A robust phylogenetic framework for members of the order Legionellales and its main genera (Legionella, Aquicella, Coxiella and Rickettsiella) based on phylogenomic analyses and identification of molecular markers demarcating different clades.

The order Legionellales contains several clinically important microorganisms. Although members of this order are well-studied for their pathogenesis, there is a paucity of reliable characteristics distinguishing members of this order and its constituent genera. Genome sequences are now available for 73 Legionellales species encompassing ≈90% of known members from different genera. With the aim of understanding evolutionary relationships and identifying reliable molecular characteristics that are specific for this order and its constituent genera, detailed phylogenetic and comparative analyses were conducted on the protein sequences from these genomes. A phylogenomic tree was constructed based on 393 single copy proteins that are commonly shared by the members of this order to delineate the evolutionary relationships among its members. In parallel, comparative analyses were performed on protein sequences from Legionellales genomes to identify novel molecular markers consisting of conserved signature indels (CSIs) that are specific for different clades and genera. In the phylogenomic tree and in an amino acid identity matrix based on core proteins, members of the genera Aquicella, Coxiella, Legionella and Rickettsiella formed distinct clades confirming their monophyly. In these studies, Diplorickettsia massiliensis exhibited a close relationship to members of the genus Rickettsiella. The results of our comparative genomic analyses have identified 59 highly specific molecular markers consisting of CSIs in diverse proteins that are uniquely shared by different members of this order. Four of these CSIs are specific for all Legionellales species, except the two deeper-branching "Candidatus Berkiella" species, providing means for identifying members of this order in molecular terms. Twenty four, 7 and 6 CSIs are uniquely shared by members of the genera Legionella, Coxiella and Aquicella, respectively, identifying these groups in molecular terms. The descriptions of these three genera are emended to include information for their novel molecular characteristics. We also describe 12 CSIs that are uniquely shared by D. massiliensis and different members of the genus Rickettsiella. Based on these results, we are proposing an integration of the genus Diplorickettsia with Rickettsiella. Three other CSIs suggest that members of the genera Coxiella and Rickettsiella shared a common ancestor exclusive of other Legionellales. The described molecular markers, due to their exclusivity for the indicated taxa/genera, provide important means for the identification of these clinically important microorganisms and for discovering novel properties unique to them.

Robust demarcation of 17 distinct Bacillus species clades, proposed as novel Bacillaceae genera, by phylogenomics and comparative genomic analyses: description of Robertmurraya kyonggiensis sp. nov. and proposal for an emended genus Bacillus limiting it only to the members of the Subtilis and Cereus clades of species.

To clarify the evolutionary relationships and classification of Bacillus species, comprehensive phylogenomic and comparative analyses were performed on >300 Bacillus/Bacillaceae genomes. Multiple genomic-scale phylogenetic trees were initially reconstructed to identify different monophyletic clades of Bacillus species. In parallel, detailed analyses were performed on protein sequences of genomes to identify conserved signature indels (CSIs) that are specific for each of the identified clades. We show that in different reconstructed trees, most of the Bacillus species, in addition to the Subtilis and Cereus clades, consistently formed 17 novel distinct clades. Additionally, some Bacillus species reliably grouped with the genera Alkalicoccus, Caldalkalibacillus, Caldibacillus, Salibacterium and Salisediminibacterium. The distinctness of identified Bacillus species clades is independently strongly supported by 128 identified CSIs which are unique characteristics of these clades, providing reliable means for their demarcation. Based on the strong phylogenetic and molecular evidence, we are proposing that these 17 Bacillus species clades should be recognized as novel genera, with the names Alteribacter gen. nov., Ectobacillus gen. nov., Evansella gen. nov., Ferdinandcohnia gen. nov., Gottfriedia gen. nov., Heyndrickxia gen. nov., Lederbergia gen. nov., Litchfieldia gen. nov., Margalitia gen. nov., Niallia gen. nov., Priestia gen. nov., Robertmurraya gen. nov., Rossellomorea gen. nov., Schinkia gen. nov., Siminovitchia gen. nov., Sutcliffiella gen. nov. and Weizmannia gen. nov. We also propose to transfer 'Bacillus kyonggiensis' to Robertmurraya kyonggiensis sp. nov. (type strain: NB22=JCM 17569T=DSM 26768). Additionally, we report 31 CSIs that are unique characteristics of either the members of the Subtilis clade (containing the type species B. subtilis) or the Cereus clade (containing B. anthracis and B. cereus). As most Bacillus species which are not part of these two clades can now be assigned to other genera, we are proposing an emended description of the genus Bacillus to restrict it to only the members of the Subtilis and Cereus clades.

Control of Legionella Contamination and Risk of Corrosion in Hospital Water Networks following Various Disinfection Procedures.

Physical and chemical disinfection methods have been proposed with the aim of controlling Legionella water contamination. To date, the most effective procedures for reducing bacterial contamination have not yet been defined. The aim of this study was to assess the long-term effectiveness of various disinfection procedures in order to reduce both culturable and nonculturable (NC) legionellae in different hospital water networks treated with heat, chlorine dioxide, monochloramine, and hydrogen peroxide. The temperature levels and biocide concentrations that proved to give reliable results were analyzed. In order to study the possible effects on the water pipes, we verified the extent of corrosion on experimental coupons after applying each method for 6 months. The percentage of positive points was at its lowest after treatment with monochloramine, followed by chlorine dioxide, hydrogen peroxide, and hyperthermia. Different selections of Legionella spp. were observed, as networks treated with chlorine-based disinfectants were contaminated mainly by Legionella pneumophila serogroup 1, hyperthermia was associated with serogroups 2 to 14, and hydrogen peroxide treatment was associated mainly with non-pneumophila species. NC cells were detected only in heat-treated waters, and also when the temperature was approximately 60°C. The corrosion rates of the coupons were within a satisfactory limit for water networks, but the morphologies differed. We confirm here that chemical disinfection controls Legionella colonization more effectively than hyperthermia does. Monochloramine was the most effective treatment, while hydrogen peroxide may be a promising alternative to chlorine-based disinfectants due to its ability to select for other, less virulent or nonpathogenic species.