16S Ribosomal DNA Research Articles

A PCR-based method for the diagnosis of Enterobius vermicularis in stool samples, specifically designed for clinical application.

Enterobius vermicularis (E. vermicularis) is a nematode that infects up to 200 million people worldwide, despite effective medications being available. Conventional diagnostic tests are hindered by low sensitivity and poor patient compliance. Furthermore, no biomolecular techniques are available for clinical application. The aim of this study was to develop a procedure specifically designed for clinical application to detect E. vermicularis by means of PCR. Two subject groups were taken into account: a group of 27 infected patients and a control group of 27 healthy subjects. A nested-PCR was performed on fecal samples to detect E. vermicularis. Due to the intrinsic difficulties of the fecal matrix, several countermeasures were adopted to ensure the efficient performance of the method: (a) a large amount of feces for the extraction process (20 g instead of 200 mg); (b) a combination of chemical and physical treatments to grind the fecal matrix; (c) an additional purification process for the negative samples after the first nested-PCR; and (d) the selection of a very specific target region for the PCR. Due to the lack of overlap with other organisms, a sequence of the 5S ribosomal DNA (rDNA) spacer region including the tract SL1 was chosen to design appropriate external and internal primers. The first nested-PCR detected E.vermicularis in 19/27 samples from infected patients. After further purification, 5/8 of the negative samples resulted positive at the second PCR. Conversely, all the samples from healthy controls resulted negative to both PCRs. Sensitivity and specificity of the method were, respectively, 88.9% and 100%. The results prove the high diagnostic accuracy of the proposed method, addressing and overcoming the challenges posed by both conventional tests and PCR-based approaches. Therefore, the method can be proposed for clinical application.

High diversity of 5S ribosomal DNA and evidence of recombination with the satellite DNA PcP190 in frogs

Distinct types of 5S rDNA repeats, differing by size and composition of its non-transcribed spacer (NTS), have been found in diverse taxa. Both concerted evolution and birth-and-death evolution have proven to play important roles in the evolution of the 5S rDNA family. In anurans, however, this subject has been underexplored as only a few anuran species had their 5S rDNA characterized and evolutionary analyzed to date. In the present study, we characterized the 5S rDNA sequences from species of two anuran families using classical molecular biology techniques and bioinformatic approaches. Based on the NTS, more than one type of 5S rDNA was identified in each analyzed species, which suggests that birth-and-death processes take part in the evolution of these sequences along the Anura tree of life. In addition, closely related species shared the same types of sequences, in accordance with the model of concerted evolution. We also found evidence of recombination between 5S rDNA and PcP190 satellite DNA, a repetitive sequence that is derived from the 5S rDNA. The interplay between 5S rDNA and PcP190 satellite DNA might favor the maintenance of the latter in the genome and respond to its presence in several species of frogs. The analysis of 5S rRNA transcripts confirmed the type I 5S rDNA of leiuperine as a functional 5S rRNA gene. Finally, the chromosome mapping of 5S rDNA sequences allowed some inferences of chromosome homology in Leiuperinae. In conclusion, our study provides additional information about the organization, differentiation and functionality of 5S rDNA in anuran species, revealing the potential participation of satellite DNA in the evolution of this family of rDNA.

The complete mitochondrial genome of the Arctic fairy shrimp Branchinectapaludosa (Müller, 1788) (Anostraca, Branchinectidae) from Sirius Passet, North Greenland.

Here we report the complete mitochondrial genome of the Arctic fairy shrimp, Branchinectapaludosa (Müller, 1788) (Anostraca, Branchinectidae), which was collected in the High Arctic of North Greenland. A complete 16,059 bp mitochondrion of B.paludosa was sequenced and assembled with the Illumina next generation sequencing platform. The B.paludosa mitogenome contains 13 PCGs, 22 tRNAs and 2 rRNA genes that are commonly observed in most metazoans and shows the conserved gene arrangement pattern of Anostraca. Our results of the phylogenomic analysis are consistent with the previous phylogenetic relationship, based on nuclear 18S ribosomal DNA. The B.paludosa mitogenome will be useful for understanding the geographical distribution and phylogenetic relationship of anostracans.

Immune and intestinal microbiota responses to heat stress in Chinese mitten crab (Eriocheir sinensis)

Temperature is an important environmental stressor that affects the metabolism and immunity of Chinese mitten crab. In this study, we investigated immune-related enzyme activity, metabolism-related substances in haemolymph and intestinal microbiota responses of Eriocheir sinensis assigned to the control (24 °C) and heat stress (32 °C) groups. Based on the data obtained, the levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and lactate dehydrogenase in the heat stress group exhibited a trend of increasing first and then decreasing, and all peaked at 12 h or 24 h. The metabolism-related substances in the haemolymph, including glucose, total cholesterol, and total protein, all showed significant differences between the control and heat stress groups from the mid to late period (P < 0.05). Under heat stress, the levels of alkaline phosphatase and acid phosphatase were both significantly higher than those of the control group from 12 h to 24 h and significantly lower at 72 h and 96 h (P < 0.05). The T-AOC of the heat stress group was significantly lower than that of the control group from 48 h to 96 h (P < 0.05). High-throughput 16S ribosomal DNA (rDNA) sequencing revealed that heat stress altered the composition of intestinal microbes of Eriocheir sinensis. Specifically, the abundance of the beneficial bacteria Candidatus, Hepatoplasma and Marinifilum decreased significantly, while the abundance of the pathogenic bacteria Rhodococcus and Morganella increased significantly (P < 0.05). The results revealed that acute heat stress exposure affected immune function and intestinal microbial community in E. sinensis. This work can help to uncover the mechanisms of heat stress responses in this species.

Dual RNA and 16S ribosomal DNA sequencing reveal arbuscular mycorrhizal fungi-mediated mitigation of selenate stress in Zea mays L. and reshaping of soil microbiota

Excessively high concentrations of selenium (Se) in soil are toxic to crop plants, and inoculation with arbuscular mycorrhizal fungi (AMF) can reverse Se stress in maize (Zea mays L.). To investigate the underlying mechanisms, maize seedlings were treated with sodium selenate (5 mg Se[VI] kg-1) and/or AMF (Funneliformis mosseae and Claroideoglomus etunicatum). Dual RNA sequencing in mycorrhiza and 16 S ribosomal DNA sequencing in soil were performed. The results showed that Se(VI) application alone decreased plant dry weight, but increased plant Se concentration, total Se content (mainly selenocysteine), and root superoxide content. Inoculation with either F. mosseae or C. etunicatum increased plant dry weight, decreased Se accumulation and selenocysteine proportion, enhanced root peroxidase activity, and alleviated oxidative stress in Se(VI)-treated plants. Inoculation also downregulated the expression of genes encoding Se transporters, assimilation enzymes, and cysteine-rich receptor-like kinases in Se(VI)-stressed plants, similar to plant–pathogen interaction and glutathione metabolism related genes. Conversely, genes encoding selenium-binding proteins and those related to phenylpropanoid biosynthesis were upregulated in inoculated plants under Se(VI) stress. Compared with Se(VI)-free plants, Se tolerance index, symbiotic feedback percentage on plant dry weight, and root colonization rate were all increased in inoculated plants under Se(VI) stress, corresponding to upregulated expression of ‘key genes’ in symbiosis. AMF inoculation increased bacterial diversity, decreased the relative abundances of selenobacteria related to plant Se absorption (e.g., Proteobacteria and Firmicutes), and improved bacterial network complexity in Se(VI)-stressed soils. We suggest that stress-mediated enhancement of mycorrhizal symbiosis contributed to plant Se(VI) tolerance, whereas AMF-mediated reshaping of soil bacterial community structure prevented excessive Se accumulation in maize.

Two new kathlaniid species (Nematoda: Cosmocercoidea) parasitic in salamanders of the genus Andrias (Amphibia: Caudata: Cryptobranchidae)

The new nematode species, Falcaustra hanzaki n. sp. and Urodelnema takanoensis n. sp. (Cosmocercoidea: Kathlaniidae), were found from the intestine of giant salamanders in Kyoto Prefecture, Japan. The first species is featured by the number and arrangement of caudal papillae (3 pairs of precloacal papillae, 8 pairs of postcloacal papillae and a single ventral precloacal papilla), the presence of a single pseudosucker, spicules equal in size (520–638 μm long), and V-shaped gubernaculum in males; vulva situated about 3/5 of a body in females. The second species is characterized by the number and arrangement of caudal papillae (5 pairs of precloacal papillae, 6 pairs of postcloacal papillae and a single ventral precloacal papilla), spicules equal in size (403–593 μm long), V-shaped gubernaculum in males; vulva situated about 3/5 of a body in females. The molecular phylogenetic analysis was performed using the partial 18S and 28S ribosomal DNA and the internal transcribed spacers 1 region in the nuclear DNA. This phylogenetic study raised a question about the validity of Family Kathlaniidae and related families of Cosmocercoidea.

A Sensitive and Portable Double-Layer Microfluidic Biochip for Harmful Algae Detection.

Harmful algal blooms (HABs) are common disastrous ecological anomalies in coastal waters. An effective algae monitoring approach is important for natural disaster warning and environmental governance. However, conducting rapid and sensitive detection of multiple algae is still challenging. Here, we designed an ultrasensitive, rapid and portable double-layer microfluidic biochip for the simultaneous quantitative detection of six species of algae. Specific DNA probes based on the 18S ribosomal DNA (18S rDNA) gene fragments of HABs were designed and labeled with the fluorescent molecule cyanine-3 (Cy3). The biochip had multiple graphene oxide (GO) nanosheets-based reaction units, in which GO nanosheets were applied to transfer target DNA to the fluorescence signal through a photoluminescence detection system. The entire detection process of multiple algae was completed within 45 min with the linear range of fluorescence recovery of 0.1 fM–100 nM, and the detection limit reached 108 aM. The proposed approach has a simple detection process and high detection performance and is feasible to conduct accurate detection with matched portable detection equipment. It will have promising applications in marine natural disaster monitoring and environmental care.

Microcystis sp. AE03 strain in Dal Lake harbors cylindrospermopsin and microcystin synthetase gene cluster

Cyanobacterial harmful algal blooms (CHABs) are increasing at an alarming rate in different water bodies worldwide. In India, CHAB events in water bodies such as Dal Lake have been sporadically reported with no study done to characterize the cyanobacterial species and their associated toxins. We hypothesized that this Lake is contaminated with toxic cyanobacterial species with the possibility of the presence of cyanotoxin biosynthetic genes. We, therefore, used some of the molecular tools such as 16S ribosomal DNA, PCR, and phylogenetic analysis to explore cyanobacterial species and their associated toxins. A 3-year (2018–2020) survey was conducted at three different sampling sites of Dal Lake namely, Grand Palace Gath (S1), Nigeen basin (S2), and Gagribal basin (S3). Two strains of Dolichospermum sp. AE01 and AE02 (S3 and S1 site) and one strain of Microcystis sp. AE03 (S2 site) was isolated, cultured, and characterized phylogenetically by 16S ribosomal DNA sequencing. The presence of cyanotoxin genes from the isolates was evaluated by PCR of microcystins (mcyB), anatoxins (anaC), and cylindrospermopsins (pks) biosynthesis genes. Results revealed the presence of both mcyB and pks gene in Microcystis sp. AE03, and only anaC gene in Dolichospermum sp. AE02 strain. However, Dolichospermum sp. AE01 strain was not found to harbor any such genes. Our findings, for the first time, reported the coexistence of pks and mcyB in a Microcystis AE03 strain. This study has opened a new door to further characterize the unexplored cyanobacterial species, their associated cyanotoxin biosynthetic genes, and the intervention of high-end proteomic techniques to characterize the cyanotoxins.

Comparative analysis of the endophytic bacteria inhabiting the phyllosphere of aquatic fern Azolla species by high-throughput sequencing

BackgroundAzolla is a small floating fern living in symbiosis with nitrogen-fixing cyanobacteria and provides a variety of important ecosystem benefits. Previous studies have presented that Azolla harbors diverse bacteria that may play a key role in host fitness and productivity. However, the characteristics of endophytic bacteria inhabiting the phyllosphere of different species of Azolla have not yet been fully understood.ResultsIn this study, the 16S ribosomal DNA (rDNA) V5-V7 region of bacteria was determined by Illumina high-throughput sequencing platform to study the diversity and richness of endophytic bacterial communities in the phyllosphere of five Azolla species collected from different countries. A total of 1150 operational taxonomic units (OTUs) were detected for the endophytic bacteria community. According to the α diversity indices, the diversity of bacteria was ordered as Azolla imbricata > A. pinnata > A. filiculoides > A. mexicana > A. caroliniana. The PCoA results displayed that the bacterial communities of A. mexicana and A. caroliniana shared the highest similarity, followed by the similarity between A. pinnata and A. imbricata, and they were significantly distinct from the community of A. filiculoides. The dominant bacteria of Azolla mainly belonged to the phylum of Proteobacteria, followed by Actinobacteria, Chlorobillobacteria, and Firmicutes. In detail, the relative abundance of Proteobacteria in A. imbricata was 52.23%, whereas it was more than 80.00% in the other four species of Azolla. Notably, Herbaspirillum (45.91%, 44.08%) and Methylophilus (29.97%, 37.96%) were the main genera inhabiting A. mexicana and A. caroliniana respectively. Ferrovibrio (18.54%) and Rhizobium (16.68%) were the dominant genera inhabiting A. filiculoides. The group of unidentified genera (41.63%, 44.92%) consisted most of the bacteria in A. imbricata and A. pinnata respectively. Further analysis suggested that the significant different bacteria identified in LDA Effect Size analysis existed Azolla species-specific patterns.ConclusionsIn summary, all results suggested that the diversity and composition of the endophytic bacterial communities were different in Azolla species.

Molecular characterization of Brucella spp. from seropositive herds of cattle farmed at the wildlife-livestock-human interface in Rwanda.

Seroprevalence studies showed that brucellosis is prevalent in cattle in Rwanda with no recent study on the characterization of Brucella spp. Therefore, this study aimed to characterize Brucella spp. in seropositive herds of cattle farmed at the wildlife–livestock–human interface. Whole blood samples (n = 118), milk (n = 41), and vaginal swabs (n = 51) were collected from 64 seropositive herds. All samples (n = 210) were inoculated onto modified Centro de Investigacion y Tecnologia Agroalimentaria (CITA) selective medium. Cultures were analyzed to detect Brucella spp. using 16S−23S ribosomal DNA interspacer region (ITS) PCR, the Brucella cultures were speciated using AMOS and Bruce-ladder PCR assays. Brucella spp. were detected in 16.7% (35/210) of the samples established from the samples using ITS-PCR. The AMOS PCR assay identified mixed Brucella abortus and B. melitensis (n = 6), B. abortus (n = 7), and B. melitensis (n = 1) from cultures from blood samples; mixed B. abortus and B. melitensis (n = 1) and B. abortus (n = 4) from cultures from milk samples; mixed B. abortus and B. melitensis (n = 6), B. abortus (n = 8), and B. melitensis (n = 1) from cultures from vaginal swabs. Bruce-ladder PCR assay confirmed B. abortus and B. melitensis cultures. The isolation of Brucella spp. was significantly associated with districts, with the Nyagatare district having more isolates than other districts (p = 0.01). This study identified single or mixed B. abortus and B. melitensis infections in cattle samples in Rwanda, which emphasizes the need to improve brucellosis control at the wildlife–livestock–human interface and raise the awareness of cattle keepers, abattoir workers, laboratory personnel, and consumers of cattle products.

Duodenal inflammation in common variable immunodeficiency has altered transcriptional response to viruses

A substantial proportion of common variable immunodeficiency (CVID) patients has duodenal inflammation of largely unknown etiology. However, because of its histologic similarities with celiac disease, gluten sensitivity has been proposed as a potential mechanism. We aimed to elucidate the role of the duodenal microenvironment in the pathogenesis of duodenal inflammation in CVID by investigating the transcriptional, proteomic, and microbial signatures of duodenal biopsy samples in CVID. DNA, total RNA, and protein were isolated from snap-frozen pieces of duodenal biopsy samples from CVID (with and without duodenal inflammation), healthy controls, and patients with celiac disease (untreated). RNA sequencing, mass spectrometry-based proteomics, and 16S ribosomal DNA sequencing (bacteria) were then performed. CVID separated from controls in regulation of transcriptional response to lipopolysaccharide and cellular immune responses. These differences were independent of mucosal inflammation. Instead, CVID patients with duodenal inflammation displayed alterations in transcription of genes involved in response to viral infections. Four proteins were differently regulated between CVID patients and healthy controls-DBNL, TRMT11, GCHFR, and IGHA2-independent of duodenal inflammation. Despite similar histology, there were major differences in CVID with duodenal inflammation and celiac disease both at the RNA and protein level. No significant difference was observed in the bacterial gut microbial signature between CVID, celiac, and healthy controls. Our findings suggest the existence of altered functions of the duodenal epithelium, particularly in response to lipopolysaccharide and viruses. The latter finding was related to duodenal inflammation, suggesting that viruses, not gluten sensitivity, could be related to duodenal inflammation in CVID.

Lysinabacillus fusiformis and Paenibacillus alvei Obtained from the Internal of Nasutitermes Termites Revealed Their Ability as Antagonist of Plant Pathogenic Fungi.

This study was performed to reveal phenotypic characters and identity of symbiont bacteria of Nasutitermes as well as investigate their potential as antagonist of plant pathogenic fungi. Isolation of the symbiont bacteria was carried out from inside the heads and the bodies of soldier and worker termite which were collected from 3 locations of nests. Identification was performed using phenotypic test and sequence of 16S ribosomal DNA (16S rDNA). Antagonistic capability was investigated in the laboratory against 3 phytopathogenic fungi i.e., Phytophthora capsici, Ganoderma boninense, and Rigidoporus microporus. Totally, 39 bacterial isolates were obtained from inside the heads and the bodies of Nasutitermes. All the isolates showed capability to inhibit growth of P. capsici, however, 34 isolates showed capability to inhibit growth of G. boninense and 32 isolates showed capability to inhibit growth of R. microporus. Two bacterial strains (IK3.1P and 1B1.2P) which showed the highest percentage of inhibition were further identified based on their sequence of 16S rDNA. The result showed that 1K3.1P strain was placed in the group of type strain and reference strains of Lysinibacillus fusiformis meanwhile 1B1.2P strain was grouped within type strain and reference strains Paenibacillus alvei. The result of this study supply valuable information on the role of symbiont bacteria of Nasutitermes, which may support the development of the control method of the three above-mentioned phytopathogenic fungi.

A Cross-section Metagenomics and 16S Ribosomal DNA Based Evaluation of the Bacterial and Archaeal Communities Resident in the Forumad Chromite Mine, Northeastern of Iran.

The Forumad chromite area from Sabzevar ophiolite belt, Northeastern Iran, is an environment with high concentration of heavy metals, particularly chromite and magnesite minerals, containing chromium and magnesium. In this study for the first time, we analyzed and report the diversity of microbial (bacterial and archaeal) community inhabiting in Forumad chromite mine environment using metagenomics approach. Samples were obtained from different areas of the mine, and total DNA was extracted from water and soil samples. 16S rDNA was amplified using universal primers and the PCR products were cloned in pTz57R/T plasmid. Then, 43% of the positive clones were randomly sequenced. BLAST program in NCBI and EzTaxon databases were used to identify similar 16S rDNA sequences. Phylogenetic analysis was performed using the MEGA5 software and multiple alignments of sequences. In the phylogenetic analyses, proteobacteria, which contains many heavy metals tolerant bacteria especially chromium, were the dominant population in bacterial libraries with Rheinheimera and Cedecaeas the most abundant genuses. Other phyla were Bacteroidetes, Firmicutes, Verrucomicrobia, Chloroflexi, Actinobacteria, Acidobacteria, Cyanobacteria, Gemmatimonadetes, and Planctomycetes. In the archaeal clone library, all the sequences were related to the phylum Thaumarchaeota. Further, 68.6% of the sequences had less than 98.7℅ similarity with the recorded strains which could represent new taxons. The results showed that there was a high microbial diversity in the Forumad chromite area. These results can be used for detoxification and bioremediation of regions contaminated with heavy metals, although more studies are needed.

Detection of Toxoplasma gondii oocysts in soil and risk mapping in an island environment in the Northeast of Brazil.

Toxoplasmosis is an emerging and re-emerging infectious disease that can be transmitted through a contaminated environment. Environmental contamination is an emergency health issue, and determining its occurrence is fundamental to a One Health approach. In this study, we addressed the extent of environmental contamination and viability of Toxoplasma gondii oocysts in soil in different environments on Fernando de Noronha Island, Brazil. In addition, we performed species distribution modelling to predict the environmental suitability for coccidia persistence in the studied area. Soil samples were collected in 14 neighbourhoods of the Island and in the four main squares, creating a total of 95 soil samples (five samples per site). The samples were analyzed by the polymerase chain reaction (PCR) technique for the presence of the 18S ribosomal DNA gene of Apicomplexan protozoa, followed by genetic sequencing. We obtained 4.2% (4/95) positive soil samples with 100% similarity for T. gondii sequences. Two out of four positive sites on PCR showed viability of T. gondii oocysts through the mouse bioassay technique. As a result of the application of the species distribution modelling, environmental adequacy for the coccidia was observed throughout the Island. The results confirm the contamination of the soil in this insular environment by T. gondii oocysts and the environmental suitability by modelling application. These findings are an alert for the possibility of infection in animals and humans by contaminated soil, and for contamination of the maritime environment in addition to water resources for consumption by the local population.

A new genus Unguispora in Kickxellales shows an intermediate lifestyle between saprobic and gut-inhabiting fungi

ABSTRACT Kickxellomycotina encompasses two fungal groups: a saprobic group in excrement and soil and an arthropod gut-inhabiting group. The evolutionary transition between these two lifestyles is unclear due to the lack of knowledge on intermediate forms and lifestyles. Here, we describe a new species, Unguispora rhaphidophoridarum, that was isolated from the excrement of cave crickets (Rhaphidophoridae) in Japan. This species has a novel lifestyle that is intermediate between the saprobic and gut-inhabiting groups. The new genus Unguispora is a member of the Kickxellales and characterized by the sterile appendages born on the sporocladium and by the claw-like ornamentation of the sporangiole. Phylogenetic analysis based on 18S and 28S nuclear ribosomal DNA showed that this fungus is distinct from all known kickxellalean genera and is sister to Linderina. The sporangiospore of the new species germinated only in anaerobiosis and grew in a yeast-like form. The yeast-like cells, defined as “secondary spores,” germinated into hyphae in aerobiosis. In the alimentary tract of cave crickets, the sporangiola are attached to the proventriculus (foregut) by the claw-like ornamentation and multiplicate in the same yeast-like form as under culture. We introduce a new term, “amphibious fungi,” to describe fungi that have two life stages, one outside and the other inside the host gut, like U. rhaphidophoridarum. The discovery of an amphibious fungus in Kickxellales, which was formerly considered to be only saprobic, suggests that Kickxellomycotina has evolved in association with the animal gut.

Commensal Fungus Candida albicans Maintains a Long-Term Mutualistic Relationship with the Host To Modulate Gut Microbiota and Metabolism.

ABSTRACTCandida albicans survives as a commensal fungus in the gastrointestinal tract, and that its excessive growth causes infections in immunosuppressed individuals is widely accepted. However, any mutualistic relationship that may exist between C. albicans and the host remains undetermined. Here, we showed that a long-term feeding of C. albicans does not cause any noticeable infections in the mouse model. Our 16S and 18S ribosomal DNA (rDNA) sequence analyses suggested that C. albicans colonizes in the gut and modulates microbiome dynamics, which in turn mitigates high-fat-diet-induced uncontrolled body weight gain and metabolic hormonal imbalances. Interestingly, adding C. albicans to a nonobesogenic diet stimulated the appetite-regulated hormones and helped the mice maintain a healthy body weight. In concert, our results suggest a mutualism between C. albicans and the host, contrary to the notion that C. albicans is always an adversary and indicating it can instead be a bona fide admirable companion of the host. Finally, we discuss its potential translational implication as a probiotic, especially in obese people or people dependent on high-fat calorie intakes to manage obesity associated complications.IMPORTANCE Candida albicans is mostly considered an opportunistic pathogen that causes fetal systemic infections. However, this study demonstrates that in its commensal state, it maintains a long-term mutualistic relationship with the host and regulates microbial dynamics in the gut and host physiology. Thus, we concluded that C. albicans is not always an adversary but rather can be a bona fide admirable companion of the host. More importantly, as several genomic knockout strains of C. albicans were shown to be avirulent, such candidate strains may be explored further as preferable probiotic isolates to control obesity.

Phenotypic and Molecular Identification of Cronobacter sakazakii Isolated from Clinical Specimens in Thi-Qar Province

Cronobacter sakazakii is a food-born pathogenic bacteria which can cause several diseases for human and animals. The present study focused on the isolation of this species from clinical specimens ( Burn humans, stool of patients with diarrhea, urine with UTI. in addition to hospital environments specimens) from different sexes and ages. The specimens have been taken from group of hospitals in Al-Nasiriyah city (center of Thi-Qar province- south of Iraq). The identification was done by both phenotypical and molecular methods with using of culture (on enriched, selective and differential media), microscopic examination, biochemical tests, API. 20 E. system and PCR. for amplification of ITS. region (the space between 16s and 23s ribosomal DNA.).Out of 400 specimens (100 from each source) there were 16 (4 %) of specimens givepositive for C. sakazakii which included: 4/100 burns specimens (4%), 6/100 stool specimens (6%), 0/100 urine (0%) and 6/100 hospital environments specimens (6%). The 16 isolates have been identified by PCR. for the presence of ITS. region with using of SG. Primer (282 bp.), all the 16 (100%) isolates were positive for this gene. This species was exist in the clinical specimens and can cause diarrhea and burn infection in the area of study with ratio equal to what obtain by other researchers. There are variation in results of biochemical tests according to its strains which leads to difficulty with diagnosis, furthermore the diagnosis with ITS. showed the same results of phenotypical methods used in the present study

Exploring the Species Limits in Balanophora subgenus Balania

Abstract— Balanophora subgen. Balania (Balanophoraceae), whose members differ from those of the other subgenus in having three-merous male flowers, includes B. flava, B. involucrata, B. tobiracola, the B. harlandii assemblage, and the agamospermic species B. japonica. Species limits of B. flava, B. involucrata, and the B. harlandii as currently circumscribed (B. harlandii assemblage) have long been controversial. Here, species limits in subgen. Balania are explored based on morphological characters and phylogenetic analysis using nuclear 18S and ITS ribosomal DNA sequences. Subgen. Balania was monophyletic if B. japonica is excluded. Balanophora harlandii assamblage was polyphyletic and three lineages, B. harlandii, B. kawakamii, and B. henryi, were recovered in the assemblage. Molecular and morphological divergence, distribution, and phenology provided strong support for the recognition of these lineages as distinct species, namely B. harlandii, B. kawakamii, and B. henryi. The findings also suggested that B. flava should be reduced to synonym under B. involucrata, thus supporting Hansen’s treatment of both dioecious and monoecious populations as members of a single species.

Characterization of Lactic Acid Bacteria from the Gut of Penaeus vannamei as Potential Probiotic

Background: The present objective of the study was to isolate and characterize the gut associated culturable lactic acid bacteria (beneficial bacteria) from the gut of Penaeus vannamei for their potential application as probiotic. Methods: Penaeus vannamei (host) gut associated bacterial isolates were obtained from ten commercial brackishwater shrimp ponds (n=10) located in Kanchipuram, Chengalpattu, Tiruvallur and Villupuram districts of Tamil Nâdu, during 2021-22 to test their efficiency as indigenous gut probiotic. Twenty-five shrimps from each pond, with salinity ranging from 5 to 25 ppt, were collected for isolation of beneficial bacterial isolates. Result: Thirty lactic acid bacteria were isolated and identified from the gut of 250 Penaeus vannamei, using a 16S ribosomal DNA sequence. Six isolates viz., Pediococcus pentosaceus (ON495586), Lactiplantibacillus plantarum (ON491817), Lactococcus lactis (ON479264), Enterococcus faecium (ON478992), Enterococcus hirae (ON478991) and Enterococcus durans (ON564885) having better enzyme activity were taken and further subjected to in vitro analysis. It was found that these isolates had antibacterial activity against shrimp pathogens V. campbelli, V. harveyi and V. parahaemolyticus with zone of inhibition ranging between 12.33 to 21.00 mm; showed better growth at pH 7.0; tolerated the bile salts up to 1% concentration and endured salt concentrations up to 6.0%. In addition, above isolates demonstrated excellent auto-aggregative activity (74.45 to 91.14%) and hydrophobicity (77 to 99.93%). No antagonist activities were detected among the strains, suggesting its use as the multiple cocktail probiotic. Hence, the investigated isolates could serve as potential probiotics in shrimp aquaculture production systems.

‘Candidatus Phytoplasma solani’ (Subgroup 16SrXII-A) Associated with Nicotiana tabacum Leaf Abnormality in Turkey

Tobacco (Nicotiana tabacum) is among the agricultural products with the highest added value in Turkey. Although frequently associated with its negative effects on human health, it also provides important contributions to the Turkish economy with the employment it creates in rural areas and continues to be a strategic product. Many postgraduate theses and studies related to the sociological and economic importance of the production of this plant, which is of great importance for our country, have been carried out. However, there are very limited studies on plant diseases in tobacco production areas in Turkey. Phytoplasma is one of the important plant pathogens that cause yield loss in tobacco. Since available data on phytoplasma diseases on tobacco was very scarce worldwide, field surveys to collect samples showing phytoplasma infection-like symptoms such as yellowish color changes, leaf blisters, proliferation, dwarfism, and other physical abnormalities were carried out in Çanakkale and Balıkesir provinces of Turkey from June to August 2021. The presence of phytoplasmas in six samples was confirmed by 16S ribosomal DNA amplification by nested-PCR using universal phytoplasma primer sets, which also suggested the pathogen associated with the symptoms on tobacco. According to phylogenetic study and virtual-RFLP analysis using AluI and MseI endonuclease enzymes, the six Turkish tobacco phytoplasma strains all belong to group 16SrXII and have more than 99% nucleotide sequence identity with some members of ‘Candidatus Phytoplasma solani’ of the taxonomic subgroup ‘stolbur’ (16SrXII-A). Genetic distances analysis indicated that group 16SrI was more closely related to 16SrXII than 16SrVI, in agreement with the groups clustering in the phylogenetic tree. Neutrality tests found that 16SrI and 16SrXII groups are experiencing expanding or bottleneck selections, probably due to new mutations in the 16S rRNA gene fragment. Meanwhile, 16SrVI populations are shown to be undergoing balancing selections, indicating that its isolates have evolved for a long time.