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Vermicomposting offers an eco-friendly solution to managing the sewage sludge generated in wastewater treatment plants. The objective of this study was to investigate the microbial community composition, structure and functionality during the vermicomposting of sewage sludge. We analyzed samples of sewage sludge, earthworm casts and vermicompost by applying high-throughput sequencing 16S and ITS rRNA. Most of the bacterial (95%) and fungal taxa (99%) were eliminated and subsequently replaced by other microbial taxa originating from earthworms. Further changes resulted in a vermicompost with a more diverse bacterial (but not fungal) community. In addition, the earthworm activity led to an increase in bacterial and a decrease in fungal alpha diversity, resulting in greater differences in beta diversity between sewage sludge, casts and vermicompost. We also found that bacterial pathways associated with amino acid and plant hormone biosynthesis and antibiotic synthesis were enriched. Vermicomposting successfully eliminated most of the 10 human bacterial pathogens found in the sewage sludge. Simultaneously, parasitic and pathogenic fungal taxa were removed. Overall, vermicompost derived from sewage sludge is safer for disposal on land than raw sludge, particularly regarding their respective microbial compositions. This indicates that it could potentially be used as a soil organic amendment and fertilizer. [ABSTRACT FROM AUTHOR]

ABSTRACT Rhesus cytomegalovirus expressing simian immunodeficiency virus (RhCMV/SIV) vaccines protect ~59% of vaccinated rhesus macaques against repeated limiting-dose intra-rectal exposure with highly pathogenic SIVmac239M, but the exact mechanism responsible for the vaccine efficacy is unknown. It is becoming evident that complex interactions exist between gut microbiota and the host immune system. Here, we aimed to investigate if the rhesus gut microbiome impacts RhCMV/SIV vaccine-induced protection. Three groups of 15 rhesus macaques naturally pre-exposed to RhCMV were vaccinated with RhCMV/SIV vaccines. Rectal swabs were collected longitudinally both before SIV challenge (after vaccination) and post-challenge and were profiled using 16S rRNA based microbiome analysis. We identified ~2,400 16S rRNA amplicon sequence variants (ASVs), representing potential bacterial species/strains. Global gut microbial profiles were strongly associated with each of the three vaccination groups, and all animals tended to maintain consistent profiles throughout the pre-challenge phase. Despite vaccination group differences, by using newly developed compositional data analysis techniques, we identified a common gut microbial signature predictive of vaccine protection outcome across the three vaccination groups. Part of this microbial signature persisted even after SIV challenge. We also observed a strong correlation between this microbial signature and an early signature derived from whole blood transcriptomes in the same animals. Our findings indicate that changes in gut microbiomes are associated with RhCMV/SIV vaccine-induced protection and early host response to vaccination in rhesus macaques.IMPORTANCEThe human immunodeficiency virus (HIV) has infected millions of people worldwide. Unfortunately, still there is no vaccine that can prevent or treat HIV infection. A promising pre-clinical HIV vaccine based on rhesus cytomegalovirus (RhCMV) expressing simian immunodeficiency virus (SIV) antigens (RhCMV/SIV) provides sustained, durable protection against SIV challenge in ~59% of vaccinated rhesus macaques. There is an urgent need to understand the cause of this protection vs non-protection outcome. In this study, we profiled the gut microbiomes of 45 RhCMV/SIV vaccinated rhesus macaques and identified gut microbial signatures that were predictive of RhCMV/SIV vaccination groups and vaccine protection outcomes. These vaccine protection-associated microbial features were significantly correlated with early vaccine-induced host immune signatures in whole blood from the same animals. These findings show that the gut microbiome may be involved in RhCMV/SIV vaccine-induced protection, warranting further research into the impact of the gut microbiome in human vaccine trials.

Avian pathogenic Escherichia coli (APEC) is a major cause of avian colibacillosis. Matrine, a natural component derived from Sophora flavescens, exhibits various pharmacological effects, including anti-inflammatory and antioxidant activities. However, its role in mitigating APEC-induced intestinal damage in chickens remains insufficiently understood. This study aimed to explore the protective effects and potential mechanisms of matrine against APEC-induced intestinal damage. Chickens were administered matrine (10 or 20 mg/kg) from 6 days old for 5 days, followed by an APEC intraperitoneal injection on day 10. After 72 h of APEC infection, tissues were collected for analysis. Results indicated that pretreatment with matrine alleviated the symptoms of APEC infection in chickens, improving survival rates and promoting weight gain. Additionally, pretreatment with matrine reduced the secretion and gene expression of IL-1β, IL-6, and TNF-α in intestinal tissues, while enhancing serum SOD, GSH, and CAT activity, as well as gene expression levels in the intestine. Pretreatment with matrine reduced the levels of TLR4, MyD88, and NF-κB in intestinal tissues. Moreover, pretreatment with matrine ameliorated intestinal inflammation and pathological damage, restoring the expression of ZO-1, Occludin, and MUC2 in the intestine during APEC infection. Furthermore, pretreatment with matrine alleviated gut microbiota dysbiosis by lowering the abundance of harmful bacteria. In summary, matrine alleviated APEC-induced intestinal inflammation and damage, potentially by inhibiting NF-κB signaling pathway and reshaping the gut microbiota. These findings provide promising insights into the prevention and treatment of avian colibacillosis.

ABSTRACT The hospital environmental microbiome, which can affect patients’ and healthcare workers’ health, is highly variable and the drivers of this variability are not well understood. In this study, we collected 37 surface samples from the neonatal intensive care unit (NICU) in an inpatient hospital before and after the operation began. Additionally, healthcare workers collected 160 surface samples from five additional areas of the hospital. All samples were analyzed using 16S rRNA gene amplicon sequencing, and the samples collected by healthcare workers were cultured. The NICU samples exhibited similar alpha and beta diversities before and after opening, which indicated that the microbiome there was stable over time. Conversely, the diversities of samples taken after opening varied widely by area. Principal coordinate analysis (PCoA) showed the samples clustered into two distinct groups: high alpha diversity [the pediatric intensive care unit (PICU), pathology lab, and microbiology lab] and low alpha diversity [the NICU, pediatric surgery ward, and infection prevention and control (IPAC) office]. Least absolute shrinkage and selection operator (LASSO) classification models identified 156 informative amplicon sequence variants (ASVs) for predicting the sample’s area of origin. The testing accuracy ranged from 86.37% to 100%, which outperformed linear and radial support vector machine (SVM) and random forest models. ASVs of genera that contain emerging pathogens were identified in these models. Culture experiments had identified viable species among the samples, including potential antibiotic-resistant bacteria. Though area type differences were not noted in the culture data, the prevalences and relative abundances of genera detected positively correlated with 16S sequencing data. This study brings to light the microbial community temporal and spatial variation within the hospital and the importance of pathogenic and commensal bacteria to understanding dispersal patterns for infection control.IMPORTANCEWe sampled surface samples from a newly built inpatient hospital in multiple areas, including areas accessed by only healthcare workers. Our analysis of the neonatal intensive care unit (NICU) showed that the microbiome was stable before and after the operation began, possibly due to access restrictions. Of the high-touch samples taken after opening, areas with high diversity had more potential external seeds (long-term patients and clinical samples), and areas with low diversity and had fewer (short-term or newborn patients). Classification models performed at high accuracy and identified biomarkers that could be used for more targeted surveillance and infection control. Though culturing data yielded viability and antibiotic-resistance information, it disproportionately detected the presence of genera relative to 16S data. This difference reinforces the utility of 16S sequencing in profiling hospital microbiomes. By examining the microbiome over time and in multiple areas, we identified potential drivers of the microbial variation within a hospital.

ABSTRACT In the wild, C. elegans are emersed in environments teeming with a veritable menagerie of microorganisms. The C. elegans cuticular surface serves as a barrier and first point of contact with their microbial environments. In this study, we identify microbes from C. elegans natural habitats that associate with its cuticle, constituting a simple “skin microbiome.” We rear our animals on a modified CeMbio, mCeMbio, a consortium of ecologically relevant microbes. We first combine standard microbiological methods with an adapted micro skin-swabbing tool to describe the skin-resident bacteria on the C. elegans surface. Furthermore, we conduct 16S rRNA gene sequencing studies to identify relative shifts in the proportion of mCeMbio bacteria upon surface-sterilization, implying distinct skin- and gut-microbiomes. We find that some strains of bacteria, including Enterobacter sp. JUb101, are primarily found on the nematode skin, while others like Stenotrophomonas indicatrix JUb19 and Ochrobactrum vermis MYb71 are predominantly found in the animal’s gut. Finally, we show that this skin microbiome promotes host cuticle integrity in harsh environments. Together, we identify a skin microbiome for the well-studied nematode model and propose its value in conferring host fitness advantages in naturalized contexts.IMPORTANCEThe genetic model organism C. elegans has recently emerged as a tool for understanding host–microbiome interactions. Nearly all of these studies either focus on pathogenic or gut-resident microbes. Little is known about the existence of native, nonpathogenic skin microbes or their function. We demonstrate that members of a modified C. elegans model microbiome, mCeMbio, can adhere to the animal's cuticle and confer protection from noxious environments. We combine a novel micro-swab tool, the first 16S microbial sequencing data from relatively unperturbed C. elegans, and physiological assays to demonstrate microbially mediated protection of the skin. This work serves as a foundation to explore wild C. elegans skin microbiomes and use C. elegans as a model for skin research.

ABSTRACT Scorpion envenoming is considered a public health problem in Brazil. A recent study described a variation in the clinical outcome of envenoming by Tityus obscurus in two populations separated by 850 km in the northeastern Amazon region. Our aim was to evaluate whether such clinical and toxinological variations are associated with underlying differences in genetic diversity between these two T. obscurus populations. We obtained DNA from five individuals of each population, in the municipalities of Belém and Santarém, located east and west of the state of Pará, Brazil, respectively. Gene regions encoding mitochondrial DNA (mtDNA) markers cytochrome oxidase subunit I (COI) and ribosomal 16S RNA (16S) were amplified and sequenced. Phylogenetic analyses were performed using maximum likelihood (ML) and Bayesian inferences (BA) for both molecular data (COI and 16S). The sampled T. obscurus populations corresponded to two distinct mtDNA lineages (genetic distance COI K2 P = 0.08 to 0.13; 16S K2 P = 0.10 to 0.11) with no shared mutations between groups and well supported by ML and BA inferences. Based on the divergence values found between eastern and western populations (COI, 0.07 to 0.12; 16S, 0.10), our study confirms the genetic heterogeneity of T. obscurus populations within the state of Pará, which correlates with observed venom and clinical differences, and reinforces the need for mapping the distribution of haplotypes throughout the geographic range of T. obscurus, to aid in future epidemiological, toxinological, and evolutionary studies.

Vermicomposting offers an eco-friendly solution to managing the sewage sludge generated in wastewater treatment plants. The objective of this study was to investigate the microbial community composition, structure and functionality during the vermicomposting of sewage sludge. We analyzed samples of sewage sludge, earthworm casts and vermicompost by applying high-throughput sequencing 16S and ITS rRNA. Most of the bacterial (95%) and fungal taxa (99%) were eliminated and subsequently replaced by other microbial taxa originating from earthworms. Further changes resulted in a vermicompost with a more diverse bacterial (but not fungal) community. In addition, the earthworm activity led to an increase in bacterial and a decrease in fungal alpha diversity, resulting in greater differences in beta diversity between sewage sludge, casts and vermicompost. We also found that bacterial pathways associated with amino acid and plant hormone biosynthesis and antibiotic synthesis were enriched. Vermicomposting successfully eliminated most of the 10 human bacterial pathogens found in the sewage sludge. Simultaneously, parasitic and pathogenic fungal taxa were removed. Overall, vermicompost derived from sewage sludge is safer for disposal on land than raw sludge, particularly regarding their respective microbial compositions. This indicates that it could potentially be used as a soil organic amendment and fertilizer.

Microbes are fundamental for biological wastewater treatment. However, most microbial species found in activated sludge (AS) from wastewater treatment plants (WWTPs) have never been isolated and grown as pure cultures, thus limiting our understanding of the underlying biological processes. To change this, we here introduce an experimental setup where the plating of dispersed AS bacteria is combined with 16S rRNA gene amplicon sequencing of the total plate biomass for rapid identification of growth conditions that allow for the isolation of key microbial species in AS. We show that agarose plates composed of AS fluid supplemented with various carbon sources support the growth of many previously uncultivated AS bacteria. To confirm that the approach can also be used to isolate previously uncultured species, we picked 200 colonies from the plates for growth in liquid medium. This resulted in 185 enriched cultures representing at least 76 strains based on unique 16S rRNA gene V1-V3 amplicon sequence variants (ASVs). Classification of the ASVs with the MiDAS 5.1 database revealed 39 distinct genera, including AAP99, Ca. Brachybacter, Ca. Proximibacter, Ellin6067, midas_g_12, and midas_29279, which all lack pure culture representatives. Among the ASVs that obtained species-level classification, we observed 26 unique species of which 16 were only classified based on the MiDAS placeholder taxonomy highlighting the potential for culturing many novel taxa. Purification by restreaking and preparation of glycerol stocks resulted in 10 pure cultures of species commonly found in WWTPs globally, including Rhodoferax midas_s_1744, Thauera midas_s_1356, Acidovorax midas_s_1484, Tessaracoccus midas_s_1151, and Sphingopyxis midas_s_983. IMPORTANCE Biological wastewater treatment relies on complex microbial communities that assimilate nutrients and break down pollutants in the wastewater. Knowledge about the physiology and metabolism of bacteria in wastewater treatment plants (WWTPs) may therefore be used to improve the efficacy and economy of wastewater treatment. Our current knowledge is largely based on 16S rRNA gene amplicon profiling, fluorescence in situ hybridization studies, and predictions based on metagenome-assembled genomes. Bacterial isolates are often required to validate genome-based predictions as they allow researchers to analyze a specific species without interference from other bacteria and with simple bulk measurements. Unfortunately, there are currently very few pure cultures representing the microbes commonly found in WWTPs. To address this, we introduce an isolation strategy that takes advantage of state-of-the-art microbial profiling techniques to uncover suitable growth conditions for key WWTP microbes. We furthermore demonstrate that this information can be used to isolate key organisms representing global WWTPs. [ABSTRACT FROM AUTHOR]

Riemerella anatipestifer (R. anatipestifer) is one of the common pathogens found in poultry flocks, resulting in serious economic losses for the poultry industry due to high mortality, reduced growth rate, poor feed conversion, increased condemnations, and high treatment costs. The aim of this study was to phenotypically characterize phylogenetic relationships and assess the presence of resistance gene strains of R. anatipestifer obtained from various poultry species in Poland. A total of 57 isolates of Riemerella were included in this study. A polymerase chain reaction (PCR) and matrix assisted laser desorption ionization mass spectrometry (MALDI-TOF MS) were used for identification of the strains. The phylogenetic relationship of the R. anatipestifer isolates was determined by analysing the rpoB gene sequence. The susceptibility to antibiotics was evaluated by minimum inhibitory concentration (MIC) in liquid media. All of the field strains of R. anatipestifer were grouped into one of two clades resulting from rpoB gene sequencing. High MIC50 and MIC90 values were obtained for gentamycin, amikacin, and colistin. Low MIC50 and MIC90 values were obtained for amoxicillin cefuroxime, cefoperazone, piperacillin, and trimethoprim/sulfamethoxazole. Among the resistance genes, tet(X) and ermF were identified most frequently. This is the first phenotypic characterization of R. anatipestifer strains obtained from poultry flocks in Poland. [ABSTRACT FROM AUTHOR]

Moonmilk is a cave deposit that was used for medical and cosmetic purposes and has lately raised interest for its antimicrobial potential. We studied five moonmilk samples from four caves with different microclimatic conditions, two temperate in north-western and northern Romania (Ferice, Fața Apei, and Izvorul Tăușoarelor caves) and one tropical in Minas Gerais, Brazil (Nestor Cave). The physicochemical and mineralogical analyses confirmed the presence of calcite and dolomite as the main phase in the moonmilk. A 16S rRNA gene-based metabarcoding approach showed the most abundant bacteria phyla Proteobacteria, GAL15, Actinobacteriota, and Acidobacteriota. The investigated caves differed in the dominant orders of bacteria, with the highest distance between the Romanian and Nestor Cave samples. Climate and, implicitly, the soil microbiome can be responsible for some differences we found between all the samples. However, other factors can be involved in shaping the moonmilk microbiome, as differences were found between samples in the same cave (Ferice). In our five moonmilk samples, 1 phylum, 70 orders (~ 36%), and 252 genera (~ 47%) were unclassified, which hints at the great potential of cave microorganisms for future uses. [ABSTRACT FROM AUTHOR]

Salinization of coastal freshwaters (FWs) due to sea-level rise is a significant stressor for FW biodiversity. However, the effect of changes in salinity on the bacterial microbiome of Neotropical FW insects is less known. We combined field collections and common garden experiments to assess how changes in salinity may affect the microbiome associated with fresh and brackish populations of the water strider Telmatometra withei in Panama. The brackish water (BW) population had higher bacterial diversity and the total communities associated with T. withei were different at each site. Common garden experiments showed that both β diversity and the relative abundance of key taxa varied significantly with changes in salinity, but α diversity remained constant. The BW treatments also showed increased diversity of taxa known to be pathogenic to both insects and humans, including Mycobacterium, Spiroplasma, and Vibrio. Our findings suggest that the host environment is an essential driver of host–microbiome interactions in T. withei. Given the expected increases in sea-level rise, salinity-driven changes in the microbiomes of aquatic insects could trigger important ecological, evolutionary, and pathogenic changes, with consequences for both natural and human populations. [ABSTRACT FROM AUTHOR]

ABSTRACT Acquisition of oral bacteria early in life is considered to have an important role in the establishment of oral microbiota. In this study, we examined tongue swab samples collected from 216 infants at the 18-month checkup, along with samples collected from them and from their mothers at the 4-month checkup. The bacterial composition of each sample was determined using PacBio single-molecule long-read sequencing of the full-length 16S rRNA gene and amplicon sequence variant (ASV) analysis. At 18 months of age, the tongue microbiota was dominated by Streptococcus salivarius, Neisseria perflava, and Granulicatella adiacens; and the overall bacterial composition was more similar to that of the mothers than at 4 months of age. The bacterial composition at 18 months of age was classified into three microbiota profiles: S. salivarius-dominant, Neisseria-dominant, and infant profile dominated by 4 months specific ASVs such as S. salivarius, Streptococcus lactarius, and Streptococcus peroris. Those with infant profiles exhibited significantly higher rates of current breastfeeding, dental caries or white spot lesions, and dental plaque accumulation compared to infants with the other profiles. Comparison between the S. salivarius- and Neisseria-dominant profiles showed that incomplete weaning, low intake of fruits, and frequent intake of sweetened beverages or sweet snacks were significantly associated with the S. salivarius-dominant profile. Our findings, based on a high-resolution profiling approach, demonstrate that the foundation of the adult tongue microbiota is established by 18 months of age, and the bacterial profile is possibly determined by dietary habits during this period. IMPORTANCE Understanding the development of oral microbiota early in life and the factors that influence it is important for preventing the establishment of dysbiotic oral microbiota later in life. This study demonstrates that the tongue microbiota undergoes early development from 4 to 18 months of age and converges into two types of microbiota showing indications of adult characteristics, with either S. salivarius or Neisseria-dominance. Interestingly, their divergence was strongly determined by their weaning status and the dietary frequencies of sweetened beverages, snacks, and fruits, suggesting that dietary habits during this period might influence the establishment of the oral microbiota. These findings may contribute to the development of novel preventive strategies against oral microbiota-related diseases.

Recent advances in genomic technologies have enabled more in-depth study of the oral microbiome. In this study, we compared the amplicons generated by primers targeting different sites of the 16S rRNA gene found in the Human Oral Microbiome Database (HOMD). Six sets of primer targeting V1–V2, V1–V3, V3–V4, V4–V5, V5–V7 and V6–V8 regions of 16S rRNA were tested via in silico simulation. Primers targeting the V1–V2, V3–V4, and V4–V5 regions generated more than 90% of the original input sequences. Primers targeting the V1–V2 and V1–V3 regions exhibited a low number of mismatches and unclassified sequences at the taxonomic level, but there were notable discrepancies at the species level. Phylogenetic tree comparisons showed primers targeting the V1–V2 and V3–V4 regions showed performances similar to primers targeting the whole 16s RNA region in terms of separating total oral microbiomes and periodontopathogens. In an analysis of clinical oral samples, V1–V2 primers showed superior performance for identifying more taxa and had better resolution sensitivity for Streptococcus than V3–V4 primers. In conclusion, primers targeting the V1–V2 region of 16S rRNA showed the best performance for oral microbiome studies. In addition, the study demonstrates the need for careful PCR primer selections. [ABSTRACT FROM AUTHOR]

Abstract Background Large-scale gut microbiome sequencing has revealed key links between microbiome dysfunction and metabolic diseases such as type 2 diabetes (T2D). To date, these efforts have largely focused on Western populations, with few studies assessing T2D microbiota associations in Middle Eastern communities where T2D prevalence is now over 20%. We analyzed the composition of stool 16S rRNA from 461 T2D and 119 non-T2D participants from the Eastern Province of Saudi Arabia. We quantified the abundance of microbial communities to examine any significant differences between subpopulations of samples based on diabetes status and glucose level. Results In this study we performed the largest microbiome study ever conducted in Saudi Arabia, as well as the first-ever characterization of gut microbiota T2D versus non-T2D in this population. We observed overall positive enrichment within diabetics compared to healthy individuals and amongst diabetic participants; those with high glucose levels exhibited slightly more positive enrichment compared to those at lower risk of fasting hyperglycemia. In particular, the genus Firmicutes was upregulated in diabetic individuals compared to non-diabetic individuals, and T2D was associated with an elevated Firmicutes/Bacteroidetes ratio, consistent with previous findings. Conclusion Based on diabetes status and glucose levels of Saudi participants, relatively stable differences in stool composition were perceived by differential abundance and alpha diversity measures. However, community level differences are evident in the Saudi population between T2D and non-T2D individuals, and diversity patterns appear to vary from well-characterized microbiota from Western cohorts. Comparing overlapping and varying patterns in gut microbiota with other studies is critical to assessing novel treatment options in light of a rapidly growing T2D health epidemic in the region. As a rapidly emerging chronic condition in Saudi Arabia and the Middle East, T2D burdens have grown more quickly and affect larger proportions of the population than any other global region, making a regional reference T2D-microbiome dataset critical to understanding the nuances of disease development on a global scale.

Chronic dust bronchitis (CDB) is one of the most common occupational diseases among employees of coal-cycle enterprises. Until recently, the lungs were considered to be sterile, but, in recent years, studies have shown that this is not the case. However, the contribution of microbiota and individual bacterial taxa to the etiology and pathogenesis of many respiratory-tract diseases remains unclear. The aim of the study to determine the taxonomic composition of the bacterial microbiota of the upper respiratory tract of patients with CDB. Sputum samples were obtained from patients with CDB (n = 22, men) and healthy auditorial donors (n = 22, men). Sequencing of variable regions V3–V4 of the 16S pRNA gene was used to study the complete taxonomic composition of the respiratory microbiome. The study revealed a significant decrease in the α diversity of bacterial microbiota in the sputum of patients with CDB compared to the Shannon index control (H = 9.795; p = 0.0017). The PERMANOVA test using a matrix of differences constructed by the Bray–Curtis method showed a significant difference in β diversity between the compared samples (pseudo-F = 2.11; p = 0.002). In the sputum microbiome of patients with CDB, compared with the control, an increase in the relative abundance of bacteria of the genus Streptococcus was found (29.97 ± 14.21 vs. 18.78 ± 11.56; p = 0.006). The taxonomic profile of the respiratory microbiome in the study group of patients with CDB differs from that of healthy people. An increase in the relative abundance of bacteria of the genus Streptococcus in the sputum microbiome of patients with CDB suggests a general dysbiotic process with the accentuation of one dominant genus of microorganisms in this pulmonary pathology. [ABSTRACT FROM AUTHOR]

ABSTRACT A substantial proportion of patients with infective endocarditis (IE) are subjected to heart valve surgery. Microbiological findings on valves are important both for diagnostics and for tailored antibiotic therapy, post-operatively. The aims of this study were to describe microbiological findings on surgically removed valves and to examine the diagnostic benefits of 16S-rDNA PCR and sequencing (16S-analysis). Adult patients who were subjected to heart valve surgery for IE between 2012 and 2021 at Skåne University Hospital, Lund, where a 16S-analysis had been performed on the valve, constituted the study population. Data were gathered from medical records, and the results from blood cultures, valve cultures, and 16S-analyses of valves were compared. A diagnostic benefit was defined as providing an agent in blood culture negative endocarditis, providing a new agent in episodes with positive blood cultures, or confirming one of the findings in episodes with a discrepancy between blood and valve cultures. 279 episodes in 272 patients were included in the final analysis. Blood cultures were positive in 259 episodes (94%), valve cultures in 60 episodes (22%), and 16S-analyses in 227 episodes (81%). Concordance between the blood cultures and the 16S-analysis was found in 214 episodes (77%). The 16S-analyses provided a diagnostic benefit in 25 (9.0%) of the episodes. In blood culture negative endocarditis, the 16S-analyses had a diagnostic benefit in 15 (75%) of the episodes. A 16S-analysis should be routinely performed on surgically removed valves in blood culture negative endocarditis. In patients with positive blood cultures, 16S-analysis may also be considered, as a diagnostic benefit was provided in some patients. IMPORTANCE This work demonstrates that it can be of importance to perform both cultures and analysis using 16S-rDNA PCR and sequencing of valves excised from patients undergoing surgery for infective endocarditis. 16S-analysis may help both to establish a microbiological etiology in cases of blood culture negative endocarditis and to provide help in situations where there are discrepancies between valve and blood cultures. In addition, our results show a high degree of concordance between blood cultures and 16S-analyses, indicating that the latter has a high sensitivity and specificity for the etiological diagnosis of endocarditis in patients who were subjected to heart valve surgery.

ABSTRACT: An in vitro model of the upper gastrointestinal tract as well as the chicken cecum was developed to have a predictive tool for estimating the production performance of animals by analyzing the feeding value of a certain diet. The upper gastrointestinal tract consists of a batch type model, whereas the cecal model is comprised of 4 semi-continuous connected vessels inoculated with cecal or fecal microbes. The upper gastrointestinal tract and cecal simulations were both run with a corn- and a wheat-based diet to simulate 2 typical feed types. Samples were collected after the 5-h cecal simulations and aliquots were frozen to assess inoculum stability. The microbiota was analyzed by 16S rRNA gene sequencing, whereas short chain fatty acids as microbial metabolites were analyzed by using gas chromatography.As expected, some significant differences in microbial abundance after simulation between the cecal and fecal slurry samples (P = 0.001) were detected, as well between the fresh and frozen status (P = 0.001), hence simulations inoculated with cecal and fresh samples being more diverse. For the measured metabolites, almost all of them increased (P < 0.05) significantly when comparing fresh and frozen inoculum. The present chicken intestinal in vitro model represents a rapid systematic screening system for studying dietary related microbial changes and reducing the need of animal sacrifice for experimentation.

IntroductionInteractions between the gut microbiome (GM) and the immune system influence host health and fitness. However, few studies have investigated this link and GM dynamics during disease in wild species. Bats (Mammalia: Chiroptera) have an exceptional ability to cope with intracellular pathogens and a unique GM adapted to powered flight. Yet, the contribution of the GM to bat health, especially immunity, or how it is affected by disease, remains unknown.MethodsHere, we examined the dynamics of the Egyptian fruit bats’ (Rousettus aegyptiacus) GM during health and disease. We provoked an inflammatory response in bats using lipopolysaccharides (LPS), an endotoxin of Gram-negative bacteria. We then measured the inflammatory marker haptoglobin, a major acute phase protein in bats, and analyzed the GM (anal swabs) of control and challenged bats using high-throughput 16S rRNA sequencing, before the challenge, 24h and 48h post challenge.ResultsWe revealed that the antigen challenge causes a shift in the composition of the bat GM (e.g., Weissella, Escherichia, Streptococcus). This shift was significantly correlated with haptoglobin concentration, but more strongly with sampling time. Eleven bacterial sequences were correlated with haptoglobin concentration and nine were found to be potential predictors of the strength of the immune response, and implicit of infection severity, notably Weissella and Escherichia. The bat GM showed high resilience, regaining the colony’s group GM composition rapidly, as bats resumed foraging and social activities.ConclusionOur results demonstrate a tight link between bat immune response and changes in their GM, and emphasize the importance of integrating microbial ecology in ecoimmunological studies of wild species. The resilience of the GM may provide this species with an adaptive advantage to cope with infections and maintain colony health.

Riemerella anatipestifer (R. anatipestifer) is one of the common pathogens found in poultry flocks, resulting in serious economic losses for the poultry industry due to high mortality, reduced growth rate, poor feed conversion, increased condemnations, and high treatment costs. The aim of this study was to phenotypically characterize phylogenetic relationships and assess the presence of resistance gene strains of R. anatipestifer obtained from various poultry species in Poland. A total of 57 isolates of Riemerella were included in this study. A polymerase chain reaction (PCR) and matrix assisted laser desorption ionization mass spectrometry (MALDI-TOF MS) were used for identification of the strains. The phylogenetic relationship of the R. anatipestifer isolates was determined by analysing the rpoB gene sequence. The susceptibility to antibiotics was evaluated by minimum inhibitory concentration (MIC) in liquid media. All of the field strains of R. anatipestifer were grouped into one of two clades resulting from rpoB gene sequencing. High MIC50 and MIC90 values were obtained for gentamycin, amikacin, and colistin. Low MIC50 and MIC90 values were obtained for amoxicillin cefuroxime, cefoperazone, piperacillin, and trimethoprim/sulfamethoxazole. Among the resistance genes, tet(X) and ermF were identified most frequently. This is the first phenotypic characterization of R. anatipestifer strains obtained from poultry flocks in Poland.

ABSTRACT Infections are relatively rare following cutaneous surgical procedures, despite the potential for wound exposure to pathogens both during surgery and throughout the healing process. Although gut commensals are believed to reduce the risk of intestinal infections, an analogous role for skin commensals has not been described. In fact, the microbiome of normally healing surgical skin wounds has not yet been profiled using culture-independent techniques. We characterized the wound microbiome in 53 patients who underwent skin cancer surgery and healed without signs or symptoms of infection. A week after surgery, several bacterial species displayed significant differences in relative abundance when compared to control, nonoperated skin from the same patient. The relative abundance of the most common bacterium found on intact skin, Cutibacterium acnes, was reduced in wounds 5-fold. Staphylococcus aureus, a frequent cause of postoperative skin infections, was enriched 6.4-fold in clinically noninfected wounds, suggesting active suppression of pathogenicity. Finally, members of the Corynebacterium genus were the dominant organism in postoperative wounds, making up 37% of the average wound microbiome. The enrichment of these bacteria in normally healing wounds suggests that they might be capable of providing colonization resistance. Future studies focused on the biological and clinical significance of the wound microbiome may shed light on normal wound healing and potential therapeutic opportunities to mitigate infection risk. IMPORTANCE Commensal bacteria on skin may limit the ability of pathogenic bacteria to cause clinically significant infections. The bacteria on healing acute wounds, which might provide such a protective effect, have not been described using culture-independent approaches in the absence of antibiotics. We compare the microbiome of wounds a week after skin cancer removal surgery with intact skin from the same patient. We find that the potentially pathogenic species S. aureus is common on these healing wounds despite the absence of symptoms or signs of infection. We report that bacteria often considered as potential skin probiotics, including Staphylococcus epidermidis, do not reach high relative abundance in wound microbiomes. In contrast, specific members of the Corynebacterium genus, rarely associated with infections, were significantly enriched in healing wounds compared to intact skin. Future work is needed to see if Corynebacterium species or derivatives thereof could be employed to lower the risk of wound infection.

ABSTRACT This study characterized compositional and functional shifts in the intestinal and oral microbiome in HIV-positive patients on antiretroviral therapy compared to HIV-negative individuals. Seventy-nine specimens were collected from 5 HIV-positive and 12 control subjects from five locations (colon brush, colon wash, terminal ileum [TI] brush, TI wash, and saliva) during colonoscopy and at patient visits. Microbiome composition was characterized using 16S rRNA sequencing, and microbiome function was predicted using bioinformatics tools (PICRUSt and BugBase). Our analysis indicated that the β-diversity of all intestinal samples (colon brush, colon wash, TI brush, and TI wash) from patients with HIV was significantly different from patients without HIV. Specifically, bacteria from genera Prevotella, Fusobacterium, and Megasphaera were more abundant in samples from HIV-positive patients. On the other hand, bacteria from genera Ruminococcus, Blautia, and Clostridium were more abundant in samples from HIV-negative patients. Additionally, HIV-positive patients had higher abundances of biofilm-forming and pathogenic bacteria. Furthermore, pathways related to translation and nucleotide metabolism were elevated in HIV-positive patients, whereas pathways related to lipid and carbohydrate metabolism were positively correlated with samples from HIV-negative patients. Our analyses further showed variations in microbiome composition in HIV-positive and negative patients by sampling site. Samples from colon wash, colon brush, and TI wash were significant between groups, while samples from TI brush and saliva were not significant. Taken together, here, we report altered intestinal microbiome composition and predicted function in patients with HIV compared to uninfected patients, though we found no changes in the oral microbiome. IMPORTANCE Over 37 million people worldwide are living with HIV. Although the availability of antiretroviral therapy has significantly reduced the number of AIDS-related deaths, individuals living with HIV are at increased risk for opportunistic infections. We now know that HIV interacts with the trillions of bacteria, fungi, and viruses in the human body termed the microbiome. Only a limited number of previous studies have compared variations in the oral and gastrointestinal microbiome with HIV infection. Here, we detail how the oral and gastrointestinal microbiome changes with HIV infection, having used 5 different sampling sites to gain a more comprehensive view of these changes by location. Our results show site-specific changes in the intestinal microbiome associated with HIV infection. Additionally, we show that while there were significant changes in the intestinal microbiome, there were no significant changes in the oral microbiome.

Ethanolamine (Etn) contained in milk is the base constituent of phosphatidylethanolamine and is required for the proliferation of intestinal epithelial cells and bacteria, which is important for maintenance of the gut microbiome and intestinal development. The present study investigated the effect of Etn on intestinal function and microbiome using 21-d-old Sprague-Dawley rats treated with 0, 250, 500 and 1000 μm Etn in drinking water for 2 weeks immediately after weaning. Growth performance, intestinal morphology, antioxidant capacity and mucosal immunity, as well as gut microbiota community composition, were evaluated. Metagenomic prediction and metabolic phenotype analysis based on 16S RNA sequencing were also carried out to assess changes in metabolic functions. We found that weaned rats administered 500 μm Etn enhanced mucosal antioxidant capacity, as evidenced by higher superoxide dismutase and glutathione peroxidase levels in the jejunum (P

Background: Large-scale gut microbiome sequencing has revealed key links between microbiome dysfunction and metabolic diseases such as type 2 diabetes (T2D). To date, these efforts have largely focused on Western populations, with few studies assessing T2D microbiota associations in Middle Eastern communities where T2D prevalence is now over 20%. We analyzed the composition of stool 16S rRNA from 461 T2D and 119 non-T2D participants from the Eastern Province of Saudi Arabia. We quantified the abundance of microbial communities to examine any significant differences between subpopulations of samples based on diabetes status and glucose level. Results: In this study we performed the largest microbiome study ever conducted in Saudi Arabia, as well as the first-ever characterization of gut microbiota T2D versus non-T2D in this population. We observed overall positive enrichment within diabetics compared to healthy individuals and amongst diabetic participants; those with high glucose levels exhibited slightly more positive enrichment compared to those at lower risk of fasting hyperglycemia. In particular, the genus Firmicutes was upregulated in diabetic individuals compared to non-diabetic individuals, and T2D was associated with an elevated Firmicutes/Bacteroidetes ratio, consistent with previous findings. Conclusion: Based on diabetes status and glucose levels of Saudi participants, relatively stable differences in stool composition were perceived by differential abundance and alpha diversity measures. However, community level differences are evident in the Saudi population between T2D and non-T2D individuals, and diversity patterns appear to vary from well-characterized microbiota from Western cohorts. Comparing overlapping and varying patterns in gut microbiota with other studies is critical to assessing novel treatment options in light of a rapidly growing T2D health epidemic in the region. As a rapidly emerging chronic condition in Saudi Arabia and the Middle East, T2D burdens have grown more quickly and affect larger proportions of the population than any other global region, making a regional reference T2D-microbiome dataset critical to understanding the nuances of disease development on a global scale. [ABSTRACT FROM AUTHOR]

Microorganisms play a vital role in the decomposition of vertebrate remains in natural nutrient cycling, and the postmortem microbial succession patterns during decomposition remain unclear. The present study used hierarchical clustering based on Manhattan distances to analyze the similarities and differences among postmortem intestinal microbial succession patterns based on microbial 16S rDNA sequences in a mouse decomposition model. Based on the similarity, seven different classes of succession patterns were obtained. Generally, the normal intestinal flora in the cecum was gradually decreased with changes in the living conditions after death, while some facultative anaerobes and obligate anaerobes grew and multiplied upon oxygen consumption. Furthermore, a random forest regression model was developed to predict the postmortem interval based on the microbial succession trend dataset. The model demonstrated a mean absolute error of 20.01 h and a squared correlation coefficient of 0.95 during 15-day decomposition. Lactobacillus, Dubosiella, Enterococcus, and the Lachnospiraceae NK4A136 group were considered significant biomarkers for this model according to the ranked list. The present study explored microbial succession patterns in terms of relative abundances and variety, aiding in the prediction of postmortem intervals and offering some information on microbial behaviors in decomposition ecology. [ABSTRACT FROM AUTHOR]

Avian pathogenic Escherichia coli (APEC) is a major cause of avian colibacillosis. Matrine, a natural component derived from Sophora flavescens, exhibits various pharmacological effects, including anti-inflammatory and antioxidant activities. However, its role in mitigating APEC-induced intestinal damage in chickens remains insufficiently understood. This study aimed to explore the protective effects and potential mechanisms of matrine against APEC-induced intestinal damage. Chickens were administered matrine (10 or 20 mg/kg) from 6 days old for 5 days, followed by an APEC intraperitoneal injection on day 10. After 72 h of APEC infection, tissues were collected for analysis. Results indicated that pretreatment with matrine alleviated the symptoms of APEC infection in chickens, improving survival rates and promoting weight gain. Additionally, pretreatment with matrine reduced the secretion and gene expression of IL-1β, IL-6, and TNF-α in intestinal tissues, while enhancing serum SOD, GSH, and CAT activity, as well as gene expression levels in the intestine. Pretreatment with matrine reduced the levels of TLR4, MyD88, and NF-κB in intestinal tissues. Moreover, pretreatment with matrine ameliorated intestinal inflammation and pathological damage, restoring the expression of ZO-1, Occludin, and MUC2 in the intestine during APEC infection. Furthermore, pretreatment with matrine alleviated gut microbiota dysbiosis by lowering the abundance of harmful bacteria. In summary, matrine alleviated APEC-induced intestinal inflammation and damage, potentially by inhibiting NF-κB signaling pathway and reshaping the gut microbiota. These findings provide promising insights into the prevention and treatment of avian colibacillosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Rhesus cytomegalovirus expressing simian immunodeficiency virus (RhCMV/SIV) vaccines protect ~59% of vaccinated rhesus macaques against repeated limiting-dose intra-rectal exposure with highly pathogenic SIVmac239M, but the exact mechanism responsible for the vaccine efficacy is unknown. It is becoming evident that complex interactions exist between gut microbiota and the host immune system. Here, we aimed to investigate if the rhesus gut microbiome impacts RhCMV/SIV vaccine-induced protection. Three groups of 15 rhesus macaques naturally pre-exposed to RhCMV were vaccinated with RhCMV/SIV vaccines. Rectal swabs were collected longitudinally both before SIV challenge (after vaccination) and post-challenge and were profiled using 16S rRNA based microbiome analysis. We identified ~2,400 16S rRNA amplicon sequence variants (ASVs), representing potential bacterial species/strains. Global gut microbial profiles were strongly associated with each of the three vaccination groups, and all animals tended to maintain consistent profiles throughout the pre-challenge phase. Despite vaccination group differences, by using newly developed compositional data analysis techniques, we identified a common gut microbial signature predictive of vaccine protection outcome across the three vaccination groups. Part of this microbial signature persisted even after SIV challenge. We also observed a strong correlation between this microbial signature and an early signature derived from whole blood transcriptomes in the same animals. Our findings indicate that changes in gut microbiomes are associated with RhCMV/SIV vaccine-induced protection and early host response to vaccination in rhesus macaques.IMPORTANCEThe human immunodeficiency virus (HIV) has infected millions of people worldwide. Unfortunately, still there is no vaccine that can prevent or treat HIV infection. A promising pre-clinical HIV vaccine based on rhesus cytomegalovirus (RhCMV) expressing simian immunodeficiency virus (SIV) antigens (RhCMV/SIV) provides sustained, durable protection against SIV challenge in ~59% of vaccinated rhesus macaques. There is an urgent need to understand the cause of this protection vs non-protection outcome. In this study, we profiled the gut microbiomes of 45 RhCMV/SIV vaccinated rhesus macaques and identified gut microbial signatures that were predictive of RhCMV/SIV vaccination groups and vaccine protection outcomes. These vaccine protection-associated microbial features were significantly correlated with early vaccine-induced host immune signatures in whole blood from the same animals. These findings show that the gut microbiome may be involved in RhCMV/SIV vaccine-induced protection, warranting further research into the impact of the gut microbiome in human vaccine trials., Competing Interests: X.P. is the Founder and CEO and has an equity interest in Depict Bio, LLC. The terms of this arrangement have been reviewed and approved by NC State University in accordance with its policy on objectivity in research.

Potentially toxic elements (PTE), such as antimony (Sb), are dangerous putative contaminants for ground and surface waters around abandoned mines and ore deposits in Slovakia. Nearby mines antimony is commonly coprecipitated in ochre sediments precipitated from Fe-rich drainage waters and, therefore, these sites function as natural scavengers of this metalloid. Bacteria are well known to contribute to the process of redox state maintenance, biosorption and bioaccumulation of antimony and, consequently, to antimony precipitation or release from iron oxides complexes. Here we isolated 48 bacterial strains from circumneutral hydrous ferric oxides (HFO) rich iron ochres accumulated in the waters running from tailing pounds nearby Budúcnosť mine, Pezinok, Slovakia and polluted with high, but fluctuating, concentrations of antimony (130 μg/l Sb in water and 2317 mg/kg Sb in iron ochre in average). The isolated strains were V1-V9 16S rRNA sequenced and the resulting taxonomic affiliations of isolated strains were compared with taxonomy assignments obtained by V4 16S rRNA next generation sequencing approach, including two independent NGS analysis pipelines and different taxonomy classifiers ((IDTAXA (RDP, GTDB, SILVA, CONTAX), MEGAN (NCBI), RDP a SILVAngs). A Sb resistant subgroup of isolated strains ( Pseudomonas A60B, Pseudomonas A59, Pseudomonas A28, Aeromonas A21, Aeromonas A13, Aeromonas A60A, Acinetobacter A14, Buttiauxella A58, Shewanella A20A a Yersinia A68), well growing at high Sb concentration (300 mg/l Sb), was tested for an ability of the strains to retain Sb from cultivation media. Based on ICP-MS measurements of the dried biomasses we concluded that all the strains can retain antimony from growth media to some extent, with strains Shewanella A20A, Buttiauxella A58, Yersinia A68 and Aeromonas A60A being the most effective., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Antimicrobial resistance gene profile characterization and dissemination offer useful detail on the possible challenge in treating bacteria. The development of aminoglycoside modifying enzymes (AMEs) is considered as the primary mechanism of resistance to aminoglycosides, in addition to the 16S rRNA methylases. This study aimed at isolation and characterization of aminoglycosides resistant clinical isolates of enterobacteriaceae family from different clinical samples. Over a period of 24 months, thirty samples were collected and 49 clinical isolates of E. coli [n=25], Klebsiella [n=13], Enterobacter species (n=7) and Proteus species (n=4) were isolated from Egyptian clinical laboratories. The identities of the cultures were confirmed following standard microbiological procedures. Resistance of the isolates to aminoglycosides was determined by the disc diffusion method and isolates with highest resistance (n=9) were selected and investigated for 16S rRNA methylase and AMES encoding genes by polymerase chain reaction (PCR) and sequencing. In general, aminoglycoside resistance was found in 95% of the isolates; the isolates displayed the highest rate of resistance to netilmicin (75%) and kanamycin (55%), while resistance to gentamycin (18%) and tobramycin (16%) was low. A total of 9 isolates have the highest aminoglycoside resistant rate, showed the highest appearance for aac(6′)-Ib as well as ant (3″)-Ia resistant genes, with aac (3)-II (44%) and ant (4′)-IIb (34%) following closely. The high prevalence of AMEs observed among resistant isolates in this study suggests the urgent need for more efficient treatment designs to mitigate the selection burden as well as improved care of patients who have been infected with these drug-resistant organisms.

Abstract Background The microbial ecosystem in the human gut varies between individuals with differences in diet. Selenium is one of most common trace elements in everyday diet, and selenium intake affects the human gut microbiota. We studied the effect of selenium intake on the gut microbiota in regions of Enshi with different distributions of selenium. Methods One hundred elderly subjects (>65 years) were recruited from high-selenium and low-selenium areas in Enshi and blood, nail, and fecal specimens were obtained. The selenium contents in these samples were determined in triplicate by hydride generation atomic fluorescence spectrometry. DNA was extracted from fecal specimens and the microbial diversity was analyzed by 16 S RNA. Results The selenium contents in the blood and nails were significantly different between the high- and low-selenium areas, and the composition of the intestinal microbiota, including abundance and extent of intestinal flora, was altered. The function and metabolic pathways of the gut microbiota showed clear differences. Conclusions As a trace element in human diet, selenium intake is an important factor that affects the intestinal microbiota and is likely involved in many human diseases. This study provides new clues and ideas for studying the correlation between selenium and human health.

ABSTRACT Fish bacterial communities provide functions critical for their host’s survival in contrasting environments. These communities are sensitive to environmental-specific factors (i.e., physicochemical parameters, bacterioplankton), and host-specific factors (i.e., host genetic background). The relative contribution of these factors shaping Amazonian fish bacterial communities is largely unknown. Here, we investigated this topic by analyzing the gill bacterial communities of 240 wild flag cichlids (Mesonauta festivus) from 4 different populations (genetic clusters) distributed across 12 sites in 2 contrasting water types (ion-poor/acidic black water and ion-rich/circumneutral white water). Transcriptionally active gill bacterial communities were characterized by a 16S rRNA metabarcoding approach carried on RNA extractions. They were analyzed using comprehensive data sets from the hosts genetic background (Genotyping-By-Sequencing), the bacterioplankton (16S rRNA) and a set of 34 environmental parameters. Results show that the taxonomic structure of 16S rRNA gene transcripts libraries were significantly different between the 4 genetic clusters and also between the 2 water types. However, results suggest that the contribution of the host’s genetic background was relatively weak in comparison to the environment-related factors in structuring the relative abundance of different active gill bacteria species. This finding was also confirmed by a mixed-effects modeling analysis, which indicated that the dissimilarity between the taxonomic structure of bacterioplanktonic communities possessed the best explicative power regarding the dissimilarity between gill bacterial communities’ structure, while pairwise fixation indexes (FST) from the hosts’ genetic data only had a weak explicative power. We discuss these results in terms of bacterial community assembly processes and flag cichlid fish ecology. IMPORTANCE Host-associated microbial communities respond to factors specific to the host physiology, genetic backgrounds, and life history. However, these communities also show different degrees of sensitivity to environment-dependent factors, such as abiotic physico-chemical parameters and ecological interactions. The relative importance of host- versus environment-associated factors in shaping teleost bacterial communities is still understudied and is paramount for their conservation and aquaculture. Here, we studied the relative importance of host- and environment-associated factors structuring teleost bacterial communities using gill samples from a wild Amazonian teleost model (Mesonauta festivus) sampled in contrasting habitats along a 1500 km section of the Amazonian basin, thus ensuring high genetic diversity. Results showed that the contribution of the host’s genetic background was weak compared to environment-related bacterioplanktonic communities in shaping gill bacterial assemblages, thereby suggesting that our understanding of teleost microbiome assembly could benefit from further studies focused on the ecological interplay between host-associated and free-living communities.

Recent advances in genomic technologies have enabled more in-depth study of the oral microbiome. In this study, we compared the amplicons generated by primers targeting different sites of the 16S rRNA gene found in the Human Oral Microbiome Database (HOMD). Six sets of primer targeting V1–V2, V1–V3, V3–V4, V4–V5, V5–V7 and V6–V8 regions of 16S rRNA were tested via in silico simulation. Primers targeting the V1–V2, V3–V4, and V4–V5 regions generated more than 90% of the original input sequences. Primers targeting the V1–V2 and V1–V3 regions exhibited a low number of mismatches and unclassified sequences at the taxonomic level, but there were notable discrepancies at the species level. Phylogenetic tree comparisons showed primers targeting the V1–V2 and V3–V4 regions showed performances similar to primers targeting the whole 16s RNA region in terms of separating total oral microbiomes and periodontopathogens. In an analysis of clinical oral samples, V1–V2 primers showed superior performance for identifying more taxa and had better resolution sensitivity for Streptococcus than V3–V4 primers. In conclusion, primers targeting the V1–V2 region of 16S rRNA showed the best performance for oral microbiome studies. In addition, the study demonstrates the need for careful PCR primer selections.

Simple Summary: Current culture methods for Campylobacter enumeration in poultry meat require at least five days. To improve this outcome, we have assembled a portable sequencing kit consisting of the Bento Lab and the MinION and developed a workflow for on-farm use that can detect and report the presence of Campylobacter in less than five hours from sampling to data. In addition, this workflow allows for the determination of microbiota profiles within those five hours. Overall, this workflow and approach can be helpful in the control of zoonotic agents, with a particular focus on poultry meat, where it takes less than 24 h from the time the animals arrive at the slaughterhouse to the time the carcasses are available on the supermarket shelves. Campylobacter is recognised as one of the most important foodborne bacteria, with a worldwide health and socioeconomic impact. This bacterium is one of the most important zoonotic players in poultry, where efficient and fast detection methods are required. Current official culture methods for Campylobacter enumeration in poultry usually include >44 h of culture and >72 h for identification, thus requiring at least five working shifts (ISO/TS 10272-2:2017). Here, we have assembled a portable sequencing kit composed of the Bento Lab and the MinION and developed a workflow for on-site farm use that is able to detect and report the presence of Campylobacter from caecal samples in less than five hours from sampling time, as well as the relationship of Campylobacter with other caecal microbes. Beyond that, our workflow may offer a cost-effective and practical method of microbiologically monitoring poultry at the farm. These results would demonstrate the possibility of carrying out rapid on-site screening to monitor the health status of the poultry farm/flock during the production chain. [ABSTRACT FROM AUTHOR]

Objective Ruminants are completely dependent on their microbiota for rumen fermentation, feed digestion, and consequently, their metabolism for productivity. This study aimed to evaluate the rumen bacteria of lactating yaks with different milk protein yields, using high-throughput sequencing technology, in order to understand the influence of these bacteria on milk production. Methods Yaks with similar high milk protein yield (high milk yield and high milk protein content, HH; n = 12) and low milk protein yield (low milk yield and low milk protein content, LL; n = 12) were randomly selected from 57 mid-lactation yaks. Ruminal contents were collected using an oral stomach tube from the 24 yaks selected. High-throughput sequencing of bacterial 16S rRNA gene was used. Results Ruminal ammonia N, total volatile fatty acids, acetate, propionate, and isobutyrate concentrations were found to be higher in HH than LL yaks. Community richness (Chao 1 index) and diversity indices (Shannon index) of rumen microbiota were higher in LL than HH yaks. Relative abundances of the Bacteroidetes and Tenericutes phyla in the rumen fluid were significantly increased in HH than LL yaks, but significantly decreased for Firmicutes. Relative abundances of the Succiniclasticum, Butyrivibrio 2, Prevotella 1, and Prevotellaceae UCG-001 genera in the rumen fluid of HH yaks was significantly increased, but significantly decreased for Christensenellaceae R-7 group and Coprococcus 1. Principal coordinates analysis on unweighted UniFrac distances revealed that the bacterial community structure of rumen differed between yaks with high and low milk protein yields. Furthermore, rumen microbiota were functionally enriched in relation to transporters, ABC transporters, ribosome, and urine metabolism, and also significantly altered in HH and LL yaks. Conclusion We observed significant differences in the composition, diversity, fermentation product concentrations, and function of ruminal microorganisms between yaks with high and low milk protein yields, suggesting the potential influence of rumen microbiota on milk protein yield in yaks. A deeper understanding of this process may allow future modulation of the rumen microbiome for improved agricultural yield through bacterial community design.

A central goal of microbial ecology is to identify and quantify the forces that lead to observed population distributions and dynamics. However, these forces, which include environmental selection, dispersal, and organism interactions, are often difficult to assess in natural environments. Here, we present a method that links microbial community structures with selective and stochastic forces through highly replicated subsampling and enrichment of a single environmental inoculum. Specifically, groundwater from a well-studied natural aquifer was serially diluted and inoculated into nearly 1,000 aerobic and anaerobic nitrate-reducing cultures, and the final community structures were evaluated with 16S rRNA gene amplicon sequencing. We analyzed the frequency and abundance of individual operational taxonomic units (OTUs) to understand how probabilistic immigration, relative fitness differences, environmental factors, and organismal interactions contributed to divergent distributions of community structures. We further used a most probable number (MPN) method to estimate the natural condition-dependent cultivable abundance of each of the nearly 400 OTU cultivated in our study and infer the relative fitness of each. Additionally, we infer condition-specific organism interactions and discuss how this high-replicate culturing approach is essential in dissecting the interplay between overlapping ecological forces and taxon-specific attributes that underpin microbial community assembly.IMPORTANCE Through highly replicated culturing, in which inocula are subsampled from a single environmental sample, we empirically determine how selective forces, interspecific interactions, relative fitness, and probabilistic dispersal shape bacterial communities. These methods offer a novel approach to untangle not only interspecific interactions but also taxon-specific fitness differences that manifest across different cultivation conditions and lead to the selection and enrichment of specific organisms. Additionally, we provide a method for estimating the number of cultivable units of each OTU in the original sample through the MPN approach.

To better understand the impacts of Corexit 9500 on the structure and activity levels of hydrocarbon-degrading microbial communities, we analyzed next-generation 16S rRNA gene sequencing libraries of hydrocarbon enrichments grown at 5 and 25°C using both DNA and RNA extracts as the sequencing templates. Oil biodegradation patterns in both 5 and 25°C enrichments were consistent with those reported in the literature (i.e., aliphatics were degraded faster than aromatics). Slight increases in biodegradation were observed in the presence of Corexit at both temperatures. Differences in community structure were observed between treatment conditions in the DNA-based libraries. The 25°C consortia were dominated by Vibrio, Idiomarina, Marinobacter, Alcanivorax, and Thalassospira species, while the 5°C consortia were dominated by several species of the genera Flavobacterium, Alcanivorax, and Oleispira Most of these genera have been linked to hydrocarbon degradation and have been observed after oil spills. Colwellia and Cycloclasticus, known aromatic degraders, were also found in these enrichments. The addition of Corexit did not have an effect on the active bacterial community structure of the 5°C consortia, while at 25°C, a decrease in the relative abundance of Marinobacter was observed. At 25°C, Thalassospira, Marinobacter, and Idiomarina were present at higher relative abundances in the RNA than DNA libraries, suggesting that they were active in degradation. Similarly, Oleispira was greatly stimulated by the addition of oil at 5°C.IMPORTANCE While dispersants such as Corexit 9500 can be used to treat oil spills, there is still debate on the effectiveness on enhancing oil biodegradation and its potential toxic effect on oil-degrading microbial communities. The results of this study provide some insights on the microbial dynamics of hydrocarbon-degrading bacterial populations in the presence of Corexit 9500. Operational taxonomic unit (OTU) analyses indicated that several OTUs were inhibited by the addition of Corexit. Conversely, a number of OTUs were stimulated by the addition of the dispersant, many of which were identified as known hydrocarbon-degrading bacteria. The results highlight the value of using RNA-based methods to further understand the impact of dispersant on the overall activity of different hydrocarbon-degrading bacterial groups.

Plant invasions often reduce native plant diversity and increase net primary productivity. Invaded soils appear to differ from surrounding soils in ways that impede restoration of diverse native plant communities. We hypothesize that invader-mediated shifts in edaphic properties reproducibly alter soil microbial community structure and function. Here, we take a holistic approach, characterizing plant, prokaryotic, and fungal communities and soil physicochemical properties in field sites, invasion gradients, and experimental plots for three invasive plant species that cooccur in the Rocky Mountain West. Each invader had a unique impact on soil physicochemical properties. We found that invasions drove shifts in the abundances of specific microbial taxa, while overall belowground community structure and functional potential were fairly constant. Forb invaders were generally enriched in copiotrophic bacteria with higher 16S rRNA gene copy numbers and showed greater microbial carbohydrate and nitrogen metabolic potential. Older invasions had stronger effects on abiotic soil properties, indicative of multiyear successions. Overall, we show that plant invasions are idiosyncratic in their impact on soils and are directly responsible for driving reproducible shifts in the soil environment over multiyear time scales. IMPORTANCE In this study, we show how invasive plant species drive rapid shifts in the soil environment from surrounding native communities. Each of the three plant invaders had different but consistent effects on soils. Thus, there does not appear to be a one-size-fits-all strategy for how plant invaders alter grassland soil environments. This work represents a crucial step toward understanding how invaders might be able to prevent or impair native reestablishment by changing soil biotic and abiotic properties.

Logging and forest conversion are occurring at alarming rates in tropical forests. These disturbances alter soil microbial community structure and functions. While direct links between changes in soil properties, such as pH and microbial community structure are well established, the indirect effects of logging and forest conversion on soil microbial community structure and functions are poorly understood. We used a space-for-time substitution to investigate the changes in soil microbial diversity and functions across a forest recovery gradient in the tropical montane forests of northern Borneo. We used surface (top 5 cm) soil to assess soil physicochemical and microbial (next-generation DNA sequencing) properties, and standardized litterbags (Tea Bag Index) to assess litter decomposition and stabilization. Our results show that bacterial and fungal diversity increases with recovery time and reaches pre-disturbance levels between 60- and 80-years post-disturbance. Litter decomposition rate constants increased linearly with increasing bacterial and fungal diversity. Litter stabilization also increased linearly with fungal diversity, but was highest at intermediate levels of bacterial diversity. Our results provide insights on the effects of forest logging and conversion on soils and highlight the tight coupling between soil microbial diversity and soil functions in tropical montane forests.

ABSTRACT The Permian Basin is the highest producing oil and gas reservoir in the United States. Hydrocarbon resources in this region are often accessed by unconventional extraction methods, including horizontal drilling and hydraulic fracturing. Despite the importance of the Permian Basin, there is no publicly available microbiological data from this region. We completed an analysis of Permian produced water samples to understand the dynamics present in hydraulically fractured wells in this region. We analyzed produced water samples taken from 10 wells in the Permian region of the Midland Basin using geochemical measurements, 16S rRNA gene sequencing, and metagenomic sequencing. Compared to other regions, we found that Permian Basin produced water was characterized by higher sulfate and lower total dissolved solids (TDS) concentrations, with a median of 1,110 mg/L and 107,000 mg/L. Additionally, geochemical measurements revealed the presence of frac hits, or interwell communication events where an established well is affected by the pumping of fracturing fluid into a new well. The occurrence of frac hits was supported by correlations between the microbiome and the geochemical parameters. Our 16S rRNA gene sequencing identified a produced water microbiome characterized by anaerobic, halophilic, and sulfur reducing taxa. Interestingly, sulfate and thiosulfate reducing taxa including Halanaerobium, Orenia, Marinobacter, and Desulfohalobium were the most prevalent microbiota in most wells. We further investigated the metabolic potential of microorganisms in the Permian Basin with metagenomic sequencing. We recovered 15 metagenome assembled genomes (MAGs) from seven different samples representing 6 unique well sites. These MAGs corroborated the high presence of sulfate and thiosulfate reducing genes across all wells, especially from key taxa including Halanaerobium and Orenia. The observed microbiome composition and metabolic capabilities in conjunction with the high sulfate concentrations demonstrate a high potential for hydrogen sulfide production in the Permian Basin. Additionally, evidence of frac hits suggests the possibility for the exchange of microbial cells and/or genetic information between wells. This exchange would increase the likelihood of hydrogen sulfide production and has implications for the oil and gas industry. IMPORTANCE The Permian Basin is the largest producing oil and gas region in the United States and plays a critical role supplying national energy needs. Previous work in other basins has demonstrated that the geochemistry and microbiology of hydrocarbon regions can have a major impact on well infrastructure and production. Despite that, little work has been done to understand the complex dynamics present in the Permian Basin. This study characterizes and analyzes 10 unique wells and one groundwater sample in the Permian Basin using geochemical and microbial techniques. Across all wells we found a high number of classic and thiosulfate reducers, suggesting that hydrogen sulfide production may be especially prevalent in the Permian Basin. Additionally, our analysis revealed a biogeochemical signal impacted by the presence of frac hits, or interwell communication events where an established well is affected by the pumping of fracturing fluid into a new well. This information can be utilized by the oil and gas industry to improve oil recovery efforts and minimize commercial and environmental costs.

Simple Summary: The gut microbiota could directly induce immune responses and affect the health of the host. In this study, we assessed changes in the gut microbiota of resistant segregated phenotypic pigs under Porcine Reproductive and Respiratory Syndrome Virus exposure. The results showed that the resistance of pigs was related to the composition of gut microbiota. The quantity and relative abundance of probiotics in resistant individuals positively affected host immunity and growth performance, whereas high levels of pathogenic bacteria in susceptible individuals were associated with poorer clinical outcomes. The results of this study suggest that gut microbiota may serve as an effective probiotic resource to provide new methods for PRRS prevention and treatment. Porcine Reproductive and Respiratory Syndrome (PRRS) is one of the serious infectious diseases that threatens the swine industry. Increasing evidence shows that gut microbiota plays an important role in regulating host immune responses to PRRS virus (PRRSV). The aim of this study was to investigate gut microbiota difference between PRRSV-resistant pigs and PRRSV-suspectable pigs derived from a Tongcheng pigs and Large White pigs crossed population. PRRSV infection induces an increase in the abundance and diversity of gut microbiota. Correlation analysis showed that 36 genera were correlated with viral loads or weight gain after PRRSV infection. Prevotellaceae-NK3B31-group, Christensenellaceae-R7-group, and Parabacteroides were highly correlated with both viral load and weight gain. Notably, the diversity and abundance of beneficial bacteria such as Prevotellaceae-NK3B31-group was high in resistant pigs, and the diversity and abundance of pathogenic bacteria such as Campylobacter and Desulfovibrio were high in susceptible pigs. Gut microbiota were significantly associated with immune function and growth performance, suggesting that these genera might be related to viremia, clinical symptoms, and disease resistance. Altogether, this study revealed the correlation of gut microbiota with PRRSV infection and gut microbiota interventions may provide an effective prevention against PRRSV infection. [ABSTRACT FROM AUTHOR]

ABSTRACT Microbial communities in the rhizosphere are distinct from those in soils and are influenced by stochastic and deterministic processes during plant development. These communities contain bacteria capable of promoting growth in host plants through various strategies. While some interactions are characterized in mechanistic detail using model systems, others can be inferred from culture-independent methods, such as 16S amplicon sequencing, using machine learning methods that account for this compositional data type. To characterize assembly processes and identify community members associated with plant growth amid the spatiotemporal variability of the rhizosphere, we grew Brassica rapa in a greenhouse time series with amended and reduced microbial treatments. Inoculation with a native soil community increased plant leaf area throughout the time series by up to 28%. Despite identifying spatially and temporally variable amplicon sequence variants (ASVs) in both treatments, inoculated communities were more highly connected and assembled more deterministically overall. Using a generalized linear modeling approach controlling for spatial variability, we identified 43 unique ASVs that were positively or negatively associated with leaf area, biomass, or growth rates across treatments and time stages. ASVs of the genus Flavobacterium dominated rhizosphere communities and showed some of the strongest positive and negative correlations with plant growth. Members of this genus, and growth-associated ASVs more broadly, exhibited variable connectivity in networks independent of growth association (positive or negative). These findings suggest host-rhizobacterial interactions vary temporally at narrow taxonomic scales and present a framework for identifying rhizobacteria that may work independently or in concert to improve agricultural yields. IMPORTANCE The rhizosphere, the zone of soil surrounding plant roots, is a hot spot for microbial activity, hosting bacteria capable of promoting plant growth in ways like increasing nutrient availability or fighting plant pathogens. This microbial system is highly diverse and most bacteria are unculturable, so to identify specific bacteria associated with plant growth, we used culture-independent community DNA sequencing combined with machine learning techniques. We identified 43 specific bacterial sequences associated with the growth of the plant Brassica rapa in different soil microbial treatments and at different stages of plant development. Most associations between bacterial abundances and plant growth were positive, although similar bacterial groups sometimes had different effects on growth. Why this happens will require more research, but overall, this study provides a way to identify native bacteria from plant roots that might be isolated and applied to boost agricultural yields.

ABSTRACT The melting temperature (Tm) mapping method is a novel technique that uses seven primer sets without sequencing to detect dominant bacteria. This method can identify pathogenic bacteria in adults within 3 h of blood collection without using conventional culture methods. However, no studies have examined whether pathogenic bacteria can be detected in clinical specimens from pediatric patients with bacterial infections. Here, we designed a new primer set for commercial use, constructed a database with more bacterial species, and examined the agreement rate of bacterial species in vitro. Moreover, we investigated whether our system could detect pathogenic bacteria from pediatric patients using the Tm mapping method and compared the detection rates of the Tm mapping and culture methods. A total of 256 pediatric clinical specimens from 156 patients (94 males and 62 females; median age, 2 years [

ABSTRACT We hereby present the first descriptions of human-invasive infections caused by Escherichia marmotae, a recently described species that encompasses the former “Escherichia cryptic clade V.” We describe four cases, one acute sepsis of unknown origin, one postoperative sepsis after cholecystectomy, one spondylodiscitis, and one upper urinary tract infection. Cases were identified through unsystematic queries in a single clinical lab over 6 months. Through genome sequencing of the causative strains combined with available genomes from elsewhere, we demonstrate Es. marmotae to be a likely ubiquitous species containing genotypic virulence traits associated with Escherichia pathogenicity. The invasive isolates were scattered among isolates from a range of nonhuman sources in the phylogenetic analyses, thus indicating inherent virulence in multiple lineages. Pan genome analyses indicate that Es. marmotae has a large accessory genome and is likely to obtain ecologically advantageous traits, such as genes encoding antimicrobial resistance. Reliable identification might be possible by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), but relevant spectra are missing in commercial databases. It can be identified through 16S rRNA gene sequencing. Escherichia marmotae could represent a relatively common human pathogen, and improved diagnostics will provide a better understanding of its clinical importance. IMPORTANCE Escherichia coli is the most common pathogen found in blood cultures and urine and among the most important pathogenic species in the realm of human health. The notion that some of these isolates are not Es. coli but rather another species within the same genus may have implications for what Es. coli constitutes. We only recently have obtained methods to separate the two species, which means that possible differences in important clinical aspects, such as antimicrobial resistance rates, virulence, and phylogenetic structure, may exist. We believe that Es. marmotae as a common pathogen is new merely because we have not looked or bothered to distinguish between the thousands of invasive Escherichia passing through microbiological laboratories each day.

ABSTRACT Sanger sequencing of the 16S rRNA gene is routinely used for the identification of bacterial isolates. However, this method is still performed mostly in more-specialized reference laboratories, and traditional protocols can be labor intensive. In this study, 99 clinical bacterial isolates were used to validate a fast, simplified, and largely automated protocol for 16S sequencing. The workflow combines real-time PCR of the first 500 bp of the bacterial 16S rRNA gene and amplicon sequencing on an automated, cartridge-based sequence analyzer. Sequence analysis, NCBI BLAST search, and result interpretation were performed using an automated R-based script. The automated workflow and R analysis described here produced results equal to those of manual sequence analysis. Of the 96 sequences with adequate quality, 90 were concordantly identified to the genus (n = 62) or species level (n = 28) compared with routine laboratory identification of the organism. One organism identification was discordant, and 5 resulted in an inconclusive identification. For sequences that gave a valid result, the overall accuracy of identification to at least the genus level was 98.9%. This simplified sequencing protocol provides a standardized approach to clinical 16S sequencing, analysis, and quality control that would be suited to frontline clinical microbiology laboratories with minimal experience. IMPORTANCE Sanger sequencing of the 16S rRNA gene is widely used as a diagnostic tool for bacterial identification, especially in cases where routine diagnostic methods fail to provide an identification, for organisms that are difficult to culture, or from specimens where cultures remain negative. Our simplified protocol is tailored toward use in frontline laboratories with little to no experience with sequencing. It provides a highly automated workflow that can deliver fast results with little hands-on time. Implementing 16S sequencing in-house saves additional time that is otherwise required to send out isolates/specimens for identification to reference laboratories. This makes results available much faster to physicians who can in turn initiate or adjust patient treatment accordingly.

Critical illness is hypothesized to associate with loss of "health-promoting" commensal microbes and overgrowth of pathogenic bacteria (dysbiosis). This dysbiosis is believed to increase susceptibility to nosocomial infections, sepsis, and organ failure. A trial with prospective monitoring of the intensive care unit (ICU) patient microbiome using culture-independent techniques to confirm and characterize this dysbiosis is thus urgently needed. Characterizing ICU patient microbiome changes may provide first steps toward the development of diagnostic and therapeutic interventions using microbiome signatures. To characterize the ICU patient microbiome, we collected fecal, oral, and skin samples from 115 mixed ICU patients across four centers in the United States and Canada. Samples were collected at two time points: within 48 h of ICU admission, and at ICU discharge or on ICU day 10. Sample collection and processing were performed according to Earth Microbiome Project protocols. We applied SourceTracker to assess the source composition of ICU patient samples by using Qiita, including samples from the American Gut Project (AGP), mammalian corpse decomposition samples, childhood (Global Gut study), and house surfaces. Our results demonstrate that critical illness leads to significant and rapid dysbiosis. Many taxons significantly depleted from ICU patients versus AGP healthy controls are key "health-promoting" organisms, and overgrowth of known pathogens was frequent. Source compositions of ICU patient samples are largely uncharacteristic of the expected community type. Between time points and within a patient, the source composition changed dramatically. Our initial results show great promise for microbiome signatures as diagnostic markers and guides to therapeutic interventions in the ICU to repopulate the normal, "health-promoting" microbiome and thereby improve patient outcomes. IMPORTANCE Critical illness may be associated with the loss of normal, "health promoting" bacteria, allowing overgrowth of disease-promoting pathogenic bacteria (dysbiosis), which, in turn, makes patients susceptible to hospital-acquired infections, sepsis, and organ failure. This has significant world health implications, because sepsis is becoming a leading cause of death worldwide, and hospital-acquired infections contribute to significant illness and increased costs. Thus, a trial that monitors the ICU patient microbiome to confirm and characterize this hypothesis is urgently needed. Our study analyzed the microbiomes of 115 critically ill subjects and demonstrated rapid dysbiosis from unexpected environmental sources after ICU admission. These data may provide the first steps toward defining targeted therapies that correct potentially "illness-promoting" dysbiosis with probiotics or with targeted, multimicrobe synthetic "stool pills" that restore a healthy microbiome in the ICU setting to improve patient outcomes.

Simple Summary: Feeding poultry with insects could reduce production costs, but the impact of this diet on their gut microbiota and growth is little known because the network of relationships between their weights, the composition of their microbiota and their diet is complex and potentially biased by confounding factors (such as the gut compartment, age and sex of the birds). In this study, we were able to unravel these relationships in local breed chickens fed or not fed with black soldier fly larvae thanks to a technique of artificial intelligence (the probabilistic structural equation model). Bacteria were grouped into few entities with distinctive metabolic attributes and were probably linked nutritionally. Birds' age influenced body weights and bacterial composition. The proposed methodology was thus able to simplify the complex dependencies among bacteria present in the gut and to highlight links potentially important in the response of chicken to insect feed. Feeding chicken with black soldier fly larvae (BSF) may influence their rates of growth via effects on the composition of their gut microbiota. To verify this hypothesis, we aim to evaluate a probabilistic structural equation model because it can unravel the complex web of relationships that exist between the bacteria involved in digestion and evaluate whether these influence bird growth. We followed 90 chickens fed diets supplemented with 0%, 5% or 10% BSF and measured the strength of the relationship between their weight and the relative abundance of bacteria (OTU) present in their cecum or cloaca at 16, 28, 39, 67 or 73 days of age, while adjusting for potential confounding effects of their age and sex. Results showed that OTUs (62 genera) could be combined into ten latent constructs with distinctive metabolic attributes. Links were discovered between these constructs that suggest nutritional relationships. Age directly influenced weights and microbiotal composition, and three constructs indirectly influenced weights via their dependencies on age. The proposed methodology was able to simplify dependencies among OTUs into knowledgeable constructs and to highlight links potentially important to understand the role of insect feed and of microbiota in chicken growth. [ABSTRACT FROM AUTHOR]

Permafrost thaw in northern peatlands often leads to increased methane (CH4) emissions, but gaps remain in our understanding of the underlying controls responsible for increased emissions and the duration for which they persist. We assessed how shifting ecological conditions affect microbial communities, and the magnitude and stable isotopic signature (δ13C) of CH4 emissions along a thermokarst bog transect in boreal western Canada. Thermokarst bogs develop following permafrost thaw when dry, elevated peat plateaus collapse and become saturated and dominated by Sphagnum mosses. We differentiated between a young and a mature thermokarst bog stage (~30 and years ~200 since thaw, respectively). The young bog located along the thermokarst edge, was wetter, warmer and dominated by hydrophilic vegetation compared to the mature bog. Using 16S rRNA gene high throughput sequencing, we show that microbial communities were distinct near the surface and converged with depth, but lesser differences remained down to the lowest depth (160 cm). Microbial community analysis and δ13C data from CH4 surface emissions and dissolved gas depth profiles show that hydrogenotrophic methanogenesis was the dominant pathway at both sites. However, the young bog was found to have isotopically heavier δ13C CH4 in both dissolved gases profiles and surface CH4 emissions, suggesting that acetoclastic methanogenesis was relatively more enhanced throughout the young bog peat profile. Furthermore, young bog CH4 emissions were three times greater than the mature bog. Our study suggests that interactions between ecological conditions and methanogenic communities enhance CH4 emissions in young thermokarst bogs, but these favorable conditions only persist for the initial decades after permafrost thaw. [ABSTRACT FROM AUTHOR]

The present study documented the first record of Euphlyctis kalasgramensis from Punjab, Pakistan. The specimens were collected during field visits from June through August 2018. Various morphometric measurements of E. kalasgramensis were taken and compared with Euphlyctis cyanophlyctis. Snout-vent length (SVL) was 38.11 ± 0.87 mm (n = 5), snout length was 3% of SVL, foot length was 55% of SVL, head length was 32% of SVL and weight was 8.01 ± 0.12 g (n = 5). A few specimens (n = 2) were euthanized and preserved for molecular analysis through mitochondrial 16S rRNA gene sequences. The newly obtained DNA sequences of E. kalasgramensis were submitted to GenBank and accession numbers were obtained (MK881165.1 and MK920114.1). The Maximum likelihood and Neighbor-joining trees based on Kimura 2-parameter distance resulted in similar phylogenetic trees. Euphlyctis kalasgramensis was out group in both phylogenetic trees. The interspecific divergence of E. kalasgramensis and E. cyanophlyctis was high ranging from 4% to 6% as compared to low intraspecific divergence 0% and 1%. The diversity and distribution ranges of many amphibians species are not well known in Pakistan due to lack of taxonomic information. In our recommendation, a large scale DNA barcoding is required to report more cryptic or new species from Pakistan.

Background: To compare the diagnostic performance of microbiological culture and 16S/18S rRNA gene polymerase chain reaction (PCR)-Sanger sequencing for infectious keratitis (IK) and to analyse the effect of clinical disease severity on test performance and inter-test concordance., Methods: This was a three-arm, diagnostic cross-sectional study. We included all eligible patients who presented with presumed bacterial/fungal keratitis to the Queen's Medical Centre, Nottingham, UK, between June 2021 and September 2022. All patients underwent simultaneous culture (either direct or indirect culture, or both) and 16S (pan-bacterial)/18S (pan-fungal) ribosomal RNA (rRNA) PCR-Sanger sequencing. The bacterial/fungal genus and species identified on culture were confirmed using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Relevant clinical data were also collected to analyze for any potential clinico-microbiological correlation. Main outcome measures included the diagnostic yield, test accuracy (including sensitivity and specificity), and inter-test agreement [including percent agreement and Cohen's kappa ( k )]., Results: A total of 81 patients (86 episodes of IK) were included in this study. All organisms identified were of bacterial origin. Diagnostic yields were similar among direct culture (52.3%), indirect culture (50.8%), and PCR (43.1%; p = 0.13). The addition of PCR enabled a positive diagnostic yield in 3 (9.7%) direct culture-negative cases. Based on composite reference standard, direct culture had the highest sensitivity (87.5%; 95% CI, 72.4-95.3%), followed by indirect culture (85.4%; 95% CI, 71.6-93.5%) and PCR (73.5%; 95% CI, 59.0-84.6%), with 100% specificity noted in all tests. Pairwise comparisons showed substantial agreement among the three tests (percent agreement = 81.8-86.2%, Cohen's k = 0.67-0.72). Clinico-microbiological correlation demonstrated higher culture-PCR concordance in cases with greater infection severity., Conclusions: This study highlights a similar diagnostic performance of direct culture, indirect culture and 16S rRNA PCR for bacterial keratitis, with substantial inter-test concordance. PCR serves as a useful diagnostic adjuvant to culture, particularly in culture-negative cases or those with lesser disease severity (where culture-PCR concordance is lower)., Competing Interests: DJIT, JC, and JH are employees of Micropathology Ltd, UK, but the company has no role in the design of the study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Hammoudeh, Suresh, Ong, Lister, Mohammed, Thomas, Cottell, Holden, Said, Dua and Ting.)

In the wild, C. elegans are emersed in environments teeming with a veritable menagerie of microorganisms. The C. elegans cuticular surface serves as a barrier and first point of contact with their microbial environments. In this study, we identify microbes from C. elegans natural habitats that associate with its cuticle, constituting a simple "skin microbiome." We rear our animals on a modified CeMbio, mCeMbio, a consortium of ecologically relevant microbes. We first combine standard microbiological methods with an adapted micro skin-swabbing tool to describe the skin-resident bacteria on the C. elegans surface. Furthermore, we conduct 16S rRNA gene sequencing studies to identify relative shifts in the proportion of mCeMbio bacteria upon surface-sterilization, implying distinct skin- and gut-microbiomes. We find that some strains of bacteria, including Enterobacter sp. JUb101 , are primarily found on the nematode skin, while others like Stenotrophomonas indicatrix JUb19 and Ochrobactrum vermis MYb71 are predominantly found in the animal's gut. Finally, we show that this skin microbiome promotes host cuticle integrity in harsh environments. Together, we identify a skin microbiome for the well-studied nematode model and propose its value in conferring host fitness advantages in naturalized contexts., Importance: The genetic model organism C. elegans has recently emerged as a tool for understanding host-microbiome interactions. Nearly all of these studies either focus on pathogenic or gut-resident microbes. Little is known about the existence of native, nonpathogenic skin microbes or their function. We demonstrate that members of a modified C. elegans model microbiome, mCeMbio, can adhere to the animal's cuticle and confer protection from noxious environments. We combine a novel micro-swab tool, the first 16S microbial sequencing data from relatively unperturbed C. elegans , and physiological assays to demonstrate microbially mediated protection of the skin. This work serves as a foundation to explore wild C. elegans skin microbiomes and use C. elegans as a model for skin research., Competing Interests: The authors declare no conflict of interest.