Patterns of Somatic Variants in Colorectal Adenoma and Carcinoma Tissue and Matched Plasma Samples from the Hungarian Oncogenome Program.

Analysis of circulating cell-free DNA (cfDNA) of colorectal adenoma (AD) and cancer (CRC) patients provides a minimally invasive approach that is able to explore genetic alterations. It is unknown whether there are specific genetic variants that could explain the high prevalence of CRC in Hungary. Whole-exome sequencing (WES) was performed on colon tissues (27 AD, 51 CRC) and matched cfDNAs (17 AD, 33 CRC); furthermore, targeted panel sequencing was performed on a subset of cfDNA samples. The most frequently mutated genes were APC, KRAS, and FBN3 in AD, while APC, TP53, TTN, and KRAS were the most frequently mutated in CRC tissue. Variants in KRAS codons 12 (AD: 8/27, CRC: 11/51 (0.216)) and 13 (CRC: 3/51 (0.06)) were the most frequent in our sample set, with G12V (5/27) dominance in ADs and G12D (5/51 (0.098)) in CRCs. In terms of the cfDNA WES results, tumor somatic variants were found in 6/33 of CRC cases. Panel sequencing revealed somatic variants in 8 out of the 12 enrolled patients, identifying 12/20 tumor somatic variants falling on its targeted regions, while WES recovered only 20% in the respective regions in cfDNA of the same patients. In liquid biopsy analyses, WES is less efficient compared to the targeted panel sequencing with a higher coverage depth that can hold a relevant clinical potential to be applied in everyday practice in the future.

Evolutionarily recent dual obligatory symbiosis among adelgids indicates a transition between fungus- and insect-associated lifestyles.

Adelgids (Insecta: Hemiptera: Adelgidae) form a small group of insects but harbor a surprisingly diverse set of bacteriocyte-associated endosymbionts, which suggest multiple replacement and acquisition of symbionts over evolutionary time. Specific pairs of symbionts have been associated with adelgid lineages specialized on different secondary host conifers. Using a metagenomic approach, we investigated the symbiosis of the Adelges laricis/Adelges tardus species complex containing betaproteobacterial ("Candidatus Vallotia tarda") and gammaproteobacterial ("Candidatus Profftia tarda") symbionts. Genomic characteristics and metabolic pathway reconstructions revealed that Vallotia and Profftia are evolutionary young endosymbionts, which complement each other's role in essential amino acid production. Phylogenomic analyses and a high level of genomic synteny indicate an origin of the betaproteobacterial symbiont from endosymbionts of Rhizopus fungi. This evolutionary transition was accompanied with substantial loss of functions related to transcription regulation, secondary metabolite production, bacterial defense mechanisms, host infection, and manipulation. The transition from fungus to insect endosymbionts extends our current framework about evolutionary trajectories of host-associated microbes.

Convergent patterns in the evolution of mealybug symbioses involving different intrabacterial symbionts.

Mealybugs (Insecta: Hemiptera: Pseudococcidae) maintain obligatory relationships with bacterial symbionts, which provide essential nutrients to their insect hosts. Most pseudococcinae mealybugs harbor a unique symbiosis setup with enlarged betaproteobacterial symbionts ('Candidatus Tremblaya princeps'), which themselves contain gammaproteobacterial symbionts. Here we investigated the symbiosis of the manna mealybug, Trabutina mannipara, using a metagenomic approach. Phylogenetic analyses revealed that the intrabacterial symbiont of T. mannipara represents a novel lineage within the Gammaproteobacteria, for which we propose the tentative name 'Candidatus Trabutinella endobia'. Combining our results with previous data available for the nested symbiosis of the citrus mealybug Planococcus citri, we show that synthesis of essential amino acids and vitamins and translation-related functions partition between the symbiotic partners in a highly similar manner in the two systems, despite the distinct evolutionary origin of the intrabacterial symbionts. Bacterial genes found in both mealybug genomes and complementing missing functions in both symbioses were likely integrated in ancestral mealybugs before T. mannipara and P. citri diversified. The high level of correspondence between the two mealybug systems and their highly intertwined metabolic pathways are unprecedented. Our work contributes to a better understanding of the only known intracellular symbiosis between two bacteria and suggests that the evolution of this unique symbiosis included the replacement of intrabacterial symbionts in ancestral mealybugs.

Happens in the best of subfamilies: establishment and repeated replacements of co-obligate secondary endosymbionts within Lachninae aphids.

Virtually all aphids maintain an obligate mutualistic symbiosis with bacteria from the Buchnera genus, which produce essential nutrients for their aphid hosts. Most aphids from the Lachninae subfamily have been consistently found to house additional endosymbionts, mainly Serratia symbiotica. This apparent dependence on secondary endosymbionts was proposed to have been triggered by the loss of the riboflavin biosynthetic capability by Buchnera in the Lachninae last common ancestor. However, an integral large-scale analysis of secondary endosymbionts in the Lachninae is still missing, hampering the interpretation of the evolutionary and genomic analyses of these endosymbionts. Here, we analysed the endosymbionts of selected representatives from seven different Lachninae genera and nineteen species, spanning four tribes, both by FISH (exploring the symbionts' morphology and tissue tropism) and 16S rRNA gene sequencing. We demonstrate that all analysed aphids possess dual symbiotic systems, and while most harbour S. symbiotica, some have undergone symbiont replacement by other phylogenetically-distinct bacterial taxa. We found that these secondary associates display contrasting cell shapes and tissue tropism, and some appear to be lineage-specific. We propose a scenario for symbiont establishment in the Lachninae, followed by changes in the symbiont's tissue tropism and symbiont replacement events, thereby highlighting the extraordinary versatility of host-symbiont interactions.

The pine bark Adelgid, Pineus strobi, contains two novel bacteriocyte-associated gammaproteobacterial symbionts.

Bacterial endosymbionts of the pine bark adelgid, Pineus strobi (Insecta: Hemiptera: Adelgidae), were investigated using transmission electron microscopy, 16S and 23S rRNA-based phylogeny, and fluorescence in situ hybridization. Two morphologically different symbionts affiliated with the Gammaproteobacteria were present in distinct bacteriocytes. One of them ("Candidatus Annandia pinicola") is most closely related to an endosymbiont of Adelges tsugae, suggesting that they originate from a lineage already present in ancient adelgids before the hosts diversified into the two major clades, Adelges and Pineus. The other P. strobi symbiont ("Candidatus Hartigia pinicola") represents a novel symbiont lineage in members of the Adelgidae. Our findings lend further support for a complex evolutionary history of the association of adelgids with a phylogenetically diverse set of bacterial symbionts.

Reproducibility of Vibrionaceae population structure in coastal bacterioplankton.

How reproducibly microbial populations assemble in the wild remains poorly understood. Here, we assess evidence for ecological specialization and predictability of fine-scale population structure and habitat association in coastal ocean Vibrionaceae across years. We compare Vibrionaceae lifestyles in the bacterioplankton (combinations of free-living, particle, or zooplankton associations) measured using the same sampling scheme in 2006 and 2009 to assess whether the same groups show the same environmental association year after year. This reveals complex dynamics with populations falling primarily into two categories: (i) nearly equally represented in each of the two samplings and (ii) highly skewed, often to an extent that they appear exclusive to one or the other sampling times. Importantly, populations recovered at the same abundance in both samplings occupied highly similar habitats suggesting predictable and robust environmental association while skewed abundances of some populations may be triggered by shifts in ecological conditions. The latter is supported by difference in the composition of large eukaryotic plankton between years, with samples in 2006 being dominated by copepods, and those in 2009 by diatoms. Overall, the comparison supports highly predictable population-habitat linkage but highlights the fact that complex, and often unmeasured, environmental dynamics in habitat occurrence may have strong effects on population dynamics.

Population genomics of early events in the ecological differentiation of bacteria.

Genetic exchange is common among bacteria, but its effect on population diversity during ecological differentiation remains controversial. A fundamental question is whether advantageous mutations lead to selection of clonal genomes or, as in sexual eukaryotes, sweep through populations on their own. Here, we show that in two recently diverged populations of ocean bacteria, ecological differentiation has occurred akin to a sexual mechanism: A few genome regions have swept through subpopulations in a habitat-specific manner, accompanied by gradual separation of gene pools as evidenced by increased habitat specificity of the most recent recombinations. These findings reconcile previous, seemingly contradictory empirical observations of the genetic structure of bacterial populations and point to a more unified process of differentiation in bacteria and sexual eukaryotes than previously thought.

Cellulomonas phragmiteti sp. nov., a cellulolytic bacterium isolated from reed (Phragmites australis) periphyton in a shallow soda pond.

An alkalitolerant and moderately halophilic strain, designated KB23(T), characterized by optimal growth at pH 8.0-9.0 and in the presence of 5-7 % (w/v) NaCl, was isolated from a reed (Phragmites australis) periphyton sample originating from an extremely shallow, alkaline soda pond located in Hungary. Cells of strain KB23(T) were Gram-stain-positive, motile straight rods. Strain KB23(T) was facultatively anaerobic, catalase-positive, oxidase-negative and contained peptidoglycan type A4ß (L-Orn-D-Asp). MK-9(H4) was the predominant isoprenoid quinone and anteiso-C(15 : 0), C(16 : 0) and anteiso-C(15 : 1) were the major cellular fatty acids. The DNA G+C content of strain KB23(T) was 74.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that this strain belongs to the genus Cellulomonas and that it is related most closely to Cellulomonas flavigena DSM 20109(T) (97.35 % similarity), Cellulomonas terrae DB5(T) (96.81 %), Cellulomonas iranensis O(T) (96.75), Cellulomonas chitinilytica X.bu-b(T) (96.60 %), Cellulomonas persica I(T) (96.53 %), Cellulomonas composti TR7-06(T) (96.45 %), Cellulomonas biazotea DSM 20112(T) (96.34 %) and Cellulomonas fimi DSM 20113(T) (96.20 %). According to these results, together with DNA-DNA hybridization and physiological data, strain KB23(T) is considered to represent a novel species of the genus Cellulomonas, for which the name Cellulomonas phragmiteti sp. nov. is proposed. The type strain is KB23(T) ( = DSM 22512(T)  = NCAIM B002303(T)).

Phylogenetic and metabolic bacterial diversity of Phragmites australis periphyton communities in two Hungarian soda ponds.

Bacterial diversity of reed (Phragmites australis) periphyton communities of Kelemen-szék and Nagy-Vadas (two Hungarian soda ponds) was investigated using molecular cloning and cultivation-based techniques. The majority of the 80 Kelemen-szék and 72 Nagy-Vadas bacterial isolates proved to be moderately halophilic and alkaliphilic. A great proportion of the isolates showed phosphatase and urease activity, utilized aesculin, citrate and certain biopolymers (e.g., gelatine and tween 80). Partial 16S rDNA sequence analysis of 33 Kelemen-szék and 20 Nagy-Vadas ARDRA group representatives showed Gram-positive (Nesterenkonia, Cellulomonas, Dietzia, Bacillus and Planococcus) dominance at both sampling sites. Species of the genera Acidovorax, Hydrogenophaga (beta-Proteobacteria) and Flavobacterium, Sphingobacterium (Bacteroidetes) were represented only from Kelemen-szék. Altogether 16 isolates showed low sequence similarity with yet described bacteria and may represent novel taxa. Screening of the 16S rRNA gene libraries of 129 Kelemen-szék and 158 Nagy-Vadas clones resulted in 30 and 28 different ARDRA groups, respectively. Sequence analysis revealed a Gram-negative (Rheinheimera, Aquimonas, Cellvibrio, Flavobacterium and Sphingobacterium) dominated phylogenetic diversity. A high number of the clones were affiliated with uncultured bacterial clones described from diverse environmental samples.

Bacillus aurantiacus sp. nov., an alkaliphilic and moderately halophilic bacterium isolated from Hungarian soda lakes.

Three alkaliphilic and moderately halophilic strains designated K1-5T, K1-10 and B1-1, characterized by optimal growth at pH 9.0-10.0 and at 3-7 % (w/v) NaCl, were isolated from extremely shallow, alkaline soda lakes located in Hungary. Cells of the strains are Gram-positive, straight rods and form a central to subterminal, ellipsoidal endospore. The isolates are strictly aerobic, catalase-positive, oxidase-negative and contain a peptidoglycan of type A1 gamma based on meso-diaminopimelic acid. In strain K1-5T, menaquinone-7 (MK-7) is the predominant isoprenoid quinone and anteiso-C15 : 0 is the major cellular fatty acid. The DNA G+C content of strain K1-5T is 42.9 mol%. 16S rRNA gene-based phylogenetic analysis revealed that the strains exhibit levels of sequence similarity of less than 95.8 % to known Bacillus species. According to the polyphasic characterization, the strains represent a novel species, for which the name Bacillus aurantiacus sp. nov. is proposed. The type strain is K1-5T (=DSM 18675T =CCM 7447T =NCAIM B002265T).

Molecular typing indicates an important role for two international clonal complexes in dissemination of VIM-producing Pseudomonas aeruginosa clinical isolates in Hungary.

VIM metallo-beta-lactamase-producing serotype O11 or O12 Pseudomonas aeruginosa isolates infecting or colonising 19 patients from seven hospitals were reported in Hungary between 2003 and 2005. In this study we characterised VIM-producing Pseudomonas spp. clinical isolates from two novel locations in Hungary; we identified three new bla(VIM) carrying integron types and the presence of the bla(VIM-2) allele in Hungary. By applying various typing techniques, including multilocus sequence typing, we revealed an important role of two international clonal complexes, CC4 and CC11, in the dissemination of bla(VIM)-positive P. aeruginosa in hospitals in Hungary. Isolate P12-Q, a representative strain from France of the major European multiresistant P12 clone, displayed ST111 which, according to eBURST analysis, is the presently calculated founder sequence type of CC4. This is in accordance with the wide geographic distribution of the P12 clone. Our data indicate that, although the CC4 clonal complex includes serotype O1 and O6 isolates as well, it also contains the P12 clone. We characterised a P. aeruginosa nosocomial clone with a singleton sequence type (ST313), that may have acquired bla(VIM-2) and bla(VIM-4) gene cassettes from a yet unidentified local gene pool in Hungary.

Diversity of reed (Phragmites australis) stem biofilm bacterial communities in two Hungarian soda lakes.

From reed biofilm samples of Kelemen-szék (Kiskunság National Park, KNP) and Nagy-Vadas (Hortobágy National Park, HNP) altogether 260 bacterial isolates were gained after serial dilutions and plating onto different media. Following a primary selection 164 strains were investigated by "traditional" phenotypic tests and clustered by numerical analysis. Fifty-six representative strains were selected to ARDRA and 16S rDNA sequence analysis for identification. Strains were identified as members of genera Agrobacterium, Paracoccus, Halomonas, Pseudomonas, Bacillus, Planococcus and Nesterenkonia. The species diversity was also investigated by a cultivation independent method. A clone library was constructed using the community DNA isolated from the biofilm sample of Kelemen-szék. Screening of the 140 bacterial clones resulted in 45 different ARDRA groups. Sequence analysis of the representatives revealed a great phylogenetic diversity. A considerable majority of the clones was affiliated with uncultured bacterial clones (with sequence similarity between 93 and 99%) originating from diverse environmental samples (for example salt marshes, compost or wastewater treatment plants). The DNA sequences of other clones showed the presence of genera Flavobacterium, Sphingobacterium, Pseudomonas and Agrobacterium.