16S rRNA Gene Analysis Research Articles

First Report of 'Candidatus Phytoplasma trifolii' Associated with Little Leaf and Floral Virescence Disease in Solanum violaceum from the North East Region of India.

Solanum violaceum Ortega, (Indian nightshade), is a medicinal plant belonging to the family Solanaceae. It grows naturally as a weed in agricultural fields, forest edges, road sides and fallow lands throughout the North East Region of India. It is mostly consumed as a vegetable by the native people of the region because of its promising therapeutic effects (Islam and Islam 2018). In November 2019, typical phytoplasma-suspected symptoms such as little leaf, yellowing and floral virescence were observed on S. violaceum plants (Figure 1) in Kaliabor, Nagaon district of Assam, India (26.3220'N, 92.5540'E), with about 8% incidence based on visual observations. To investigate the possibility of a phytoplasma association with the symptoms, total DNA was isolated from collected leaf samples (symptomatic-6 and asymptomatic-9) by following the CTAB protocol (Kollar et al. 1990). The DNAs isolated were assayed for the presence of phytoplasma using polymerase chain reaction (PCR) assays performed with P1/P6 primer pair for 16S rRNA gene (Deng and Hiruki 1991) and SecAfor1/SecArev3 for secA gene (Hodgetts et al. 2008). The direct PCR targeting the 16S rRNA gene and secA gene amplified a product of about 1.5 kb and 840 bp, respectively, from all the symptomatic plant samples but not from any of the asymptomatic plant samples. All amplicons were double strand sequenced and corresponding high quality sequences were deposited in the GenBank with accession numbers MW261863 for 16S rRNA gene and MW885174 for secA gene with a sequence length of 1406 bp and 532 bp, respectively. Pairwise sequence comparison of 16S rRNA gene sequence of S. violaceum phytoplasma isolate revealed 100% sequence identity with strain of 'Candidatus Phytoplasma trifolii' (Accession number, EF186820) and secA gene showed up to 95% sequence identity with the same organism (Accession number, KX784498). Further analyses of the 16S rRNA and secA genes based phylogenetic tree (Figure 2A and B) and the iPhyClassifier-based virtual RFLP analysis of 16S rRNA gene revealed that the phytoplasma-associated with little leaf and floral virescence of S. violaceum belongs to 16SrIV-D subgroup with a similarity coefficient of 1.0. The 16S rRNA and secA gene sequences comparison confirmed the close association of phytoplasma strain associated with S. violaceum with 16SrVI-D subgroup phytoplasma. Earlier, the 16SrVI-D subgroup of phytoplasma has been reported to be associated with many horticultural and other agricultural crops in India (Rao 2021). Recently, the 16SrVI-D strain was reported in eggplant from Assam, India (Accession number, MW261866), showing up to 100% sequence identity with S. violaceum strain (Accession number, MW261863) (Dutta et al. 2020). To the best of our knowledge, this is the first report of the association of the 16SrVI-D subgroup of phytoplasma strain with S. violaceum plant, which is an important medicinal plant widely used in folk and traditional Indian systems of medicine. Since this study has confirmed S. violaceum as a new host of 16SrVI-D group of phytoplasma, further studies on the disease epidemiology and insects vectoring the phytoplasma would help formulate effective management strategies in preventing further spread to other hosts.

Effect of Humin and Chemical Factors on CO2-Fixing Acetogenesis and Methanogenesis.

Acetogenesis and methanogenesis have attracted attention as CO2-fixing reactions. Humin, a humic substance insoluble at any pH, has been found to assist CO2-fixing acetogenesis as the sole electron donor. Here, using two CO2-fixing consortia with acetogenic and methanogenic activities, the effect of various parameters on these activities was examined. One consortium utilized humin and hydrogen (H2) as electron donors for acetogenesis, either separately or simultaneously, but with a preference for the electron use from humin. The acetogenic activity was accelerated 14 times by FeS at 0.2 g/L as the optimal concentration, while being inhibited by MgSO4 at concentration above 0.02 g/L and by NaCl at concentrations higher than 6 g/L. Another consortium did not utilize humin but H2 as electron donor, suggesting that humin was not a universal electron donor for acetogenesis. For methanogenesis, both consortia did not utilize extracellular electrons from humin unless H2 was present. The methanogenesis was promoted by FeS at 0.2 g/L or higher concentrations, especially without humin, and with NaCl at 2 g/L or higher concentrations regardless of the presence of humin, while no significant effect was observed with MgSO4. Comparative sequence analysis of partial 16S rRNA genes suggested that minor groups were the humin-utilizing acetogens in the consortium dominated by Clostridia, while Methanobacterium was the methanogen utilizing humin with H2.

Cultivation and metabolic insights of an uncultured clade, Bacteroidetes VC2.1 Bac22 (Candidatus Sulfidibacteriales ord. nov.), from deep-sea hydrothermal vents.

Bacteroidetes VC2.1 Bac22 (referred to as VC2.1) is an uncultured clade that is widely distributed in marine ecosystems, including hydrothermal vents, oxygen-minimum zones and other anoxic, sulfide-rich environments. However, the lack of cultured representatives and sequenced genomes of VC2.1 limit our understanding of its physiology, metabolism and ecological functions. Here, we obtained a stable co-culture of VC2.1 with autotrophic microbes by establishing an autotrophy-based enrichment from a hydrothermal vent chimney sample. We recovered a high-quality metagenome-assembled genome (MAG) that belonged to VC2.1. Phylogenetic analyses of both 16S rRNA genes and conserved protein markers suggested that VC2.1 belongs to a novel order in the Bacteroidetes phylum, which we named Candidatus Sulfidibacteriales. The metabolic reconstruction of this MAG indicated that VC2.1 could utilize polysaccharides, protein polymers and fatty acids as well as flexibly obtain energy via NO/N2 O reduction and polysulfide reduction. Our results reveal the ecological potential of this novel Bacteroidetes for complex organic carbons mineralization and N2 O sinks in deep-sea hydrothermal vents. Furthermore, guided by the genome information, we designed a new culture medium in which starch, ammonium and polysulfide were used as the carbon source, nitrogen source and electron acceptor respectively, to isolate VC2.1 successfully.

Removal of Antimony in Wastewater by Antimony Tolerant Sulfate-Reducing Bacteria Isolated from Municipal Sludge.

Antimony (Sb), a global and priority controlled pollutant, causes severe environmental issues. Bioremediation by microbial communities containing sulfate-reducing bacteria (SRB) is considered to be among the safest, economical, and environmentally friendly methods to remove Sb from wastewater. However, the roles of SRB species in these communities remain uncertain, and pure cultures of bacteria that may be highly efficient have not yet been developed for Sb removal. In this study, an Sb tolerant community was enriched from municipal sludge, and molecular ecological analysis showed that Escherichia (40%) and Desulfovibrio (15%) were the dominant bacteria. Further isolation and identification showed that the enriched SRB strains were closely related to Cupidesulfovibrio oxamicus, based on the molecular analyses of 16S rRNA and dsrB genes. Among them, a strain named SRB49 exhibited the highest activity in removal of Sb(V). SRB49 was able to remove 95% of Sb(V) at a concentration of 100 mg/L within 48 h under optimum conditions: a temperature of 37–40 °C, an initial pH value of 8, 4 mM of sulfate, and an initial redox potential of 145–229 mV. SEM-EDX analysis showed that SRB49 did not adsorb Sb(V) but reduced and precipitated Sb(V) via the formation of Sb2S3. The results demonstrated the potential roles that pure cultures of SRB species may play in Sb removal and the use of Sb-tolerant SRB strains for Sb remediation.

Diversity and Distribution of Anaerobic Ammonium Oxidation Bacteria in Hot Springs of Conghua, China.

Anaerobic ammonium oxidation (anammox) is an important process of the nitrogen cycle, and the anammox bacteria have been studied in a wide variety of environments. However, the distribution, diversity, and abundance of anammox bacteria in hot springs remain enigmatic. In this study, the anammox process was firstly investigated in hot springs of Conghua, China. Anammox-like bacterial sequences that closely affiliated to “Candidatus Brocadia,” “Candidatus Kuenenia,” “Candidatus Scalindua,” “Candidatus Anammoxoglobus,” and “Candidatus Jettenia” were detected. Several operational taxonomic units (OTUs) from this study shared low sequence identities to the 16S rRNA gene of the known anammox bacteria, suggesting that they might be representing putative novel anammox bacteria. A quantitative PCR analysis of anammox-specific 16S rRNA gene confirmed that the abundance of anammox bacteria ranged from 1.60 × 104 to 1.20 × 107 copies L–1. Nitrate was a key environmental factor defining the geographical distribution of the anammox bacterial community in the hot spring ecosystem. Dissolved inorganic carbon had a significant influence on anammox bacterial biodiversity. Our findings for the first time revealed that the diverse anammox bacteria, including putative novel anammox bacterial candidates, were present in Conghua hot spring, which extended the existence of anammox bacteria to the hot springs in China and expands our knowledge of the biogeography of anammox bacteria. This work filled up the research lacuna of anammox bacteria in Chinese hot spring habitat and would guide for enrichment strategies of anammox bacteria of Conghua hot springs.

In-Depth Analysis of Bacillus anthracis 16S rRNA Genes and Transcripts Reveals Intra- and Intergenomic Diversity and Facilitates Anthrax Detection.

ABSTRACTAnalysis of 16S rRNA (rRNA) genes provides a central means of taxonomic classification of bacterial species. Based on presumed sequence identity among species of the Bacillus cereus sensu lato group, the 16S rRNA genes of B. anthracis have been considered unsuitable for diagnosis of the anthrax pathogen. With the recent identification of a single nucleotide polymorphism in some 16S rRNA gene copies, specific identification of B. anthracis becomes feasible. Here, we designed and evaluated a set of in situ, in vitro, and in silico assays to assess the unknown 16S state of B. anthracis from different perspectives. Using a combination of digital PCR, fluorescence in situ hybridization, long-read genome sequencing, and bioinformatics, we were able to detect and quantify a unique 16S rRNA gene allele of B. anthracis (16S-BA-allele). This allele was found in all available B. anthracis genomes and may facilitate differentiation of the pathogen from any close relative. Bioinformatics analysis of 959 B. anthracis SRA data sets inferred that abundances and genomic arrangements of the 16S-BA-allele and the entire rRNA operon copy numbers differ considerably between strains. Expression ratios of 16S-BA-alleles were proportional to the respective genomic allele copy numbers. The findings and experimental tools presented here provide detailed insights into the intra- and intergenomic diversity of 16S rRNA genes and may pave the way for improved identification of B. anthracis and other pathogens with diverse rRNA operons.IMPORTANCE For severe infectious diseases, precise pathogen detection is crucial for antibiotic therapy and patient survival. Identification of Bacillus anthracis, the causative agent of the zoonosis anthrax, can be challenging when querying specific nucleotide sequences such as in small subunit rRNA (16S rRNA) genes, which are commonly used for typing of bacteria. This study analyzed on a broad genomic scale a cryptic and hitherto underappreciated allelic variant of the bacterium’s 16S rRNA genes and their transcripts using a set of in situ, in vitro, and in silico assays and found significant intra- and intergenomic heterogeneity in the distribution of the allele and overall rRNA operon copy numbers. This allelic variation was uniquely species specific, which enabled sensitive pathogen detection on both DNA and transcript levels. The methodology used here is likely also applicable to other pathogens that are otherwise difficult to discriminate from their less harmful relatives.

Isolation and Complete Genome Sequence Analysis of Kosakonia cowanii Pa82, a Novel Pathogen Causing Bacterial Wilt on Patchouli.

Pogostemon cablin (patchouli), an important medicinal and aromatic plant, is widely used in traditional Chinese medicine as well as in perfume industry. Patchouli plants are susceptible to bacterial wilt disease, which causes significant economic losses by reduction in yield and quality of the plant products. However, few studies focus on the pathogens causing bacterial wilt on patchouli. In this study, strain Pa82 was isolated from diseased patchouli plants with typical bacterial wilt symptoms in Guangdong province, China, and was confirmed to be a highly virulent pathogen of patchouli bacterial wilt. Comparative sequence analysis of 16S rRNA gene showed that the strain was closely related to Kosakonia sp. CCTCC M2018092 (99.9% similarity) and Kosakonia cowanii Esp_Z (99.8% similarity). Moreover, phylogenetic tree based on 16S rRNA gene sequences showed that the strain was affiliated with genus Kosakonia. Further, the whole genome of strain Pa82 was sequenced, and the sequences were assembled and annotated. The complete genome of the strain consists of one chromosome and three plasmids. Average nucleotide identity (ANI) and phylogenetic analysis revealed that the strain belongs to Kosakonia cowanii (designated Kosakonia cowanii Pa82). Virulence-related genes of the strain involved in adherence, biofilm formation, endotoxin and other virulence factors were predicted. Among them, vgrG gene that encodes one of the type VI secretion system components was functionally validated as a virulence factor in Kosakonia cowanii Pa82 through construction of Tn5 insertion mutants and identification of mutant defective in virulence.

Irritable bowel syndrome therapy in Vietnam with probiotic enterococci

In this work, we compared composition of the intestinal microbiota of healthy volunteers and patients with irritable bowel syndrome (IBS) in Hanoi residents before and after therapy with a probiotic starter culture based on the bacteriocinogenic strain Enterococcus faecium L3 (L3), which is successfully used to correct dysbiosis and treat IBS in Russia.In IBS patients (IBS group) α-diversity was higher than in the control group (Healthy) and in IBS patients after probiotic therapy (IBS + L3 group). Phylogenetic analysis of the studied samples refl ected the division of the studied communities into 2 clusters, one of which grouped samples, mainly taken for IBS before therapy. Principal component analysis (PCoA) showed that most of the samples from the IBS group are located on the graph in a separate sector, far from the adjacent IBS + L3 and Нealthy samples.A study of the microbiome of Vietnamese patients with IBS after taking probiotic at the level of types (phyls) revealed a tendency towards an increase in the representation of Firmicutes. The introduction of L3 led to an increase in the population of members of the family Lachnospiraceae, such as butyrate- producing Roseburia and acetate-producing Blautia. At the same time, the percentage of staphylococci and bacteria belonging to the Enterobacteriaceae family was decreased. In addition to positive changes in the functioning of the gastrointestinal tract, revealed on the basis of clinical data, the features of the intestinal microbiota of IBS residents of Hanoi compared with healthy volunteers and in the same patients after probiotic therapy were revealed by metagenomic analysis of 16S rRNA genes. Positive shifts in the intestinal microbiome of IBS patients in Vietnam after therapy with a probiotic starter culture based on L3 do not fundamentally diff er from the previously described eff ects noted by the authors when using this probiotic in Russia, which creates the preconditions for expanding the possibility of its use in the Asian region.

A new genus of soil green microalgae Edaphochloris gen. nov. (Chlorellaceae, Trebouxiophyceae)

The problem of studying the species diversity of small single-celled green coccoid microalgae (Chlorophyta) is still relevant, since their simple and uniform cellular morphology makes it extremely difficult to correctly identify them. It is now generally accepted that the best approach for creating a new, more natural classification of “little green balls” is an approach that combines morphological, ecological, physiological and molecular phylogenetic methods. Strains ACSSI 104 and ACSSI 144 were isolated from solonetz and chestnut soil (Volgograd region, Russia). Their cells have a typical Nannochloris-like morphology: single spherical or subspherical cells, chloroplast single, parietal, cup-shaped, without pyrenoid, but with starch grains in it. Old cells accumulate numerous oil droplets in cytoplasm. Reproduction by 2−4 autospores. Autospores of both strains are equal size and exhibit liberation by rupture of sporangium cell wall. Zoospores and sexual reproduction not observed. Both strains are part of independent cluster closely related to genera Marvania, Pumiliosphaera, Nannochloris and Gloeotila. A comparative analysis of morphological (cell size, chloroplast type) and ultrastructural characteristics (structure of cell wall and lamellar system), features of reproduction (asexual reproduction by autospores only) and habitat (soil) with other members of the Nannochloris-clade, as well as results of analysis of 18S rRNA gene and ITS2 secondary structure of the studied strains, allowed us to propose a new genus – Edaphochloris with the type species – E. andreyevae. The report was prepared based on the materials of the V All-Russian Scientific Conference with international participation "Algae: problems of systematics, ecology and use in monitoring", 2021.

Investigation of the microbial community structure and diversity in the environment surrounding a veterinary antibiotic production factory.

The ecological toxicity caused by antibiotic residues and resistance genes in the environment affects the community structures and activities of environmental microorganisms; the ecological toxicity effects of a long-term exposure to low doses antibiotic residues on microorganisms have not however been well-studied. In this work, sequence analysis and species annotation of the full-length 16S rRNA gene were carried out on the extracted whole genome by a 3-generation sequencing method to analyze the diversity of the microbial populations and the population differences among different sampling sites in the environment surrounding a veterinary antibiotic production factory. A total of 1720 OTUs (Operational Taxonomic Unit, OTU) were found, of which 1055 OTUs were in the river samples and 993 OTUs were in the soil samples. 643 and 438 bacterial strains were identified in the river water and soil samples respectively. The bacterial populations are classified into 29 phylum, 612 genus, and 849 species. The dominant phylum of bacteria was Proteobacteria, which was also the absolutely dominant phylum. Shannon diversity index of the bacteria showed that the bacterial abundance in downstream river was significantly higher than that in an upstream non-polluted area (P < 0.001), but the difference of bacterial abundance between soil samples was not significant. There were 61 biomarkers in the river water samples from different sampling points, and 14 biomarkers in soil samples. It was found by the difference statistics of the microbial community that there are multiple biomarkers between this veterinary drug production site and the upstream non-polluted area. Significant differences between multiple functional genes were also found in metabolic pathways of the microorganisms. A similar trend was found for the distribution of antibiotic resistance genes (ARGs). It is concluded that the population composition of microorganisms and diversity are likely related to antibiotic residues and to the distributions of ARGs in the environment surrounding the antibiotic production factory.

ACTIVITY OF SERRATIA SPP. AND BACILLUS SPP. AS BIOCONTROL AGENTS AGAINST MELOIDOGYNE INCOGNITA INFECTING TOMATO

Background: Tomato )Solanum lycopersicum( is one of the most popular vegetable crops in Egypt. It is affected by a wide range of pests; prominent among them is root-knot nematode (RKN) (Meloidogyne spp.) which is considered as one of the major limiting factors affecting tomato productions worldwide, especially in Egypt. Aim of study: The present study was performed to evaluate the quantitative and qualitative chitionolytic potentials of Serratia, Bacillus megaterium and B. subtilis as well as their nematicidal effect against Meloidogyne incognita under both in vitro and in vivo conditions. Methodology: Qualitative and quantitative assays of bacterial chitinase of the applied bacteria were determined using Chitinase assay agar and liquid media, as well as the nelson-somogyi method. Nematicidal and ovicidal activities of bacterial treatments were evaluated by Test Tube Bio-assay technique. A greenhouse experiment was conducted for in vivo evaluation of bacterial treatments on nematode parameters. The most effective bacterial isolate was molecular identified based on the nucleotide sequencing of its 16S rRNA gene. Results: Serratia sp. (S2) showed the highest total and specific chitinase activities (0.620 U/mL and 0.079 U/mg), respectively, followed by S1 (0.560 U/mL and 0.061 U/mg) and S3 (0.434 U/mL and 0.057 U/mg). B. megaterium appeared the minimum total and specific chitinase activities (0.320 U/mL and 0.042 U/mg), and no chitinase activities was shown for B. subtilis. Serratia sp. (S2) recorded the highest ovicidal activity (53.61%) even when applied at low concentration (1%). Among greenhouse experiment, Serratia sp. (S2) recorded the minimum number of galls and egg masses. The nucleotide sequence analysis of 16S rRNA gene of Serratia sp. (S2) proved that this isolate could be a strain related to S. marcescens, therefore, it was documented in GenBank as S. marcescens strain ARC3 under the accession number MN533708. Conclusion: The results indicated that S. marcescens strain ARC3 showed the maximum nematicidal potentiality against M. incognita and its significant chitinolytic activity play an essential role in such bio-control effect. Recommendation: Chitonlytic bacteria could be suggested as effective and eco-friendly alternative approach for controlling the M. incognita.

Investigation of Sources, Diversity, and Variability of Bacterial Aerosols in Athens, Greece: A Pilot Study

We characterized the composition, diversity, and potential bacterial aerosol sources in Athens’ urban air by DNA barcoding (analysis of 16S rRNA genes) during three seasons in 2019. Air samples were collected using the recently developed Rutgers Electrostatic Passive Sampler (REPS). It is the first field application of REPS to study bacterial aerosol diversity. REPS samplers captured a sufficient amount of biological material to demonstrate the diversity of airborne bacteria and their variability over time. Overall, in the air of Athens, we detected 793 operational taxonomic units (OTUs), which were fully classified into the six distinct taxonomic categories (Phylum, Class, Order, etc.). These OTUs belonged to Phyla Actinobacteria, Firmicutes, Proteobacteria, Bacteroidetes, Cyanobacteria, and Fusobacteria. We found a complex community of bacterial aerosols with several opportunistic or potential pathogens in Athens’ urban air. Referring to the available literature, we discuss the likely sources of observed airborne bacteria, including soil, plants, animals, and humans. Our results on bacterial diversity are comparable to earlier studies, even though the sampling sites are different or geographically distant. However, the exact functional and ecological role of bioaerosols and, even more importantly, their impact on public health and the ecosystem requires further air monitoring and analysis.

Isolation and probiotic potential of lactic acid bacteria from swine feces for feed additive composition

Animal microbiota is becoming an object of interest as a source of beneficial bacteria for commercial use. Moreover, the escalating problem of bacterial resistance to antibiotics is threatening animals and humans; therefore, in the last decade intensive search for alternative antimicrobials has been observed. In this study, lactic acid bacteria (LAB) were isolated from suckling and weaned pigs feces (376) and characterized to determine their functional properties and usability as pigs additives. Selection of the most promising LAB was made after each stage of research. Isolates were tested for their antimicrobial activity (376) and susceptibility to antibiotics (71). Selected LAB isolates (41) were tested for the production of organic acids, enzymatic activity, cell surface hydrophobicity and survival in gastrointestinal tract. Isolates selected for feed additive (5) were identified by MALDI-TOF mass spectrometry and partial sequence analysis of 16S rRNA gene, represented by Lentilactobacillus, Lacticaseibacillus (both previously classified as Lactobacillus) and Pediococcus genus. Feed additive prototype demonstrated high viability after lyophilization and during storage at 4 °C and − 20 °C for 30 days. Finally, feed additive was tested for survival in simulated alimentary tract of pigs, showing viability at the sufficient level to colonize the host. Studies are focused on obtaining beneficial strains of LAB with probiotic properties for pigs feed additive.

The fish pathogen Flavobacterium columnare represents four distinct species: Flavobacterium columnare, Flavobacterium covae sp. nov., Flavobacterium davisii sp. nov. and Flavobacterium oreochromis sp. nov., and emended description of Flavobacterium columnare

Flavobacterium columnare is the causative agent of columnaris disease in freshwater fish and four discrete genetic groups exist within the species, suggesting that the species designation requires revision. The present study determined the taxonomic status of the four genetic groups of F. columnare using polyphasic and phylogenomic approaches and included five representative isolates from each genetic group (including type strain ATCC 23463T; genetic group 1). 16S rRNA gene sequence analysis revealed genetic group 2 isolate AL-02-36T, genetic group 3 isolate 90-106T, and genetic group 4 isolate Costa Rica 04-02-TNT shared less than <98.8 % sequence identity to F. columnare ATCC 23463T. Phylogenetic analyses of 16S rRNA and gyrB genes using different methodologies demonstrated the four genetic groups formed well-supported and distinct clades within the genus Flavobacterium. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (GGDC) values between F. columnare ATCC 23463T, genetic group 2 isolate AL-02-36T, genetic group 3 isolate 90-106T, and genetic group 4 isolate Costa Rica 04-02-TNT were less than 90.84% and 42.7%, respectively. Biochemical and physiological characteristics were similar among the four genetic groups; however, quantitative differences in fatty acid profiles were detected and MALDI-TOF analyses demonstrated numerous distinguishing peaks unique to each genetic group. Chemotaxonomic, MALDI-TOF characterization and ANI/GGDC calculations afforded differentiation between the genetic groups, indicating each group is a discrete species. Herein, the names F. covae sp. nov. (AL-02-36T), F. davisii sp. nov. (90-106T), and F. oreochromis sp. nov. (Costa Rica 04-02-TNT) are proposed to represent genetic groups 2, 3, and 4, respectively.

Detection, in silico analysis and molecular diversity of phytoplasmas from solanaceous crops in Turkey

Phytoplasma-like symptoms of leaf yellowing and calyx malformation were observed in eggplant (Solanum melongena L.), upward leaves and fruit malformation in pepper (Capsicum annuum L.), and aerial tuber formation in potato (S. tuberosum L.) during the survey performed in the late season (August to September) of 2015 and 2016 in Van province (Turkey). A total of 100 samples were tested by nested-PCR using universal primer pairs to assess the sanitary status of the solanaceous crops and to characterise the phytoplasma isolates. Among them, seven samples resulted in a 1.25 kb DNA fragment, and five (two eggplants, two peppers, and one potato) were molecularly characterised (Accession No.: KY579357, KT595210, MF564267, MF564266, and MH683601). BLAST and the virtual restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes revealed the presence of two distinct phytoplasma infections in solanaceous crops: ‘Candidatus Phytoplasma trifolii’ a member of the clover proliferation group (16SrVI) and subgroup A and ‘Candidatus P. solani’ a member of the stolbur group (16SrXII) and subgroup A. The virtual RFLP analysis and calculated coefficients of RFLP pattern similarities further revealed a remarkable genetic diversity among the ‘Candidatus P. solani’ isolates infecting pepper (similarity coefficient of 0.90) and eggplant (similarity coefficients of 0.98 and 1.00) at the same geographical area. This is the first report of the natural occurrence of ‘Candidadtus P. trifolii’ in potato from the Eastern Anatolia region, Turkey.

Prevotella illustrans sp. nov., derived from human oropharyngeal abscess puncture fluid.

An obligately anaerobic strain, designated as A2931T, was isolated from oropharyngeal abscess puncture fluid of a patient sampled during routine care at a hospital and further characterized both phenotypically, biochemically and genotypically. This Gram-negative rod-shaped bacterium was moderately saccharolytic and proteolytic. Phylogenetic analyses of full-length 16S rRNA gene and whole-genome sequences revealed it to be best placed in the genus Prevotella, but to be only comparatively distantly related to recognized species, with the closest relationship to Prevotella baroniae (average nucleotide identity and digital DNA-DNA hybridization values both well below the generally accepted thresholds). Strain A2931T had a genomic DNA G+C content of 47.7 mol%. Its most abundant cellular long-chain fatty acids were anteiso-C15 : 0, iso-C15 : 0 and C16 : 0. Taken together, this polyphasic data suggests strain A2931T to represent a novel species within the genus Prevotella, for which the name Prevotella illustrans sp. nov. is proposed. The type strain is A2931T (=DSM 108028T=CCOS 1232T=CCUG 72806T). Interestingly, we found strain A2931T to correspond to the oral taxon Prevotella HMT-820 in the Human Oral Microbiome Database, as supported by overall genome relatedness index analyses >99 %. Thus, our work not only closes one of the gaps of knowledge about hitherto unnamed species isolated from humans, but also will facilitate identification of this taxon both in the clinical microbiology context and in research alike.

Parvimonas parva sp. nov., derived from a human genito-urinary lesion.

A strain of obligately anaerobically growing Gram-positive cocci was isolated from a human genito-urinary sample and characterized by a polyphasic approach. Analyses of 16S rRNA gene and whole-genome sequences of this strain S3374T indicated that it belonged to the genus Parvimonas. Overall genome relatedness index calculations confirmed it to be phylogenetically distinct from Parvimonas micra (NCTC 11808T) as its most closely related species with standing in nomenclature, with average nucleotide identity and genome-to-genome distance values of 85.8 and 30.2 %, respectively. Biochemically, strain S3374T was strongly proteolytic and can be differentiated from P. micra (DSM 20468T) by absence of phosphatase activity. The DNA G+C content of strain S3374T was 28.6 mol%. Based on the phenotypical, biochemical and genetic findings, strain S3374T is considered to represent a novel species within the genus Parvimonas, for which the name Parvimonas parva sp. nov. is proposed. The type strain is S3374T (=DSM 110786T=CCOS 1934T=CCUG 74294T). This description adds strain S3374T as a second species to the genus Parvimonas which has so far been monotypic. While the type strain of this genus, P. micra, has a long standing in nomenclature and its role in human health and disease has been studied to some extent, this description of the proposed novel species represented by strain S3374T will allow microbiologists worldwide to identify isolates of P. parva sp. nov., a prerequisite for further investigation of its relevance in the clinical context and beyond.

Anisakis pegreffii Larvae in Sphyraena viridensis and Description of Granulomatous Lesions.

Simple SummaryFish-borne zoonoses are caused by bacteria and parasites, while no viral fish-borne zoonoses have been reported, to date. Regarding zoonoses caused by parasites, Anisakiasis is one of the most important, with Anisakis simplex and Anisakis pegreffii agents in the central Mediterranean Sea. Humans can be infected by accidental ingestion of third-stage larvae in raw, undercooked or improperly processed fish or cephalopods. After ingestion, the larvae migrate from the gastrointestinal tract to gastrointestinal tissue, causing pain and, subsequently, inflammatory reaction leading to eosinophilic granuloma. This kind of reaction has not been described to date, in fish, and the aim of this study is to describe gastric wall lesions caused by A. pegreffii in Sphyraena viridensis and to compare them to those reported in humans, which appear macroscopically identical, albeit showing significant microscopic differences.The aim of the present study was to describe gastric granuloma caused by Anisakis pegreffii in Sphyraena viridensis caught in the central Mediterranean Sea. Sixty-eight S. viridensis specimens were collected from different fish markets on the east coast of Sicily. Coelomic organs were observed both macroscopically and with the aid of stereomicroscope. Parasite specimens and lesioned tissues were collected for identification, histological and molecular analyses. Twelve specimens (p = 17.6%) were positive for the presence of nematode larvae, morphologically identified as larvae of Anisakis sp., with values of mean abundance and mean intensity of 0.9 and 4.8, respectively. One large female specimen showed massive parasite infection associated with nodular lesions of the gastric wall. By histology, several nematode larvae encysted through the gastric wall were found. The parasite bodies were surrounded by a granulomatous reaction made up of macrophages, epithelioid cells, some lymphocytes and an external connective sheet. Molecular analysis of 18S rRNA and cox2 genes from Anisakis sp. collected larvae, identified them as A. pegreffii. The lesions here described, though macroscopically superimposable on human eosinophilic granuloma, microscopically showed significant differences in the inflammatory cells involved and in the type of immune reaction.

Corynebacterium zhongnanshanii sp. nov. isolated from trachea of Marmota himalayana, Corynebacterium lujinxingii sp. nov. and Corynebacterium wankanglinii sp. nov. from human faeces.

Six novel facultatively anaerobic, Gram-stain-positive, rod-shaped, non-haemolytic bacteria (zg-320T/zg-336, zg-917T/zg-910 and zg-913T/zg-915) isolated from animal tissues and human faeces were found to belong to the genus Corynebacterium based on the phylogenetic analyses of 16S rRNA gene and 262 core genes set. Based on the greatest degree of 16S rRNA similarity, zg-320T/zg-336 had the highest 16S rRNA gene similarity to Corynebacterium falsenii DSM 44353T (97.51 %), zg-917T/zg-910 to Corynebacterium coyleae DSM 44184T (98.68 %), and zg-913T/zg-915 to Corynebacterium afermentans subsp. lipophilum CIP 103500T (98.79 %). The three novel type strains had a relatively high DNA G+C content (61.2-64.4 mol%), low DNA relatedness and ANI values with their respective neighbours: 23.5/72.7 %, 25.0/72.3%and 22.6/73.1 % (zg-320T vs. Corynebacterium auriscanis CIP 106629T, Corynebacterium resistens DSM 45100T and Corynebacterium suicordis DSM 45110T); 24.4/82.3% and 23.7/81.3 % (zg-917T vs. C. coyleae DSM 44184T and Corynebacterium jeddahense JCBT); 26.8/83.7% and 27.7/84.4 % (zg-913T vs. Corynebacterium mucifaciens ATCC 700355T and C. afermentans subsp. lipophilum CCUG 32105T). The three novel species had C16 : 0, C18 : 0, C18 : 1 ω9c and C18 : 0 ante/C18 : 2 ω6,9c as the major cellular fatty acids; MK-8(H2) in strain zg-917T and MK-9(H2) in strains zg-320T and zg-913T were found to be the major respiratory quinones. For the three novel species, the detected major polar lipids included diphosphatidylglycerol, phosphatidyl inositol mannoside, phosphatidylglycerol and phosphatidylinositol, the cell-wall peptidoglycan was based on meso-DAP, and the whole-cell sugars mainly included ribose, arabinose and galactose. The three novel species grew optimally at 35-37 °C, 0.5 % (w/v) NaCl and pH 7.0-8.0; notably, they were tolerant of 10.5 % (w/v) NaCl. Based on the results of these comprehensive analyses, three novel species in the genus Corynebacterium are proposed, aptly named Corynebacterium zhongnanshanii sp. nov. (zg-320T = GDMCC 1.1719T = JCM 34106T), Corynebacterium lujinxingii sp. nov. (zg-917T = GDMCC 1.1707T = JCM 34094T) and Corynebacterium wankanglinii sp. nov. (zg-913T = GDMCC 1.1706T = JCM 34398T).

A morphological and molecular reinvestigation of Janickina pigmentifera (Grassi, 1881) Chatton 1953 - an amoebozoan parasite of arrow-worms (Chaetognatha).

Amoebozoan parasites of arrow-worms (Chaetognatha) were isolated from their hosts living in plankton of the Bay of Villefranche (Mediterranean Sea). Based on the light microscopic characters, the amoebae were identified as Janickina pigmentifera (Grassi, 1881) by their limax locomotive form and due to the presence of the intracellular symbiont, Perkinsela amoebae, surrounded by a layer of pigment granules. Sequences of the 18S rRNA gene of both J. pigmentifera and its symbiont were obtained for the first time. The molecular phylogenetic analyses of 18S rRNA gene placed J. pigmentifera within the genus Neoparamoeba, a taxon also characterized by the presence of a symbiont, known as Perkinsela amoebae-like organism (PLO). The 18S rRNA gene sequence of P. amoebae from J. pigmentifera grouped with the sequences of 18S rRNA genes of PLOs from Neoparamoeba branchiphila and Neoparamoeba invadens. The first photo documentation of the light microscopic features of J. pigmentifera, such as locomotive form, the morphology of the nucleus and P. amoebae have been provided. The new results support the affinity of J. pigmentifera with the family Paramoebidae suggested previously based on the presence of PLO. In contrast to Janickina, typical members of Paramoebidae (Neoparamoeba and Paramoeba) have a flattened, dactylopodial locomotive form. This discrepancy in morphology can be explained by the obligate parasitic lifestyle of Janickina.