16S Ribosomal Gene Research Articles

Thermo-resistant enzyme-producing microorganisms isolated from composting.

Organo-mineral fertilizers supplemented with biological additives are an alternative to chemical fertilizers. In this study, thermoresistant microorganisms from composting mass were isolated by two-step procedures. First, samples taken at different time points and temperatures (33 days at 52 ºC, 60 days at 63 ºC, and over 365 days at 26 ºC) were pre-incubated at 80 oC for 30 minutes. Second, the microbial selection by in vitro culture-based methods and heat shock at 60 oC and 100 oC for 2h and 4h. Forty-one isolates were able to grow at 60 °C for 4h; twenty-seven at 100 °C for 2h, and two at 100 °C for 4h. The molecular identification by partial sequencing of the 16S ribosomal gene using universal primers revealed that thirty-five isolates were from eight Bacillus species, one Brevibacillus borstelensis, three Streptomyces thermogriseus, and two fungi (Thermomyces lanuginosus and T. dupontii). Data from amylase, phytase, and cellulase activity assays and the enzymatic index (EI) showed that 38 of 41 thermo-resistant isolates produce at least one enzyme. For amylase activity, the highest EI value was observed in Bacillus licheniformis (isolate 21C2, EI= 4.11), followed by Brevibacillus borstelensis (isolate 6C2, EI= 3.66), Bacillus cereus (isolate 18C2, EI= 3.52), and Bacillus paralicheniformis (isolate 20C2, EI= 3.34). For phytase, the highest EI values were observed for Bacillus cereus (isolate 18C2, EI= 2.30) and Bacillus licheniformis (isolate 3C1, EI= 2.15). Concerning cellulose production, B. altitudinis (isolate 6C1) was the most efficient (EI= 6.40), followed by three Bacillus subtilis (isolates 9C1, 16C2, and 19C2) with EI values of 5.66, 5.84, and 5.88, respectively, and one B. pumilus (isolate 27C2, EI= 5.78). The selected microorganisms are potentially useful as a biological additive in organo-mineral fertilizers and other biotechnological processes.

Fermentation box cleaning can impair cacao seed fermentation

The quality of the raw material for chocolate manufacture depends on the control of cacao seed fermentation. Practices affect the diversity and metabolic activities of spontaneous yeasts in fermentations. The control of the initial inoculum is important to standardize fermentations and to improve the final seed quality. We conducted fermentations using non-cleaned or cleaned fermentation boxes to evaluate the dynamics of the microorganism populations and their effect in seed composition in two consecutive years. The isolated yeasts were identified by sequencing the D1-domain of the 26S ribosomal genes. Pulp and cotyledon parameters were monitored during fermentations. Wickerhamomyces anomalus was the dominant yeast in the fermentations, but the species was less represented in cleaned boxes. In the cleaned-box fermentation, there was a delay in temperature rise of the fermentation, which retarded the suitable moment for seed-mass turning. The reduction in seed-pulp quality caused by restricted rainfall in one year limited the seed-mass heating and voided the seed turning. Thus, under substrate restriction, the initial reduced inoculum caused by cleaning the boxes impaired fermentation, mainly under environmental conditions that compromise the quality of the pulp. However, this variation had little effect on the desired degradation of cotyledon components, substrates for flavor precursors.

Results of a Clinical Trial Showing Changes to the Faecal Microbiome in Racing Thoroughbreds after Feeding a Nutritional Supplement

Next-generation sequencing (NGS) has been used to evaluate the effect of various interventions on the equine microbiome. The aim of this randomised blinded clinical trial was to determine if a prebiotic nutritional supplement would result in a change from baseline in the faecal microbiome composition of racing Thoroughbred horses in training being fed a high concentrate/grain-based diet to be more similar to that found in forage fed/pasture grazed horses. Thirty-two horses on one training yard were randomised to either receive the supplement or not. Faecal samples were collected at baseline, 6 and 12 weeks for NGS of the 16S ribosomal subunit gene. Twenty-two horses completed the trial, met the inclusion criteria and were included in the intention to treat analysis; 20 horses were included in the per protocol analysis. The mean and median percent decreases in Bacteroidetes, increases in Firmicutes and the Firmicutes:Bacteroidetes ratio were significantly greater than zero for the treated horses only. Supplemented horses (8/10) were more likely than control horses (2/10) to show an increase in Firmicutes of a ≥9% with ≥24% increase in Clostridia, ≥5% decrease in Bacteroidetes, ≥16% increase in the F:B ratio and ≥2% increase in Actinobacteria (RR = 4, 95% CI: 1.1-14.4, p = 0.01). This provides useful information for further investigations on long-term effects on the microbiome and on health and racing-related outcomes.

Physical activity and diet associations with the gut microbiota in the Coronary Artery Risk Development in Young Adults (CARDIA) study

BackgroundGut microbiota may influence metabolic pathways related to chronic health conditions. Evidence for physical activity and diet influences on gut microbial composition exists, but data from diverse population-based cohort studies are limited. ObjectivesWe hypothesized that gut microbial diversity and genera are associated with physical activity and diet quality. MethodsData were from 537 participants in the Coronary Artery Risk Development in Young Adults (CARDIA) Study, a prospective cohort, who attended the year 30 follow-up examination (2015–2016; aged 47–61 y; 45% Black race/55% White race; 45% men/55% women). The 16S ribosomal RNA marker gene was sequenced from stool DNA, and genus-level taxonomy was assigned. Within-person microbial diversity (α-diversity) was assessed with Shannon diversity index and richness scores; between-person diversity (β-diversity) measures were generated with principal coordinates analysis (PCoA). Current and long-term physical activity and diet quality measures were derived from data collected over 30 y of follow-up. Multivariable-adjusted regression analysis controlled for: sociodemographic variables (age, race, sex, education, and field center), other health behaviors (smoking, alcohol consumption, and medication use), and adjusted for multiple comparisons with the false discovery rate (<0.20). ResultsBased on PCoA β-diversity, participants’ microbial community compositions differed significantly (P < 0.001), with respect to both current and long-term physical activity and diet quality. α-Diversity was associated only with current physical activity (positively) in multivariable-adjusted analysis. Multiple genera (n = 45) were associated with physical activity and fewer with diet (n = 5), including positive associations with Lachnospiraceae UCG-001 and Ruminococcaceae IncertaeSedis with both behaviors. ConclusionsPhysical activity and diet quality were associated with gut microbial composition among 537 participants in the CARDIA study. Multiple genera were associated with physical activity. Physical activity and diet quality were associated with genera consistent with pathways related to inflammation and short-chain fatty acid production.

Comparative Analysis of Grapevine Epiphytic Microbiomes among Different Varieties, Tissues, and Developmental Stages in the Same Terroir

There is limited knowledge about the relationships of epiphytic microbiomes associated with the phyllosphere of different Vitis vinifera cultivars in the same vineyard and terroir. To address this research gap, we investigated the microbiome compositionof 36 grapevine genotypes grown in the same vineyard in different plant sections during the growing season. Using high-throughput NGS-based metagenomic analysis targeting the ITS2 and the V4 regions of the 16S ribosomal gene of fungal and bacterial communities, respectively, weassessed the impact of grapevine genotypes on microbial assemblages in various parts of the phyllosphere. The results indicated that different phyllosphere tissues display high microbial diversity regardless of the cultivars’ identity and use. The selected three phyllosphere parts representing three distinct phenological stages, namely bark and bud, berry set, and fruit harvest, had almost a similar number of fungal OTUs, while a difference was recorded for the bacterial species. The fruit harvest stage hosted the highest number of bacterial OTUs, whereas the bark and bud stage contained the lower. Bacterial dominant phyla were Proteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes, and the genera were Gluconacetobacter, Erwinia, Gluconobacter, Zymobacter, Buchnera, Pseudomonas, Pantoea, Hymenobacter, Pedobacter, Frigoribacterium, Sphingomonas, and Massilia. For fungi, the dominant phyla were Ascomycota and Basidiomycota, and the genera were Aureobasidium, Cladosporium, Alternaria, Aspergillus, Davidiella, Phoma, Epicoccum, Rhodosporidium, Glomerella, Botryosphaeria, Metschnikowia, Issatchenkia, and Lewia. Both the genotype of the cultivar and the phenological stage appeared to considerably impact the shape of microbial diversity and structure within the same terroir. Taken together, these results indicate that microbiome analysis could be proved to be an important molecular fingerprint of cultivars and provide an efficient management tool for the traceability of wine and grape end products. Moreover, the unique identity of cultivars’ microbial signatures highlights the need for further development of precision management to support viticulture sustainability in the face of climate change.

Endodontic Microbial Communities in Apical Periodontitis

IntroductionApical periodontitis (AP) represents an inflammatory condition of peri-radicular tissues due to invasion and colonization of bacteria in the root canals. Primary apical periodontitis (PAP) is associated with untreated necrotic root canal and can be efficiently treated with endodontic treatment to remove bacteria. Persistent/secondary apical periodontitis (SAP) is a perpetual periapical lesion due to unsuccessfully treated root canals after an initial apparent healing of the tooth. The aim of the study was evaluating the microbial communities associated with root canals using Nanopore sequencing. MethodsSeventeen samples from the root canals of 15 patients with AP were Polymerase Chain Reaction-amplified for 16s ribosomal DNA gene and sequenced. Information regarding the presence or absence of AP symptoms, PAP and SAP, and periapical index of patients were recorded. ResultsFirmicutes, Bacteroidetes, and Actinobacteria were the most abundant phyla detected and Phocaeicola, Pseudomonas, Rothia, and Prevotella were the most prominent genera. In samples of patients with AP symptoms, the most frequent detected genera were Cutibacterium, Lactobacillus, Pseudomonas, Dialister, Prevotella, and Staphylococcus. In PAP samples, the most represented genera were Cutibacterium, Lactobacillus, Pseudomonas, and Prevotella, whilst in SAP cases were Cutibacterium, Prevotella, Atopobium, Capnocytophaga, Fusobacterium, Pseudomonas, Solobacterium, and Streptococcus. ConclusionsThe results provide additional information on the microbiota of root-canals. These data evidence the complexity of the microbiota and the relationship with many clinical and endodontic conditions. Future studies must evaluate these conditions and identify their role in inducing bone damage and local and systemic disease, aiming to better elucidate the relationship between microbes and endodontic pathologies.

Exploring the Core Microbiota of Four Different Traditional Fermented Beverages from the Colombian Andes

Fermentation is an ancient process used to prepare and preserve food. Currently, fermented beverages are part of the culture of people living in the Colombian Andean Region, and they are a vital part of their cosmology and ancestral vision. Chicha, Forcha, Champús, and Masato are some of the most common Colombian Andes region’s traditional fermented beverages. These drinks come from the fermentation of maize (Zea maize), but other cereals such as wheat or rye, could be used. The fermentation is carried out by a set of bacteria and yeasts that provide characteristic organoleptic properties of each beverage. In this work, the information collected from the metagenomics analyses by sequencing ITS 1-4 (Internal Transcriber Spacer) and the 16S ribosomal gene for fungi and the V3-V4 region of the rDNA for bacteria allowed us to identify the diversity present in these autochthonous fermented beverages made with maize. The sequencing analysis showed the presence of 39 bacterial and 20 fungal genera. In addition, we determined that only nine genera of bacteria and two genera of fungi affect the organoleptic properties of smell, colour, and flavour, given the production of compounds such as lactic acid, alcohol, and phenols, highlighting the critical role of these microorganisms. Our findings provide new insights into the core microbiota of these beverages, represented by Lactobacillus fermentum, Acetobacter pasteurianus, and Saccharomyces cerevisiae.

Mitochondrial DNA of Sardinian and North-West Italian Populations Revealed a New Piece in the Mosaic of Phylogeography and Phylogeny of Salariopsis fluviatilis (Blenniidae)

The genus Salariopsis (Blenniidae) comprises freshwater blenny fish that inhabits Mediterranean Sea, Black Sea, and north-east Atlantic areas. Three species were formally described to date: Salariopsis fluviatilis. S. economidisi, and S. atlantica. In this study, 103 individuals were collected from different Italian regions (Sardinia, Liguria, Piedmont, Lombardy) and analyzed using the mtDNA Control Region and the ribosomal 16s gene. We aimed (i) to depict the phylogeographic patterns of S. fluviatilis in northern Italy and Sardinia and (ii) to compare the genetic structure of Italian samples with those from other Mediterranean regions. Results obtained showed the presence of a well-supported genetic structuring among Italian S. fluviatilis populations, shedding new light on the phylogeographic patterns of northern Italian populations of S. fluviatilis sensu stricto across the Ligurian Alpine ridge and the Sardinia Island-mainland dispersal patterns. Furthermore, our species delimitation analysis was consistent in supporting results of previous research about the presence of genetic differentiation among S. fluviatilis, evidencing: (i) a large group of S. fluviatilis sensu stricto that includes two sub-groups (Occidental and Oriental), (ii) one group comprising populations from the Middle East of a taxonomic entity corresponding to Salariopsis cf. fluviatilis, and (iii) one group of Iberian individuals from the Guadiana River.

Description and Phylogenetic Relationships of Anhingatrema n. gen. (Digenea: Diplostomidae) with Two New Species from New World Anhingas (Aves: Anhingidae).

The Diplostomidae is a globally distributed family of digeneans that parasitize a wide variety of tetrapod definitive hosts. Recent molecular phylogenetic studies have revealed unknown diplostomid diversity in avian hosts throughout the New World. Herein, we provide descriptions of a novel genus of diplostomids with two new species. Two species of diplostomids belonging to the new genus were collected from anhinga birds in Mississippi (USA) and Brazil. Partial nuclear 28S ribosomal and mitochondrial cox1 genes were sequenced. Ribosomal data were used for phylogenetic inference. Both species of Anhingatrema n. gen. were positioned in a 100% supported, monophyletic clade in the phylogenetic tree. The molecular phylogenetic position and a combination of morphological features (e.g., presence of pseudosuckers, testes shape and orientation) supported erection of the new genus. Anhingatrema overstreeti n. sp. and Anhingatrema cararai n. sp. are morphologically similar, but differ in size of and ratios associated with pseudosuckers. The two species differ by 2% of 28S sequences and 13.8% of cox1 sequences. Comparison of DNA sequences revealed that Diplostomidae gen. sp. in GenBank (MZ314151) is conspecific with An. overstreeti n. sp. Anhingatrema n. gen. is the sixth genus of diplostomids known from anhingas worldwide. Anhingatrema cararai n. sp. is the first diplostomid to be reported from anhingas in South America. Combined with previous studies, the molecular phylogenies revealed at least two host switches to anhingas from other birds during the evolutionary history of the Diplostomidae.

Remarkable population structure in the tropical Atlantic lace corals Stylaster roseus (Pallas, 1766) and Stylaster blatteus (Boschma, 1961)

Biodiversity on coral reefs depends not only on primary reef-builders, but also on associated taxa that create microhabitats for other species. Hydrocorals of the genus Stylaster, commonly known as lace corals, form small branching colonies that enhance three-dimensional complexity on reefs and are known to support a variety of commensal species. Furthermore, the genus is highly speciose, further increasing biodiversity. Despite their important ecological roles, little is known about the evolutionary history and the intraspecific diversity and structure in these broadly distributed hydrocorals. Here, we assessed the phylogenetic relationships among Atlantic species in the genus Stylaster and examined the genetic structure of S. roseus in the Tropical Western Atlantic (Caribbean and Brazil) and of S. blatteus in the Tropical Eastern Atlantic (Africa), using DNA sequences from the 16S ribosomal gene. Time-calibrated phylogenetic analyses showed that S. roseus and S. blatteus diverged at ~ 24.6 Ma. A well-supported Brazilian clade within S. roseus indicates a possible cryptic species that diverged at ~ 11.6 Ma, consistent with the formation of the Amazon River at 9 Ma (Hoorn et al. in Glob Planet Change 153:51–65, 2017). Strong genetic structure was observed even over moderate distances, with ΦST values over all populations being 0.98 for S. roseus and 0.90 for S. blatteus. Nearly, all haplotypes were private (found in a single location) and diverged by many mutational steps from one another. In contrast, genetic diversity was low at the local scale for both species, with most sites showing no variation (a single haplotype). These results are coherent with the reproductive strategy of Stylasteridae, where larvae are brooded and are highly developed at the time of release, often settling near the parental colony. Limited dispersal coupled with possible clonal reproduction have likely contributed to the high levels of genetic differentiation observed here. Lace corals show unusual reproductive and population dynamics compared to other reef inhabiting cnidarians. Future work may reveal additional cryptic diversity in this poorly studied family.

Abstract 13266: Sex Specific Differences in the Gut Microbiome in Patients With Pulmonary Arterial Hypertension

Introduction: Pulmonary arterial hypertension (PAH) patients exhibit sexual dimorphism in pulmonary vascular remodeling and right ventricular (RV) dysfunction. While female PAH patients have severe pulmonary vascular remodeling, male PAH patients have worse RV function. Inflammation, a key component of PAH pathobiology, has been implicated as a potential mechanism for sexual dimorphism. Gut dysbiosis causes systemic inflammation. However, it is unknown whether gut dysbiosis contributes to sexual dimorphism in PAH. Hypothesis: There are sex-specific differences in the gut microbiome and microbial metabolites of PAH patients. Methods: 16S ribosomal ribonucleic acid gene sequencing was performed on stool samples from PAH patients (n=73). Markers of inflammation and circulating microbial metabolites were measured using plasma samples. Results: There was no significant difference in the Shannon diversity indices or species richness (Chao1 indices) between the gut microbiomes of male and female PAH patients. Moreover, principal component analysis of pairwise Bray-Curtis dissimilarity indices revealed no distinct microbiome compositions dependent on sex. There was no difference in serum claudin-3, a marker of gut permeability, and serum interleukin 6 levels between male and female PAH patients. When examining microbial metabolites, including trimethylamine N-oxide, short chain fatty acids, and secondary bile acids, no sex-specific differences were observed. Linear discriminant analysis of effect size revealed that there were no taxonomic differences based on sex. Conclusions: Male and female PAH patients do not possess distinct differences in their gut microbiomes or metabolites. There are also no sex-specific taxonomic differences. These findings do not support our hypothesis that gut dysbiosis may serve as a possible explanation for the sexual dimorphism observed in PAH.

Mesozooplankton biodiversity, vertical assemblages, and diel migration in the western tropical Pacific Ocean revealed by eDNA metabarcoding and morphological methods

Zooplankton plays a crucial role in marine ecosystems. However, due to issues with morphological identification and sampling methods, knowledge of their biodiversity and community structure in the vast pelagic zone of the western tropical Pacific Ocean is still limited compared with other areas in the North Pacific Ocean. In this study, we used environmental DNA (eDNA) metabarcoding and morphological identification approaches to investigate mesozooplankton’s biodiversity and vertical assemblages in the western tropical Pacific Ocean from the surface to 1000 m deep. Two different barcodes were used in metabarcoding: mitochondrial cytochrome oxidase I (COI) and small subunit ribosomal RNA genes (18S). Our results showed that eDNA metabarcoding revealed 2-4 times more medusae taxa than morphological processing. Both methods detected that small copepod species were dominant in the community and six of the top-10 most abundant copepod genera overlapped. Morphological results showed that mesozooplankton communities were classified into three groups: epipelagic (0–200 m), upper mesopelagic (200-500 m), and lower mesopelagic (500–1000 m). And eDNA results showed distinct vertical variations of mesozooplankton communities within the epipelagic. Both methods showed that the dominant genera Clausocalanus, Paracalanus, and Calocalanus exhibited diel patterns in relative abundance, demonstrating that the eDNA method can capture diel vertical migration (DVM) signals of copepods. Overall, our results indicate that eDNA metabarcoding is an effective and efficient tool for the study and monitoring of mesozooplankton diversity and vertical assemblages in the open sea.

High-resolution phylogenetic and population genetic analysis of microbial communities with RoC-ITS

Microbial communities are inter-connected systems of incredible complexity and dynamism that play crucial roles in health, energy, and the environment. To better understand microbial communities and how they respond to change, it is important to know which microbes are present and their relative abundances at the greatest taxonomic resolution possible. Here, we describe a novel protocol (RoC-ITS) that uses the single-molecule Nanopore sequencing platform to assay the composition of microbial communities at the subspecies designation. Using rolling-circle amplification, this methodology produces long-read sequences from a circular construct containing the complete 16S ribosomal gene and the neighboring internally transcribed spacer (ITS). These long reads can be used to generate a high-fidelity circular consensus sequence. Generally, the ribosomal 16S gene provides phylogenetic information down to the species-level, while the much less conserved ITS region contains strain-level information. When linked together, this combination of markers allows for the identification of individual ribosomal units within a specific organism and the assessment of their relative stoichiometry, as well as the ability to monitor subtle shifts in microbial community composition with a single generic assay. We applied RoC-ITS to an artificial microbial community that was also sequenced using the Illumina platform, to assess its accuracy in quantifying the relative abundance and identity of each species.

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.

Ionizing Radiation from Radiopharmaceuticals and the Human Gut Microbiota: An Ex Vivo Approach

This study aimed to determine the effect of three widely used radiopharmaceuticals with intestinal excretion on selected relevant bacteria that are part of the human gut microbiota, using an ex vivo approach. Fecal samples obtained from healthy volunteers were analyzed. Each sample was divided into four smaller aliquots. One served as the non-irradiated control. The other three were homogenized with three radiopharmaceutical solutions ([131I]NaI, [99mTc]NaTcO4, and [223Ra]RaCl2). Relative quantification of each taxa was determined by the 2−ΔΔC method, using the ribosomal gene 16S as an internal control (primers 534/385). Twelve fecal samples were analysed: three controls and nine irradiated. Our experiment showed fold changes in all analyzed taxa with all radiopharmaceuticals, but results were more significant with I-131, ranging from 1.87–83.58; whereas no relevant differences were found with Tc-99m and Ra-223, ranging from 0.98–1.58 and 0.83–1.97, respectively. This study corroborates limited existing research on how ionizing radiation changes the gut microbiota composition, providing novel data regarding the ex vivo effect of radiopharmaceuticals. Our findings justify the need for future larger scale projects.

Novel CRISPR-based detection of Leishmania species.

Tegumentary leishmaniasis, a disease caused by protozoan parasites of the genus Leishmania, is a major public health problem in many regions of Latin America. Its diagnosis is difficult given other conditions resembling leishmaniasis lesions and co-occurring in the same endemic areas. A combination of parasitological and molecular methods leads to accurate diagnosis, with the latter being traditionally performed in centralized reference and research laboratories as they require specialized infrastructure and operators. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems have recently driven innovative tools for nucleic acid detection that combine high specificity, sensitivity and speed and are readily adaptable for point-of-care testing. Here, we harnessed the CRISPR-Cas12a system for molecular detection of Leishmania spp., emphasizing medically relevant parasite species circulating in Peru and other endemic areas in Latin America, with Leishmania (Viannia) braziliensis being the main etiologic agent of cutaneous and mucosal leishmaniasis. We developed two assays targeting multi-copy targets commonly used in the molecular diagnosis of leishmaniasis: the 18S ribosomal RNA gene (18S rDNA), highly conserved across Leishmania species, and a region of kinetoplast DNA (kDNA) minicircles conserved in the L. (Viannia) subgenus. Our CRISPR-based assays were capable of detecting down to 5 × 10−2 (kDNA) or 5 × 100 (18S rDNA) parasite genome equivalents/reaction with PCR preamplification. The 18S PCR/CRISPR assay achieved pan-Leishmania detection, whereas the kDNA PCR/CRISPR assay was specific for L. (Viannia) detection. No cross-reaction was observed with Trypanosoma cruzi strain Y or human DNA. We evaluated the performance of the assays using 49 clinical samples compared to a kDNA real-time PCR assay as the reference test. The kDNA PCR/CRISPR assay performed equally well as the reference test, with positive and negative percent agreement of 100%. The 18S PCR/CRISPR assay had high positive and negative percent agreement of 82.1% and 100%, respectively. The findings support the potential applicability of the newly developed CRISPR-based molecular tools for first-line diagnosis of Leishmania infections at the genus and L. (Viannia) subgenus levels.

The Lung Allograft Microbiome Associates with Pepsin, Inflammation, and Primary Graft Dysfunction.

Rationale: Primary graft dysfunction (PGD) is the principal cause of early morbidity and mortality after lung transplantation. The lung microbiome has been implicated in later transplantation outcomes but has not been investigated in PGD. Objectives: To define the peritransplant bacterial lung microbiome and relationship to host response and PGD. Methods: This was a single-center prospective cohort study. Airway lavage samples from donor lungs before organ procurement and recipient allografts immediately after implantation underwent bacterial 16S ribosomal ribonucleic acid gene sequencing. Recipient allograft samples were analyzed for cytokines by multiplex array and pepsin by ELISA. Measurements and Main Results: We enrolled 139 transplant subjects and obtained donor lung (n = 109) and recipient allograft (n = 136) samples. Severe PGD (persistent grade 3) developed in 15 subjects over the first 72 hours, and 40 remained without PGD (persistent grade 0). The microbiome of donor lungs differed from healthy lungs, and recipient allograft microbiomes differed from donor lungs. Development of severe PGD was associated with enrichment in the immediate postimplantation lung of oropharyngeal anaerobic taxa, particularly Prevotella. Elevated pepsin, a gastric biomarker, and a hyperinflammatory cytokine profile were present in recipient allografts in severe PGD and strongly correlated with microbiome composition. Together, immediate postimplantation allograft Prevotella/Streptococcus ratio, pepsin, and indicator cytokines were associated with development of severe PGD during the 72-hour post-transplantation period (area under the curve = 0.81). Conclusions: Lung allografts that develop PGD have a microbiome enriched in anaerobic oropharyngeal taxa, elevated gastric pepsin, and hyperinflammatory phenotype. These findings suggest a possible role for peritransplant aspiration in PGD, a potentially actionable mechanism that warrants further investigation.

Genetic characterization and antimicrobial profiling of bacterial isolates collected from Nile tilapia (Oreochromis niloticus) affected by summer mortality syndrome.

In recent years, Egyptian tilapia aquaculture has experienced mortality episodes during the summer months. The causative agents responsible for such mortalities have not been clearly identified. A total of 400 fish specimens were collected from affected tilapia farms within five Egyptian governorates. A total of 344 bacterial isolates were identified from the examined fish specimens. Bacterial isolates were grouped into seven genera based on API 20E results. The most prevalent pathogens were Aeromonas spp. (42%), Vibrio spp. (21%), and Streptococcus agalactiae (14.5%). Other emerging infections like, Plesiomonas shigelloides (10%), Staphyloccocus spp. (8%), Pseudomonas oryzihabitans, and Acinetobacter lwoffii (2.3%) were also detected. Sequence analysis of the 16S ribosomal RNA bacterial gene of some isolates, confirmed the phenotypic identification results. The analysis of antibiotic resistance genes revealed the presence of aac(6')-Ib-cr (35.7%), blaCTX gene (23.8%), qnrS (19%), ampC (16.7%), floR (14.3%), sul1, tetA, and van.C1 (2.4%) genes in some isolates. The antimicrobia resistance gene, qac was reported in 46% of screened isolates. Bacterial strains showed variable virulence genes profiles. Aeromonas spp. harboured (act, gcat, aerA, lip, fla, and ser) genes. All Vibrio spp. possessed the hlyA gene, while cylE, hylB, and lmb genes, were detected in S. agalactiae strains. Our findings point to the possible role of the identified bacterial pathogens in tilapia summer mortality syndrome and highlight the risk of the irresponsible use of antibiotics on antimicrobial resistance in aquaculture.

The Microbiome and Metabolomic Profile of the Transplanted Intestine with Long-Term Function.

We analyzed the fecal microbiome by deep sequencing of the 16S ribosomal genes and the metabolomic profiles of 43 intestinal transplant recipients to identify biomarkers of graft function. Stool samples were collected from 23 patients with stable graft function five years or longer after transplant, 15 stable recipients one-year post-transplant and four recipients with refractory rejection and graft loss within one-year post-transplant. Lactobacillus and Streptococcus species were predominant in patients with stable graft function both in the short and long term, with a microbiome profile consistent with the general population. Conversely, Enterococcus species were predominant in patients with refractory rejection as compared to the general population, indicating profound dysbiosis in the context of graft dysfunction. Metabolomic analysis demonstrated significant differences between the three groups, with several metabolites in rejecting recipients clustering as a distinct set. Our study suggests that the bacterial microbiome profile of stable intestinal transplants is similar to the general population, supporting further application of this non-invasive approach to identify biomarkers of intestinal graft function.

Heterosigma akashiwo in Patagonian Fjords: Genetics, Growth, Pigment Signature and Role of PUFA and ROS in Ichthyotoxicity.

Heterosigma akashiwo is the only raphidophyte described for Chilean waters. A recent 2021 fish-killing bloom event of this raphidophyte ignited scientific research, but the ichthyotoxic mechanism and environmental conditions that promote its growth are still unclear. This is the first study confirming the occurrence of H. akashiwo in Chilean waters on the basis of the region D1/D2 of the 28S ribosomal gene. The pigment signature of the CREAN_HA03 strain revealed chlorophyll-a, fucoxanthin, and violaxanthin as the most abundant pigments, but profiles were variable depending on culture and field conditions. A factorial temperature–salinity growth experiment showed a maximal growth rate of 0.48 d−1 at 17 °C and 35 in salinity, but reached a maximal cell abundance of ~50,000 cells mL−1 at 12 °C and 25 in salinity. The fatty acid profile included high levels of saturated (16:0) and polyunsaturated (18:4 ω3; 20:5 ω3) fatty acids, but superoxide production in this strain was low (~0.3 pmol O2– cell−1 h−1). The RTgill-W1 bioassay showed that the H. akashiwo strain was cytotoxic only at high cell concentrations (>47,000 cells mL−1) and after cell rupture. In conclusion, salmon mortality during H. akashiwo bloom events in Patagonian fjords is likely explained by the high production of long-chain PUFAs at high cell densities, but only in the presence of high ROS production.