated with either the genus Legionella (Birtles et al, 1996;Michel et al, 1998), the Chlamydiales Fritsche et al, submitted) or the Rickettsiales (Fritsche et al, 1999).…”

“…Parasitic and actual endosymbiotic host±endocytobiont relationships have been observed (Birtles et al, 1996;Fritsche et al, 1999). Naturally occurring endocytobionts of acanthamoebae that have previously been characterized phylogenetically are af®liated with either the genus Legionella (Birtles et al, 1996;Michel et al, 1998), the Chlamydiales Fritsche et al, submitted) or the Rickettsiales (Fritsche et al, 1999). Here, we report the existence of a novel fourth group of Acanthamoeba endosymbionts, which are members of the a-subclass of Proteobacteria and are clearly af®liated to a monophyletic cluster of exclusively obligate intracellular bacteria distinct from, but related to, the Rickettsiaceae.…”

“…In contrast to these transient interrelationships, which result in lysis of the infected amoebae, the presence of stably infecting, obligate bacterial endosymbionts has been reported for about 25% of all Acanthamoeba isolates (Fritsche et al, 1993). Using the rRNA approach, the relationship of some of these endosymbionts to the genera Rickettsia and Chlamydia has been demonstrated Fritsche et al, 1999;T. R. Fritsche et al, submitted).…”