MiSeq Platform Research Articles

Soil Fertilization with Palm Oil Mill Effluent Has a Short-Term Effect on the Bacterial Diversity of an Amazonian Agricultural Land Area.

Palm oil derived from the fruits of Elaeis guineensis Jacq. has global economic importance and is largely produced in tropical regions. The palm oil production process leads to a highly polluting waste called palm oil mill effluent (POME). A strategy commonly used by producers to overcome environmental issues and to improve soil fertility is the reuse of POME as a fertilizer due to the chemical and biological characteristics of the effluent. In this research, three groups were analyzed: soil without POME application (control group) and soil samples after 4 and 9 days of POME application. An environmental DNA metabarcoding approach was used. eDNA was extracted, and the V4 region of the 16S rRNA gene was amplified and sequenced in the Illumina MiSeq platform. The abundance of Proteobacteria (48.1%) and Firmicutes (9.0%) was higher in fertilized soil, while Bacteroidetes (20.3%) and Verrucomicrobia (7.8%) were more abundant in control soil. Additionally, the effluent seemed to modify soil characteristics favoring taxa responsible for the mineralization of organic compounds and nitrogen fixation such as species of Gammaproteobacteria class. Our study highlights the influence of POME on soil biological components and contributes to the sustainable production of palm oil in the Amazon.

Comparison of ruminal microbiota, IL-1β gene variation, and tick incidence between Holstein × Gyr and Holstein heifers in grazing system.

The variation in bacterial communities among breeds has been previously reported and may be one of the reasons why Holstein × Gyr dairy heifers have better development in grazing systems in tropical conditions. This study aimed to explore the ruminal microbiota composition, the IL-1β gene variation, tick incidence, and blood parameters of Holstein × Gyr (½ Holstein × ½ Gyr) and Holstein heifers grazing intensely managed Guinea grass (Panicum maximum Jacq. cv. Mombaça). Sixteen heifers were divided into two groups consisting of 8 Holstein × Gyr and 8 Holstein heifers. The experimental period was comprised of 3 periods of 21 days. Ruminal samples were taken via the stomach tube technique. The sequencing of the V4 hypervariable region of the 16S rRNA gene was performed using the Illumina MiSeq platform. Counting and collection of ticks were conducted each 21 days. Blood and skeletal muscle tissue biopsies were performed at the end of the experiment. Firmicutes were the most abundant phyla present in both breed rumen samples and Bacteroidota showed differences in relative abundance between breed groups, with greater values for Holstein heifers (p < 0.05 with FDR correction). The 10 most abundant unique OTUs identified in each breed included several OTUs of the genus Prevotella. Holstein heifers had a greater tick count and weight (9.8 ticks/animal and 1.6 g/animal, respectively) than Holstein × Gyr (2.56 ticks/animal and 0.4 g/animal, respectively). We found nucleotide substitutions in the IL-1β gene that might be related to adaptation and resistance phenotypes to tick infestation in Holstein × Gyr heifers. Blood concentrations of urea, albumin, insulin-like growth factor 1, triiodothyronine, and thyroxine were greater in Holstein × Gyr than in Holstein heifers. Adaptations in Holstein × Gyr heifers such as ruminal microbiota, tick resistance, nucleotide substitutions in IL-1β gene, and hormone concentration suggest a better energy metabolism and thermoregulation resulting in better performance in tropical grazing systems.

The impact of diets containing Hermetia illucens meal on the growth, intestinal health, and microbiota of gilthead seabream (Sparus aurata)

The present study investigated the effect of replacing fishmeal (FM) with insect meal of Hermetia illucens (HI) in the diet of Sparus aurata farmed inshore on growth, gut health, and microbiota composition. Two isolipidic (18% as fed) and isoproteic (42% as fed) diets were tested at the farm scale: a control diet without HI meal and an experimental diet with 11% HI meal replacing FM. At the end of the 25-week feeding trial, final body weight, specific growth rate, feed conversion rate, and hepatosomatic index were not affected by the diet. Gross morphology of the gastrointestinal tract and the liver was unchanged and showed no obvious signs of inflammation. High-throughput sequencing of 16S rRNA gene amplicons (MiSeq platform, Illumina) used to characterize the gut microbial community profile showed that Proteobacteria, Fusobacteria, and Firmicutes were the dominant phyla of the gut microbiota of gilthead seabream, regardless of diet. Dietary inclusion of HI meal altered the gut microbiota by significantly decreasing the abundance of Cetobacterium and increasing the relative abundance of the Oceanobacillus and Paenibacillus genera. Our results clearly indicate that the inclusion of HI meal as an alternative animal protein source positively affects the gut microbiota of seabream by increasing the abundance of beneficial genera, thereby improving gut health and maintaining growth performance of S. aurata from coastal farms.

Optimization of HIV Sequencing Method Using Vela Sentosa Library on Miseq Ilumina Platform.

Genotypic testing is often recommended to improve the management of patients infected with human immunodeficiency virus (HIV). To help combat this major pandemic, next-generation sequencing (NGS) techniques are widely used to analyse resistance to antiretroviral drugs. In this study, we used a Vela Sentosa kit (Vela Diagnostics, Kendall, Singapore), which is usually used for the Ion Torrent personal genome machine (PGM) platform, to sequence HIV using the Illumina Miseq platform. After RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR), minor modifications were applied to the Vela Sentosa kit to adapt it to the Illumina Miseq platform. Analysis of the results showed the same mutations present in the samples using both sequencing platforms. The total number of reads varied from 185,069 to 752,343 and from 642,162 to 2,074,028 in the Ion Torrent PGM platform and the Illumina Miseq platform, respectively. The average depth was 21,955 and 46,856 for Ion Torrent PGM and Illumina Miseq platforms, respectively. The cost of sequencing a run of eight samples was quite similar between the two platforms (about USD 1790 for Illumina Miseq and about USD 1833 for Ion Torrent PGM platform). We have shown for the first time that it is possible to adapt and use the Vela Sentosa kit for the Illumina Miseq platform to obtain high-quality results with a similar cost.

Identification of keystone taxa in root canals and periapical lesions of post-treatment endodontic infections: Next generation microbiome research.

The aim of this study was to analyse and compare the microbiome present in root canals and periapical lesions of teeth with post-treatment infections, and to identify the presence of keystone taxa in both habitats using next-generation sequencing analysis. Apices and periapical lesions of patients with post-treatment apical periodontitis were surgically extracted. Specimens were cryo-pulverized, bacterial DNA was extracted, and the V3-V4 hypervariable regions of the 16S rRNA gene were sequenced using the Illumina Miseq platform. Bioinformatic analysis was carried out with Mothur software, whilst diversity indices were obtained using operational taxonomic units (OTUs). The diversity indices were compared with the Kruskal-Wallis test, and community composition differences were explored with Permutational Multivariate Analysis of Variance (PERMANOVA). A bacterial functional study was performed with the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis. Co-occurrence network analyses were performed using the Sparse Correlations for Compositional data (SparCC). Eigencentrality, clr-based abundance and ubiquitousness were applied to infer keystone taxa. P values <.05 were considered statistically significant. Thirty-two apices and thirty-nine periapical lesions were sequenced and analysed. A similar alpha-diversity (p < .05) and community composition (p = .91) was observed for apices and lesion samples. The most abundant OTUs identified amongst all samples included Fusobacterium nucleatum, Prevotella loescheii, Streptococcus intermedius, Porphyromonas gingivalis, Parvimonas micra, Synergistetes bacterium, Tannerella forsythia and Peptostreptococcus stomatis. The metabolic pathways with >0.81% abundances included membrane transport, genetic information processing and metabolic pathways. F. nucleatum was identified as a keystone taxon as it showed ubiquitousness, an eigenvector centrality value of 0.83 and a clr-based abundance >4. The microbiome in apices and periapical lesions of post-treatment endodontic infections showed a similar diversity and taxonomic composition. Co-occurrence network analyses at OTU level identified F. nucleatum as a keystone taxon candidate in these infections.

A185 A GUT MICROBIOME TRANSITION: THE JOURNEY FROM INDIA TO CANADA

Abstract Background Young Indian immigrants and Indo-Canadians face a significantly higher risk of inflammatory bowel disease (IBD) in westernized countries. While the etiology of IBD remains unclear, a gut microbiome that is no longer symbiotic with its host is a key player. However, Indians are underrepresented in microbiome research, therefore we cannot accurately assess the role of their gut microbiome in IBD. To understand why Indians are at a greater risk for IBD in Canada, we must first characterize their gut microbiome. Aims This study explores how immigration to Canada impacts the gut microbiome of Indian populations, potentially elevating their risk to IBD. Methods Stool samples from healthy volunteers (ages 17-53) were collected from Indians in India, Indo-Immigrants, Indo-Canadians, Euro-Canadians and Euro-Immigrants. DNA was extracted for 16S sequencing on the Illumina MiSeq platform and shotgun sequencing on the Illumina NovaSeq platform. Dietary data was processed in ESHA. Results Weighted Unifrac revealed distinct clusters of Indian and Indo-Immigrant samples from the westernized cohorts. The most pronounced difference was between Indian and Euro-Canadians with a pseudo-F value of 49.18 (q = 0.00125). Indian versus Indo-Immigrants resulted in a pseudo-F value of 7.707 (q = 0.00125), and Indian versus Indo-Canadian had a pseudo-F value of 15.95 (q = 0.00125). BugBase estimated a significantly higher stress tolerance in the Indian gut, with Proteobacteria driving this phenotypic prediction. Compared to Euro-Canadians, a significantly higher pathogenic potential was predicted in Indians (pFDR= 1.04e-11) and Indo-Immigrants (pFDR= 1.41e-04), driven mainly from Proteobacteria and Bacteroidetes. Multiple comparisons with LDA revealed Prevotella was over 5 times more abundant in Indians residing in India. The gut microbiome in Indians were also enriched with taxa associated with a plant-based diet. Dietary data showed that 59% of Indians do not consume meat, and they also had the lowest consumption (12% caloric intake) of group 4 ultra-processed food (UPF), whereas the highest consumption of UPF was in Indo-Canadians (61% caloric intake) (pbonf ampersand:003C 0.0001). A frequent trend was observed in Indians living in Canada, which was a reduction in key nutrients including soluble fiber, zinc, iron, and folate, some of which are commonly found in plant-based foods. Conclusions Overall findings reveal that a loss of Prevotella abundance was observed in Indo-Immigrants and Indo-Canadians, which was associated with a reduction in key nutrients that are common to a plant-based diet, indicating a transition away from their traditional diet. Their dietary changes may be a driver to the displacement of Prevotella and other taxa commonly found in the Indian gut microbiome. Funding Agencies NSERC, Killam

High-throughput analysis using 16S rRNA gene of bacterial communities present in selected bivalves and gastropods species from Bayug Island, Iligan City, Philippines

Abstract. Albarido NA, Tabugo SR. 2024. High-throughput analysis using 16S rRNA gene of bacterial communities present in selected bivalves and gastropods species from Bayug Island, Iligan City, Philippines. Biodiversitas 25: 431-438. Seashells, which include bivalves and gastropods, have global recognition for their significant contributions to the economy, ecology, and medicine. They hold value as a food source and are highly regarded as effective biological indicators. The objective of this study is to identify the bacterial communities present in selected edible species of bivalves (Pinctada margaritifera Linnaeus, 1758 and Anadara granosa Linnaeus, 1758) and gastropods (Canarium urceus Linnaeus, 1758 and Conus stercusmuscarum Linnaeus, 1758), through high-throughput sequencing metabarcoding. Bacterial samples were collected via a swabbing technique on the surface and inside parts of selected mollusc species, which were then placed on sterilized seawater for DNA extraction. Genomic DNA was isolated from the samples, and the V3-V4 region of the 16S rRNA gene was amplified and sequenced using the Illumina MiSeq platform. Four amplicon libraries were generated, representing the two bivalve and two gastropod species in the study area. Data analysis was conducted using the Parallel Meta Suite software. Upon quality control and processing, 173,489 amplicon sequence variants (ASVs) were obtained. Within the bacterial community, the most abundant genera included Stenotrophomonas, Vibrio, Serratia, Photobacterium, and Shewanella. The assessment of alpha diversity, using the Shannon index, indicated a higher diversity in A. granosa. Furthermore, the analysis using the PICRUSt algorithm within the Parallel Meta Suite unveiled the involvement of specific bacteria found in the selected gastropod and bivalve species in various functions. These functions encompass protein production, xenobiotic metabolism, biodegradation, and other metabolism-related processes, supporting these organisms' ecological and physiological roles.

Metabarcoding mosquitoes: MinION sequencing of bulk samples gives accurate species profiles for vector surveillance (Culicidae)

Mosquitoes (Family: Culicidae) are dominant vectors of pathogens, and their surveillance has been incorporated into major disease control programs worldwide. However, routine, species-level identification of mosquitoes is often a bottleneck for management, and Next Generation Sequencing (NGS) platforms and DNA metabarcoding can revolutionize this process. MinION nanopore technologies promise on-site sequencing and rapid sample processing rates ideal for time-sensitive biosurveillance. Here, we benchmark the results of DNA metabarcoding on the MinION against the Illumina MiSeq platform, which is known for its higher sequencing accuracy. We used metazoan COI mini-barcode primers to carry out DNA metabarcoding of mosquito bulk samples caught during a real vector survey, then compared the mosquito species profiles recovered on each sequencing platform. We also tested the influence of using different trap lures, storage methods, and pooling different specimen body parts on the number of species recovered. We report that mosquito species-level identifications were highly congruent between MinION and Illumina (93% overlap). We also find that CO2 gas cylinders outperformed biogenic CO2 sources significantly, by two-fold. Notably, we demonstrated the feasibility of detecting zoonotic reservoirs and pathogen signals from mosquito bulk samples. We present the first use of DNA metabarcoding on the MinION for vector surveillance and discuss future applications.

Diversity of Omicron sublineages and clinical characteristics in hospitalized patients in the southernmost state of Brazil

BackgroundOmicron has become the dominant variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since first reported in November 2021. From the initially detected Wuhan lineage, sublineages BA.2, BA.4, BA.5, BQ, XAG, and XBB have emerged over time and are dominant in many countries. Therefore, the aim is to evaluate which variants are circulating and the clinical characteristics of inpatients infected with the Omicron variant.MethodsThis retrospective cohort study selected hospitalized patients admitted with respiratory symptoms to a hospital in the state of Rio Grande do Sul, Brazil, between June and July 2022. SARS-CoV-2 results were analyzed together with clinical outcomes and vaccination status. A viral genome library was prepared and forwarded to the Illumina MiSeq Platform for sequencing.ResultsIn total, 37 genomes were sequenced. Concerning the Omicron sublineages, our study detected: BA.1 (21 K), BA.2 (21 L), BA.4 (22A), BA.5 (22B), BA.2.12.1 (22C), BQ.1 (22E), XBB (22F), and XAG recombinant. Omicron BA.5 (30%), BA.2 (19%), and BQ.1 (19%) were the most frequent sublineages, respectively. In total, 38% of patients present hypertension, and the most common symptoms were coughing (62%). Analyzing the COVID-19 vaccination, 30% of patients were fully vaccinated, 49% had a partial vaccination status, and 21% were unvaccinated (no dose).ConclusionsBA.5 was the most prevalent sublineage in our study and surpassed the predominance of BA.2, as reported by the national genomic surveillance program. BQ.1 was diagnosed earlier in this study than it was officially reported in the state. Current data have demonstrated that the Omicron variant causes less severe infections, with the high rate of transmissibility and mutational landscape causing the rapid emergence of new sublineages.

Emergence and Genomic Characterization of a spa Type t4407 ST6-SCCmec Type IVa Methicillin-Resistant Staphylococcus aureus Strain Isolated from Al-Karak Hospital, Jordan.

Background and Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) is a major concern in Jordanian hospitals in terms of infection control. The purpose of this study was to identify the resistance patterns of Staphylococcus aureus strains isolated from surfaces of critical locations within the Al-Karak Governmental Hospital in 2019. Additionally, the study aimed to conduct whole-genome sequencing on the isolates. Materials and Methods: In February 2019, fourteen S. aureus strains were isolated from surfaces in critical sites in the Al-Karak Governmental Hospital. These isolates underwent antibiogram testing to determine their resistance profile. Genome sequencing using the Illumina MiSeq platform was applied to the extracted DNA from these isolates. The genomic data, including coding sequences, were analyzed to identify lineage, resistance genes, and plasmids. Results: The antibiogram results revealed that 11 of the 14 isolates were resistant to oxacillin, 6 to linezolid, and 1 to rifampicin, while none showed resistance to chloramphenicol. Eleven isolates were identified as MRSA, with a novel spa type (t4407) not previously reported in Jordan. High-quality sequencing data were obtained for only one isolate, i.e., A29, the genome showed 2,789,641 bp with a 32.7% GC content and contained 2650 coding sequences. Genomic analysis indicated the ST6 lineage, mecA gene (SCCmec type IVa(2B)), and a hybrid plasmid (pJOR_blaZ) carrying the blaZ gene for β-lactam resistance. Genomic data were deposited in NCBI (CP104989). The A29 genome closely resembled an MRSA genome isolated from a Danish hospital in 2011. The SNP analysis revealed identical antimicrobial resistance genes in these two genomes. Conclusions: This study unveils the first genomic sequence of an MRSA isolate from Jordan, marked by distinctive genotypic traits. The findings enhance our understanding of the MRSA types circulating in Jordan and the region and substantiate the phenomenon of intercontinental MRSA transmission.

Gut microbiota of preterm infants in the neonatal intensive care unit: a study from a tertiary care center in northern India.

Disruptions of the gut microbiota of preterm infants admitted to the neonatal intensive care unit (NICU) during the first 2 weeks of life are of critical importance. These infants are prone to various complications, including necrotizing enterocolitis (NEC) and sepsis. Studying the gut microbiota will improve outcomes in preterm infants. In the present study, we examined the gut microbiota of preterm infants admitted to the NICU in the first month of life. Neonates admitted to the NICU were recruited, and stool samples were collected weekly from the seventh day of the infant's life until the 30th day of life. DNA was extracted using a DNeasy Powersoil DNA isolation kit. 16S rRNA gene sequencing targeting the V3-V4 region was performed using the MiSeq platform. Sequenced reads were processed on DADA2 pipeline to obtain an amplicon sequence variant (ASV) table. All bioinformatic and statistical analyses were performed using different packages in the R statistical framework. Fourteen preterm infants were recruited, and 48 samples were collected. Alpha diversity metrics, observed ASV count, and Shannon index were found to have no differences in any clinical variables. Permutational multivariate analysis of variance (PERMANOVA) showed discrimination of neonates by gestational age and administration of probiotics. Differential abundance analysis showed a decreased abundance of Bifidobacterium Breve in extremely preterm infants (gestational age <28 weeks) compared to moderate preterm infants (gestational age 29-32 weeks). Supplementation with probiotics decreased Acinetobacter and increased Bifidobacterium in the gut of preterm neonates regardless of gestational age. Gestational age and probiotic supplementation alter the gut microbiota of preterm infants admitted to the NICU.

Difference of Microbial Community in the Stream Adjacent to the Mixed Antibiotic Effluent Source.

Released antibiotics from source to stream can influence bacterial communities and potentially alter the ecosystem. This research provides a comprehensive examination of the sources, distribution, and bacterial community dynamics associated with varied antibiotic release sources adjacent to the stream. The residual of antibiotics from different sources was determined, and the bacterial community structure was examined to reveal the differences in the bacteria community in the stream. The residual of antibiotics was quantified with liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the Illumina MiSeq platform was utilized to sequence bacterial 16S rRNA genes, providing comprehensive insights into the bacterial community structure in the sediment across five different sites. Results indicated that the presence and distribution of antibiotics were significantly influenced by released sources. In the case of the bacterial community, the Proteobacteria and Firmicutes were the most dominant phyla in the sediment, and especially, the Firmicutes showed higher abundance in sites mostly affected by livestock sources. Additionally, livestock gut bacteria such as Clostridium saudiense, Proteiniclasticum ruminis, and Turicibacter sanguinis were prevalent in antibiotic-contaminated sites adjacent to livestock facilities. Overall, this study provides critical insights into the effect of antibiotic contamination by verifying the relationship between the occurrence of antibiotic residuals and the alteration in the bacterial community in the stream.

(044) Does Time of Intraoperative Exposure to the Aerobiome Increase Microbial Growth on Inflatable Penile Prostheses?

Abstract Introduction The use of inflatable penile prostheses (IPP) is a fundamental approach in managing erectile dysfunction that does not respond to medical management. Approximately 28,000 IPPs are implanted annually in the United States. Device infection, with an incidence up to 3%, is a particularly concerning complication as management generally necessitates device removal. This can result in significant cost burden, requiring inpatient admission, return to operative room and extensive postoperative care. One important risk factor for IPP infection is operative time, with the suggestion that increased operative time facilitates a colonization of microbes from the skin flora and ambient air within the operating room (referred to as the aerobiome). Objective To evaluate whether increased exposure to the operating room (OR) aerobiome results in increased microbial colonization of IPPs. Methods We performed an ex vivo study focusing on the reservoir and tips of Coloplast Titan IPP. Conditions of an IPP surgery were attempted to be closely replicated. Setup included a dedicated table within the same institutional OR where IPPs are routinely performed, facilitating sample collection, and with standard intra-operative conditions-pressure 0.024”WC, temperature 66.80F, and humidity 49.2% RH-to emulate surgical conditions. Stringent surgical hygiene was observed to obtain samples in a sterile manner; this including surgical scrub, sterile field, and double-glove technique. A sterile swab was collected at 30-minute intervals over a total duration of 3 hours, from each individual component of the IPP, resulting in a total of 6 samples per component. To prevent any potential interference from previous samples, care was taken to collect each swab from distinct areas. The obtained swabs were subjected to quantitative polymerase chain reaction (qPCR) and next generation sequencing (NGS) analyses of the V12 16S rRNA conservative region using the MiSeq platform (Illumina, San Diego, CA) with 2x250 paired read chemistry. Subsequent bioinformatic processing was carried out utilizing a clustering-based method with operational taxonomic units set at a 97% similarity threshold, and taxonomic assignment was accomplished using an internally maintained taxonomic classifier. Results The results of the study, obtained through both standard culture and next-generation sequencing (NGS) analyses, demonstrated consistent and significant findings. Through rigorous examination, it was determined that no microbial growth was detected on any component of the inflatable penile prostheses (IPPs) throughout the entire duration of the experiment, which lasted up to 3 hours. This dual approach of standard culture and NGS provided comprehensive and robust evidence of the absence of microbial colonization on the IPP devices. Conclusions These findings suggest that the duration of exposure to the OR aerobiome alone does not appear to increase the risk of microbial colonization on IPP devices. Further ex vivo studies are needed to support these findings and evaluate whether factors such as time of skin-to-IPP contact or changes in OR conditions contribute to risk of IPP microbial colonization. Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Coloplast, Boston Scientific.

High-throughput analysis using 16S rRNA sequencing of bacterial communities associated in selected mangrove species from Bayug Island, Iligan City, Philippines

Abstract. Siblos SKV, Tabugo SR. 2023. High-throughput analysis using 16S rRNA sequencing of bacterial communities associated in selected mangrove species from Bayug Island, Iligan City, Philippines. Biodiversitas 25: 53-61. Mangrove ecosystems are recognized globally as highly productive ecosystems, that play a crucial role in carbon sequestration, erosion control, water purification, and as essential breeding grounds for diverse aquatic life. They also offer specialized ecological niches, accommodating a diverse range of organisms, including bacterial communities. This study aimed to investigate bacterial communities of Bayug Island in Iligan City, Philippines, inhabiting six mangrove species, namely Rhizophora stylosa Griffith, Rhizophora apiculata Blume, Rhizophora mucronata Lam., Sonneratia alba Sm., Ceriops tagal (Perr.) C.B.Rob., and Nypa fruticans Wurmb and their possible functions within the mangrove forests. Genomic DNA was extracted from the six pooled soil samples, and the V3-V4 region of the 16S rRNA gene was sequenced using the Illumina MiSeq platform. Six amplicon libraries, corresponding to the mangrove species were analyzed with Parallel Meta Suite software, yielding 173,270 amplicon sequence variants (ASVs) after quality control. The study identified the top five most abundant ASVs in the mangrove rhizosphere, linked to the genera Vibrio, Stenotrophomonas, Serratia, Pseudoalteromonas, and Achromobacter. Among the mangrove species, S. alba exhibited higher alpha diversity according to the Shannon index. PICRUSt analysis revealed that microorganisms are involved in biodegradation, xenobiotic metabolism, and other metabolic processes within the mangroves. These bacteria could find applications in environmental cleanup, bioremediation, waste treatment, and soil health improvement. This result contributes to the ongoing ecological restoration of mangrove forests at the research site.

Chronic intestinal pseudo-obstruction: associations with gut microbiota and genes expression of intestinal serotonergic pathway

BackgroundPediatric chronic intestinal pseudo-obstruction (PIPO) is a rare disease characterized by symptoms and radiological signs suggestive of intestinal obstruction, in the absence of lumen-occluding lesions. It results from an extremely severe impairment of propulsive motility. The intestinal endocrine system (IES) jointly with the enteric nervous system (ENS) regulates secreto-motor functions via different hormones and bioactive messengers/neurotransmitters. The neurotransmitter 5-hydroxytryptamine (5-HT) (or serotonin) is linked to intestinal peristalsis and secretory reflexes. Gut microbiota and its interplay with ENS affect 5-HT synthesis, release, and the subsequent serotonin receptor activation. To date, the interplay between 5-HT and gut microbiota in PIPO remains largely unclear. This study aimed to assess correlations between mucosa associated microbiota (MAM), intestinal serotonin-related genes expression in PIPO. To this purpose, biopsies of the colon, ileum and duodenum have been collected from 7 PIPO patients, and 7 age-/sex-matched healthy controls. After DNA extraction, the MAM was assessed by next generation sequencing (NGS) of the V3-V4 region of the bacterial RNA 16 S, on an Illumina Miseq platform. The expression of genes implicated in serotoninergic pathway (TPH1, SLC6A4, 5-HTR3 and 5-HTR4) was established by qPCR, and correlations with MAM and clinical parameters of PIPO have been evaluated.ResultsOur results revealed that PIPO patients exhibit a MAM with a different composition and with dysbiosis, i.e. with a lower biodiversity and fewer less connected species with a greater number of non-synergistic relationships, compared to controls. qPCR results revealed modifications in the expression of serotonin-related intestinal genes in PIPO patients, when compared to controls. Correlation analysis do not reveal any kind of connection.ConclusionsFor the first time, we report in PIPO patients a specific MAM associated to underlying pathology and an altered intestinal serotonin pathway. A possible dysfunction of the serotonin pathway, possibly related to or triggered by an altered microbiota, may contribute to dysmotility in PIPO patients. The results of our pilot study provide the basis for new biomarkers and innovative therapies targeting the microbiota or serotonin pathways in PIPO patients.

Understanding the long-lasting associations among humans, neglected dogs, tick-borne diseases, and core blood bacteria-related pathogenic taxa using next-generation sequencing

Background and Aim: Dogs are long-lasting companion animals, and ticks are the most common external parasites in dogs. An increase in the population of neglected domestic dogs has increased the risk of contact with ticks, especially in places where tick-borne diseases (TBDs) are endemic. We aimed to characterize the bacterial blood profiles of people who were either exposed (HE) or not exposed (HC) to tick bites using next-generation sequencing (NGS). Materials and Methods: In the present study, the bacteria observed in the blood of people exposed to tick bites were compared with those in the blood of people not exposed to tick bites in Northern Mexico. Human blood samples (n = 12) were analyzed, DNA was extracted, and the V3–V4 region of the 16S ribosomal RNA gene was amplified. In addition, NGS was performed on a MiSeq platform (Illumina), and the data were analyzed through Quantitative Insights into Microbial Ecology. Results: Differences in beta diversity were significant. In HEs, several potentially pathogenic bacterial taxa were found to be the most abundant: Kocuria ( x = 14.59%), Staphylococcus ( x = 3.05%), and Treponema ( x = 2.93%), in addition to Chlamydia, Clostridium, and Ehrlichia, which are considered TBDs. Conclusion: This study identified important differences in the bacterial composition of the HE and HC groups. In addition to Ehrlichia (a TBD considered a taxon), other bacterial pathogenic taxa, such as Chlamydia, Clostridium, Kocuria, Staphylococcus, and Treponema, were also observed in the tick bite-exposed group. Future studies with larger sample sizes should provide an improved understanding of the human blood microbiome profile by providing additional evidence of tick exposure, associated TBDs, and other pathogenic bacterial taxa. Keywords: Blood, Ehrlichia, microbiome, tick bites, tick-borne diseases, ticks.

An improved and extended dual-index multiplexed 16S rRNA sequencing for the Illumina HiSeq and MiSeq platform.

Recent advancements in next-generation sequencing (NGS) technology have ushered in significant improvements in sequencing speed and data throughput, thereby enabling the simultaneous analysis of a greater number of samples within a single sequencing run. This technology has proven particularly valuable in the context of microbial community profiling, offering a powerful tool for characterizing the microbial composition at the species level within a given sample. This profiling process typically involves the sequencing of 16S ribosomal RNA (rRNA) gene fragments. By scaling up the analysis to accommodate a substantial number of samples, sometimes as many as 2,000, it becomes possible to achieve cost-efficiency and minimize the introduction of potential batch effects. Our study was designed with the primary objective of devising an approach capable of facilitating the comprehensive analysis of 1,711 samples sourced from diverse origins, including oropharyngeal swabs, mouth cavity swabs, dental swabs, and human fecal samples. This analysis was based on data obtained from 16S rRNA metagenomic sequencing conducted on the Illumina MiSeq and HiSeq sequencing platforms. We have designed a custom set of 10-base pair indices specifically tailored for the preparation of libraries from amplicons derived from the V3-V4 region of the 16S rRNA gene. These indices are instrumental in the analysis of the microbial composition in clinical samples through sequencing on the Illumina MiSeq and HiSeq platforms. The utilization of our custom index set enables the consolidation of a significant number of libraries, enabling the efficient sequencing of these libraries in a single run. The unique array of 10-base pair indices that we have developed, in conjunction with our sequencing methodology, will prove highly valuable to laboratories engaged in sequencing on Illumina platforms or utilizing Illumina-compatible kits.

Comparison of mucosal microbiomes from biliary tract among patients with extrahepatic cholangiocarcinoma, patients with chronic cholecystitis, and healthy controls.

550 Background: Cholangiocarcinoma (CCA) is a malignancy associated with a poor prognosis. Gut microbiome dysbiosis is considered to be related with multiple disease, including and cancer. However, relationship between mucosal microbiome dysbiosis and CCA remains unclear. In this study, we evaluated mucosal microbiome of patients with extrahepatic-CCA (EH-CCA) and compared it with patients with chronic cholecystitis (CC) and living liver donors who served as healthy control (HC), to understand the role of microbiome in development EH-CCA. Methods: A total of 18 patients with EH-CCA, 21 patients with CC, and 17 HC were included. During operation, swab sample were obtained from cancer tissue of patient with EH-CCA and from gallbladder in other participants, respectively. The bacterial 16S rRNA gene (V3/V4 region) was amplified using PCR and sequenced on the Illumina MiSeq platform. The QIIME 2 pipelines were used to analyze the raw data. Results: The median age were 71, 53, and 29 years for patients with EH-CCA, patients with CC, and HC, respectively. The mean number of operational taxonomic unit were significantly higher in the CCA (13,037 [SD 9,676]) compared to HC (5,045 [12,679]; p&lt;0.001). Among the sample from HC, the most abundant taxa at phylum level was Firmicutes (74.47%), followed by Bacteroidota (14.8%), and Proteobacteria accounted for only 2%. In the CC group, Firmicutes is most abundant (63.15%) phylum, followed by Proteobacteria (22.09%). In contrast, among the samples from CCA, Proteobacteria were the most common phylum (50.43%). Pairwise comparison of α-diversity measured by Chao1 index showed that richness was higher in samples from CC compared to other groups (vs HC, p=0.02; vs CCA, p&lt;0.001). In terms of α-diversity measured by Shannon index, samples from cancer have lower diversity compared to healthy controls ( p&lt;0.001), however, no significant differences were found between samples from cancer and CC ( p=0.1). The Principal Coordinate Analysis plot based on Bray-Curtis distance showed that all three groups formed significantly separate clusters (p=0.001). The linear discriminant analysis effect size showed that the most abundant taxa from samples from CCA were Enterobacter, Klebsiella, and Fusobacterium. In the samples from HC, Limosilactobacillus, Lachnospiraceae_NK4A136_group, and Faecalibacterium were most significant, while Lactobacillus and Pseudomonas were most abundant among the CC group. Conclusions: This study compared the mucosal microbiome from biliary tract among patients with CCA, patients with CC, and HC. All three groups showed significantly different microbiome profiles. In CCA sample, the proportion of Proteobacteria is high, and diversity were low, suggesting mucosal microbial dysbiosis. Further studies are needed to investigate the clinical implications of microbial dysbiosis in CCA.

Sex-specific differences in intestinal microbiota associated with cardiovascular diseases

BackgroundCardiovascular diseases (CVD), including coronary heart disease (CHD), display a higher prevalence in men than women. This study aims to evaluate the variations in the intestinal microbiota between men and women afflicted with CHD and delineate these against a non-CVD control group for each sex.MethodsOur research was conducted in the framework of the CORDIOPREV study, a clinical trial which involved 837 men and 165 women with CHD. We contrasted our findings with a reference group of 375 individuals (270 men, 105 women) without CVD. The intestinal microbiota was examined through 16S metagenomics on the Illumina MiSeq platform and the data processed with Quiime2 software.ResultsOur results showed a sex-specific variation (beta diversity) in the intestinal microbiota, while alpha-biodiversity remained consistent across both sexes. Linear discriminant analysis effect size (LEfSe) analysis revealed sex-centric alterations in the intestinal microbiota linked to CVD. Moreover, using random forest (RF) methodology, we identified seven bacterial taxa—g_UBA1819 (Ruminococcaceae), g_Bilophila, g_Subdoligranulum, g_Phascolarctobacterium, f_Barnesiellaceae, g_Ruminococcus, and an unknown genus from the Ruminococcaceae family (Ruminococcaceae incertae sedis)—as key discriminators between men and women diagnosed with CHD. The same taxa also emerged as critical discriminators between CHD-afflicted and non-CVD individuals, when analyzed separately by sex.ConclusionOur findings suggest a sex-specific dysbiosis in the intestinal microbiota linked to CHD, potentially contributing to the sex disparity observed in CVD incidence.Trial registration Clinical Trials.gov.Identifier NCT00924937.Graphical

Genome analysis of Pseudomonas strain 4B with broad antagonistic activity against toxigenic fungi.

Pseudomonas sp. 4B isolated from the effluent pond of a bovine abattoir was investigated as antifungal against toxigenic fungi. The complete genome of Pseudomonas 4B was sequenced using the Illumina MiSeq platform. Phylogenetic analysis and genome comparisons indicated that the strain belongs to the Pseudomonas aeruginosa group. In silico investigation revealed gene clusters associated with the biosynthesis of several antifungals, including pyocyanin, rhizomide, thanamycin, and pyochelin. This bacterium was investigated through antifungal assays, showing an inhibitory effect against all toxigenic fungi tested. Bacterial cells reduced the diameter of fungal colonies, colony growth rate, and sporulation of each indicator fungi in 10-day simultaneous growing tests. The co-incubation of bacterial suspension and fungal spores in yeast extract-sucrose broth for 48h resulted in reduced spore germination. During simultaneous growth, decreased production of aflatoxin B1 and ochratoxin A by Aspergillus flavus and Aspergillus carbonarius, respectively, was observed. Genome analysis and in vitro studies showed the ability of P. aeruginosa 4B to reduce fungal growth parameters and mycotoxin levels, indicating the potential of this bacterium to control toxigenic fungi. The broad antifungal activity of this strain may represent a sustainable alternative for the exploration and subsequent use of its possible metabolites in order to control mycotoxin-producing fungi.