Targeting 16S rRNA Research Articles

A CRISPR-Cas12a-based universal rapid scrub typhus diagnostic method targeting 16S rRNA of Orientia tsutsugamushi.

Scrub typhus is caused by Orientia tsutsugamushi infection and occurs frequently in an area called the Tsutsugamushi Triangle. Currently, there is no vaccine for O. tsutsugamushi, and its infection is treated with antibiotics such as doxycycline. Scrub typhus responds to effective treatment, and early treatment shortens the course of the disease, reduces mortality, and accelerates recovery. Therefore, it is important to rapidly diagnose O. tsutsugamushi infection to ensure successful outcomes. Here, we developed a CRISPR-Cas12a-based diagnostic method targeting the bacterial 16S rRNA to detect O. tsutsugamushi infection of all known genotypes. To reduce the possibility of contamination and increase field applicability, we designed the one-pot assay system in addition to conventional two-pot assay system. Using this method, we successfully detected up to 100 copies of in vitro transcribed O. tsutsugamushi 16S rRNA within 1 hour under isothermal conditions. In blood samples from patients confirmed to be infected with O. tsutsugamushi by nested PCR, the developed method exhibited a clinical sensitivity of 98% and high specificity. These data demonstrate that the presented method is applicable for the rapid and universal diagnosis of scrub typhus to facilitate timely and appropriate treatment.

Long-term and seasonal evaluation on environmental microbiology and water quality of Shanmei reservoir in southeast China.

As a crucial source of potable water, the quality of water in Shanmei reservoir strongly and directly impacts the safety and well-being of downstream residents. Microorganisms play a pivotal role in the reservoir's resource and energy cycle. However, ecological protection efforts for the Shanmei reservoir have encountered numerous challenges in recent years. This study conducted an extensive visual analysis of microbial communities in sediment from the Shanmei reservoir between 2022 and 2024 using amplicon sequencing technology targeting 16S rRNA gene. The results showed that the microbial diversity of sediment showed an obvious seasonal pattern. At the same time, microbial composition also changes with the long-term evolution of time, which may be closely related to the change in environmental conditions. In addition, we have also carried out long-term multi-dimensional monitoring of the water quality of the Shanmei reservoir, and the results show that the water quality has reached the national drinking water grade. In conclusion, this study not only unveiled the interseasonal dynamics and long-term evolutionary characteristics of sediment microbial communities but also elucidated the significant influence of environmental factors on their composition, structure and function. These findings offer a fresh perspective for understanding the freshwater ecosystem microbial dynamics and provide a scientific foundation for reservoir management and water quality protection.

Explore Alteration of Lung and Gut Microbiota in a Murine Model of OVA-Induced Asthma Treated by CpG Oligodeoxynucleotides.

We sought to investigate the impact of CpG oligodeoxynucleotides (CpG-ODN) administration on the lung and gut microbiota in asthmatic mice, specifically focusing on changes in composition, diversity, and abundance, and to elucidate the microbial mechanisms underlying the therapeutic effects of CpG-ODN and identify potential beneficial bacteria indicative of its efficacy. HE staining were used to analyze inflammation in lung, colon and small intestine tissues. High-throughput sequencing technology targeting 16S rRNA was employed to analyze the composition, diversity, and correlation of microbiome in the lung, colon and small intestine of control, model and CpG-ODN administration groups. (1) Histopathologically, both lung and intestinal tissue in asthmatic mice exhibited significant structural damage and inflammatory response, whereas the structure of both lung and intestinal tissue approached normal levels, accompanied by a notable improvement in the inflammatory response after CpG-ODN treatment. (2) In the specific microbiota composition analysis, bacterial dysbiosis observed in the asthmatic mice, accompanied by enrichment of Proteobacteria found to cause lung and intestinal epithelial damage and inflammatory reaction. After CpG-ODN administration, bacterial dysbiosis was improved, and a notable enrichment of beneficial bacteria, indicating a novel microecology. Meanwhile Oscillospira and Clostridium were identified as two biomarkers of the CpG-ODN treatment. (3) Heatmap analysis revealed significant correlations among lung, small intestine, and colon microbiota. CpG-ODN treatment can ameliorate OVA-induced asthma in mice. One side, preserving the structural integrity of the lung and intestine, safeguarding the mucosal physical barrier, the other side, improving the dysbiosis of lung and gut microbiota in asthmatic mice. Beneficial bacteria and metabolites take up microecological advantages, regulate immune cells and participate in the mucosal immune response to protect the immune barrier. Meanwhile, Oscillospira and Clostridium as biomarkers for CpG-ODN treatment, has reference significance for exploring precise Fecal microbiota transplantation treatment for asthma.

Detection of Potato Pathogen Clavibacter sepedonicus by CRISPR/Cas13a Analysis of NASBA Amplicons.

The ring rot of potato caused by the bacterial pathogen Clavibacter sepedonicus is a quarantine disease posing a threat to the potato industry worldwide. The sensitive and selective detection of C. sepedonicus is of a high importance for its effective control. Here, the detection system is reported to determine viable bacteria of C. sepedonicus in potato tubers, based on the coupling of CRISPR/Cas13a nuclease with NASBA (Nucleic Acid Sequence Based Amplification)-the method of isothermal amplification of RNA. Detection can be conducted using both instrumental and non-instrumental (visual inspection of test tubes under blue light) modes. When NASBA and Cas13a analyses were carried out in separate test tubes, the limit of detection (LOD) for the system was 1000 copies of purified target 16S rRNA per NASBA reaction or about 24 colony-forming units (CFUs) of C. sepedonicus per 1 g of tuber tissue. The testing can also be conducted in the "one-pot" format (a single test tube), though with lower sensitivity: LOD was 10,000 copies of target RNA or about 100 CFU per 1 g of tuber tissue for both instrumental and visual detection modes. The overall time of NASBA/Cas13a analysis did not exceed 2 h. The developed NASBA/Cas13a detection system has the potential to be employed as a routine test of C. sepedonicus, especially for on-site testing.

Characterizing the urobiome in geriatric males with chronic indwelling urinary catheters: an exploratory longitudinal study.

The impact of chronic indwelling urinary catheters (IUCs) on the composition and stability of the urinary microbiota remains unknown. The primary aim of this study was to describe the urinary microbiomes of geriatric males with chronic IUCs. A secondary aim was to explore clinical catheter-associated urinary tract infection (CAUTI) courses of the participants. Geriatric male patients with chronic IUCs were followed longitudinally. Catheterized urine, catheter tips, and both urethral and periurethral swabs were collected from participants at monthly intervals. Microbes were isolated and identified from each specimen using an enhanced culture method called expanded quantitative urine culture (EQUC) and targeted 16S rRNA gene DNA sequencing. Microbial outcomes were examined both in the absence of urinary symptoms and in the context of clinical diagnosis of CAUTI. Ten male participants (mean age 86 years) were enrolled. Urinary microbiomes differed for each participant. However, within each individual, microbiomes were similar over time and across niches (bladder, catheter, urethra, and periurethra). Within-niche microbiomes differed across individuals, and this was observed over time. The most abundant bacteria isolated from all niches were known uropathogens. Six of 10 individuals met diagnostic criteria for CAUTI at least once during the 12-month observation period, but no evidence of this or antibiotic treatment/response was discernable in our monthly samples. The microbiomes of each participant were unique and remained similar over time and across niches. Longitudinal EQUC or 16S rRNA gene sequencing data could be useful to clinicians when diagnosing or treating possible CAUTI.IMPORTANCECatheter-associated urinary tract infections (CAUTIs) are serious but preventable nosocomial infections. The most common risk factor for developing CAUTI is prolonged use of indwelling urinary catheters (IUCs). This study provides the first longitudinal description of the urinary microbiomes of geriatric males with chronic IUCs, in the absence of urinary signs and symptoms, as a first step toward enhancing our knowledge of the impact of chronic IUCs on the composition and stability of the urinary microbiota. This is an understudied area, particularly for males.

Links between Nosocomial Endodontic Infections and Bacteremia Associated with Apical Periodontitis and Endodontic Treatment

IntroductionNosocomial endodontic infections can cause treatment failure and are potential risk of bacteremia. This study is aimed to investigate bacteremia associated with apical periodontitis and immediate postendodontic treatment using next-generation sequencing. The study will also elucidate if the endodontic nosocomial pathogens are present in the blood microbiome. MethodsSaliva, blood, and intracanal samples were collected from patients undergoing endodontic treatment (n = 65), while gloves samples were collected from the operator. Bacterial DNA was extracted and the targeted 16S rRNA gene was sequenced using the Illumina Miseq 300 platform for the V1-V2 hypervariable region. The alpha and beta diversities were carried out using the phyloseq package (version-1.36.0) in R (version-4.1). Differences in abundance and diversity were calculated using Mann-Whitney test. Linear discriminant analysis Effect Size was used to compare the biological relevance between different sample sources. ResultsThe phyla firmicutes, actinobacteria, proteobacteria, and bacteroidetes were identified from all sample sources. In blood and glove samples, Cutibacterium genus was highly abundant (14.6%, 17.7%, respectively), while in intracanal samples, Enterococcus took precedence (18.2%). Saliva samples exhibited Prevotella as the most abundant genera (17.9%). Common genera were present between intracanal and postoperative blood samples. Circos graph showed that all the phyla identified from a patient were consistently present in all the samples investigated from the same patient. ConclusionsNosocomial endodontic pathogens being predominant in both; gloves and blood samples, along with common genera present in all sample sources, suggests a potential pathway of transfer of pathogens from saliva to gloves, intracanal, and blood, thus providing evidence of nosocomial endodontic bacteremia.

A Molecular Approach to Understand the Involvement of Mycoplasma in Suppurative Otitis Cases of Cattle in Chittoor District of Andhra Pradesh

In bovines the economic losses mainly due to the outbreak of diseases, leads to mortality, morbidity, treatment cost and reduced production. Recently in August month of 2024, received 13 bovine suppurativeotitis samples from outbreak in KuppamMandal of Chittoor district. The history having the purulent discharges with foul smell from ear infections of cattle that to crossbreed cattle of all age groups are affected. After 5 days of appearing aural discharges the animals were also shown the nervous signs, head tilting and other clinical findings includes unilateral or bilateral ear droop, epiphora, ptosis, abnormal nystagmus, strabismus, regurgitation, stiff neck, opisthotonus, facial hyperesthesia, purulent aural discharge, nasopharyngeal collapse, recumbency and finally death were noticed in severely affected animals. All the samples were processed for cultural tests by inoculating in PPLO selective broth and selective media PPLO agar then incubated anaerobically at 370C and observed the color development in broth pink to yellow color after 3- 5 days of incubation indicative of positive growth and fried egg micro colonies on selective PPLO agar media after 8-14 days of incubation observed under low power and 40X. The DNA was isolated from all the samples and screened for presence of Mycoplasma by targeting 16s rRNA gene and found that out of 13 samples 10 were positive for genus Mycoplasma and produced the predicted 280bp size product in all positive samples. The suppurativeotitis in cattle usually is caused by Actinomyces spp., Corynebacterium pseudotuberculosis, E. coli, Heamophilus, P.multocida, Pseudomonas spp, Streptococcus spp., and Mycoplasma bovis. This study was targeted only the emerging pathogen i.e the cellwall deficient bacteria Mycoplasma because of infections is hampered by a lack of effective vaccines and specific treatments, leads to increasing trends in antimicrobial resistance. Concluded that isolation and identification of the pathogenic ........

Molecular Epidemiology and Phylogeny of Theileria ovis and Theileria lestoquardi in Sheep and Goats from Southern Punjab, Pakistan.

Background: Theileria spp. are responsible for ovine and caprine theileriosis, leading to significant morbidity and mortality in small ruminants.The present study aims to investigate Theileria spp. infections in small ruminants from Southern Punjab in Pakistan, and genetic characterize revealed Theileria spp. isolates. Methods: A total of 93 sheep and 107 goats were sampled between May and August 2022. Blood smears were examined microscopically, and PCR amplification targeting the 18S rRNA gene was performed to detect Theileria spp. Additionally, specific PCR assays targeting 18S rRNA and ms1 partial sequences were used to identify Theileria ovis and T. lestoquardi, respectively. Results: The prevalence of Theileria spp. was significantly higher using PCR (13.5%) compared to microscopic screening (5%). Sheep showed a higher prevalence rate (19.4%) compared to goats (8.4%) (p = 0.024). Young sheep aged ≤ 1 year were more commonly infected with Theileria spp. (41%) compared to older sheep (p = 0.006). The prevalence of Theileria spp. was higher in sheep-only herds (37.3%) compared to goat-only herds (18%) or mixed-species herds (8.1%) (p = 0.015). The prevalence rates of T. ovis and T. lestoquardi were 9% and 2.5%, respectively, with four animals (2 goats and 2 sheep) showing co-infection. Phylogenetic analysis revealed that our T. ovis 18S rRNA sequence clustered with previously reported sequences from sheep in Turkey, China, Spain, and goats in Tanzania. The obtained T. lestoquardi ms1 partial sequence formed a distinct cluster from other T. lestoquardi isolates in Pakistan and neighboring countries. Conclusion: Theileria spp. co-circulation in Pakistani small ruminants, particularly the presence of T. ovis and T. lestoquardi, highlights the need for attention from animal health decision-makers due to their financial and health impacts.

Viability discrimination of bacterial microbiomes in home kitchen dish sponges using propidium monoazide treatment.

Dish sponges are known to support the proliferation of human bacterial pathogens, yet they are commonly used by consumers. Exposure to foodborne pathogens via sponge use may lead to illness, a serious concern among susceptible populations. The extent of exposure risks from sponge use has been limited by constraints associated with culture-independent or dependent methods for bacterial community characterization. Therefore, five used dish sponges were characterized to evaluate the presence of viable bacterial foodborne pathogens using the novel application of propidium monoazide (PMA) treatment and targeted 16S rRNA gene amplicon sequencing. Select pathogen viability was confirmed using targeted selective enrichment. The taxonomic abundance profiles of total and viable sponge microbiomes did not vary significantly. The numbers of unique bacterial species (p = 0.0465) and foodborne pathogens (p = 0.0102) identified were significantly lower in viable sponge microbiomes. Twenty unique bacterial foodborne pathogens were detected across total and viable sponge microbiomes, and three to six viable foodborne pathogens were identified in each sponge. Escherichia coli and Staphylococcus aureus were identified in each viable sponge microbiome, and viable E. coli were recovered from two sponges via targeted selective enrichment. These findings suggest that sponge-associated bacterial communities are primarily viable and contain multiple viable bacterial foodborne pathogens.

Overlap of gallbladder dysfunction and irritable bowel syndrome: clinical and microbiological features and therapeutic possibilities

Objective — to analyse the clinical and microbiological features in patients with an overlap between gallbladder dysfunction (GBD) and irritable bowel syndrome (IBS) and a complex assessment of the effectiveness and safety of using ursodeoxycholic acid (Ursis) in these patients. Materials and methods. Under our observation were 115 patients with the overlap of GBD and IBS aged from 19 to 52. The majority of them were women (62.6%). All patients complained of abdominal pain, mostly without clear localization or in the right hypochondrium and along the course of the large intestine. Constipation was diagnosed in 83 (72.2%) patients, and meteorism in 89 (77.4%) patients. Therefore, 62 patients with the overlap of GBD and IBS, who had abdominal pain, constipation and meteorism, were selected for further study. During the study, all patients kept a diary in which the frequency and nature of bowel movements, the severity of abdominal pain, and the degree of meteorism were recorded. All patients before and after treatment underwent an ultrasound examination of the abdominal cavity, which included the study of the contractile function of the gallbladder, the emptying coefficient of the gallbladder, the thickness and density of its wall, and the density of bile. Changes in the main bacterial enterotypes were analyzed by qRT‑PCR using primers targeting 16S rRNA. We studied Bacteroidetes, Firmicutes, Actinobacteria, as well as the content of butyrate‑producing bacteria (Faecalibacterium prausnitzii and Akkermansia muciniphila) and methanogens (Methanobrevibacter smithii and Methanosphaera stadmanae). Results. Before treatment, all patients had abdominal pain of moderate intensity (6.9±0.7 points), mostly without clear localization, constipation, with a frequency of defecation (1.4±0.5) times a week, and meteorism. Before treatment, patients with an overlap of GBD and IBS had changes in the contractile function of the gallbladder with a decrease in the emptying ratio, a thickening of the gallbladder wall and an increase in its density and bile density, as well as a disturbance of the intestinal microbiome (an increase in the number of Firmicutes, Actinobacteria and methanogenic archaea and a decrease in Bacteroidetes and butyrate‑producing bacteria). Conclusions. The use of Ursis® medication in the complex therapy of patients with a GBD and IBS overlap is effective and safe. The aplication of Ursis® led to a more pronounced positive clinical dynamics (a decrease in the intensity of abdominal pain, normalization of the frequency of bowel movements, a decrease in meteorism and an improvement in general well‑being) than in the patients of the comparison group. After treatment with Ursis® the coefficient of emptying of the gallbladder increased, the thickness and density of its wall decreased. The use of the drug Ursis® in the complex therapy of patients with the overlap of GBD and IBS contributed to the improvement of intestinal flora: an increase in the number of Bacteroidetes and butyrate‑producing bacteria (Faecalibacterium prausnitzii and Akkermansia muciniphila), and a decrease in the content of methanogens (Methanobrevibacter smithii and Methanosphaera stadmanae).

Microbial community profiling in intestinal tract of indigenous chickens from different villages

Village chickens (Gallus gallus domesticus) are commonly reared in rural households of South Africa and other developing countries. They play a vital role as a primary source of protein through the provision of meat and eggs. The chicken gut microbiota plays an important role in chicken’s immune system, its health, physiological development of the gut, digestion of food, nutrient absorption and productivity. Thus, it is imperative to critically investigate the chicken microbial composition in order to develop effective disease control measures and increase production. In this present study, microbial DNA was isolated from 34 non-descript mixed gender matured village chickens’ intestinal contents followed by high throughput Illumina sequencing targeting 16S rRNA gene. Senwamokgope village had the largest microbiota composition as compared to Itieleni and Thakgalang villages. Overall, Firmicutes (74%) was the most abundant phylum observed, followed by Proteobacteria (8%), Actinobateria (5%), and Bacteroidota (3%). At the genus level, Lactobacillus was the dominant bacteria. Other genera found included Sphingomonas (7%), Cutibacterium (4%), and Clostridium_sensu_stricto_1 (2%). The richness of female intestinal microbiota was higher compared to the male microbiota. The findings of this study provide baseline information that can assist to better understand the chicken gut microbiota and its interaction with diseases and parasites.

Molecular Survey of Tick-Borne Haemoparasites of Dogs by Multiplex Polymerase Chain Reaction from Punjab, India.

Tick-transmitted parasites as Babesia gibsoni, Babesia vogeli, Ehrlichia canis, and Hepatozoon canis are major health concern for dogs. Owing to prevalence and infection severity, there is need of sensitive, specific, and affordable test for their simultaneous detection. Prevalence of B. gibsoni, B. vogeli, E. canis, and H. canis infections was assessed on 719 blood samples by microscopy and multiplex PCR assay targeting 18S rRNA (B. gibsoni & H. canis), ITS1 & 5.8S rRNA (B. vogeli) and VirB9 gene (E. canis). An internal control (canine-actin) was also included to increase the accuracy of assay and effect of associated risk factors with disease prevalence was also studied. Microscopic prevalence of B. gibsoni, B. vogeli, E. canis and H. canis was 5.0%, 0.1%, 1.4% and 1.0%, respectively, whereas with multiplex PCR assay, the corresponding values were 8.9%, 1.1%, 2.6% and 5.1% besides concurrent infections of B. gibsoni & H. canis (0.4%), B. gibsoni & E. canis (0.4%), E. canis & H. canis (0.3%) and B. gibsoni & B. vogeli (0.1%). Analytical sensitivity of developed assay was 0.1pg (B. gibsoni & H. canis), 0.01pg (B. vogeli), and 1.0pg (E. canis). A ″fair″ (B. vogeli & H. canis) to ″substantial″ (B. gibsoni & E. canis) agreement between two tests was observed with data as statistically significant. Breed, sex and location were significantly associated with B. gibsoni infection. The developed multiplex PCR assay offers a potential solution to detect these pathogens simultaneously, aiding in timely diagnosis and effective disease management in suspected dogs.

Diagnostic Accuracy of Polymerase Chain Reaction Against Giemsa Staining on Tissue Biopsy for Cutaneous Leishmaniasis.

To evaluate the diagnostic accuracy of a commercial real-time polymerase chain reaction (PCR) kit targeting 18S rRNA against Giemsa-stained tissue slides in patients clinically suspected of cutaneous leishmaniasis (CL). Cross-sectional analytical study. Place and Duration of the Study: Department of Microbiology, Armed Forces Institute of Pathology / National University of Medical Sciences, Rawalpindi, Pakistan, from July to December 2022. Samples of skin tissue in 98 patients suspected of CL were evaluated. These samples were subjected to Giemsa-staining for microscopy and real-time PCR. Sensitivity, specificity, and accuracy of the PCR were calculated keeping Giemsa-stained tissue slide microscopy as gold standard. Out of the 98 tissue samples, 37 were found positive for leishmaniasis on PCR while 13 were found Leishmania positive on microscopy of Giemsa-stained slides. The sensitivity, specificity, and accuracy of the PCR for the detection of Leishmania species were 100%, 71.8%, and 91.8%, respectively with 100% negative predictive value. This study demonstrates that the commercial PCR is a reliable diagnostic test for the diagnosis of CL. The ease, rapidity, and reliability of the PCR make it a dependable tool in diagnostic repertoire of CL. Giemsa stain, Leishmania spp., Polymerase chain reaction, Viasure.

Seasonal variation in airborne microbial communities of the Akiyoshido Cave: Lampenflora dispersed by phototrophic bioaerosols

Lampenflora, such as phototrophic microorganisms, multiply on speleothem surfaces under the show-cave lighting, causing deterioration of the natural and cultural heritages of the caves. Speleothems change color from white to green through microbial photosynthesis and are destroyed by microbial metabolisms. Aerosol transmission are suspected to disperses photosynthetic microorganisms. However, the mechanism underlying the process remains unclear, because the complexity of air ventilation affects the cave atmosphere. In the present study, we collected aerosol samples from the Akiyoshido Cave, which is a special natural monument in Japan and possesses various types of speleothems, to analyze microbial concentrations and communities in the cave atmosphere. Under a fluorescence microscope, phototrophic and heterotrophic microorganisms were observed in the aerosols with in the cave. The aerosol concentrations showed seasonal changes depending on air-flow variations in cave ventilation. High-throughput DNA sequencing targeting 16S rRNA genes revealed that the airborne bacterial communities inside the cave were dominated by members of the phyla Actinomycetota, Bacillota, and Pseudomonadota, originating from the external terrestrial, phyllosphere or freshwater environments, as well as its interior (guano). Cyanobacteria showed small relative abundances (from 1.0 to 10%) randomly in the aerosol samples at several locations in the cave, suggesting that the lampenflora-derived cyanobacteria were dispersed throughout the cave by the ventilation. Additionally, phototrophic microorganisms closely related to the relatives of the genera Leptolyngbya, Calothrix, and Chroococcidiopsis from the phylum Cyanobacteria were isolated from the aerosol samples. These results confirm that Cyanobacteria are one of the candidate microorganisms responsible for lampenflora dispersion and are “alive and airborne” in caves.

Circulating Bacterial DNA in Colorectal Cancer Patients: The Potential Role of Fusobacterium nucleatum.

Intestinal dysbiosis is a major contributor to colorectal cancer (CRC) development, leading to bacterial translocation into the bloodstream. This study aimed to evaluate the presence of circulated bacterial DNA (cbDNA) in CRC patients (n = 75) and healthy individuals (n = 25). DNA extracted from peripheral blood was analyzed using PCR, with specific primers targeting 16S rRNA, Escherichia coli (E. coli), and Fusobacterium nucleatum (F. nucleatum). High 16S rRNA and E. coli detections were observed in all patients and controls. Only the detection of F. nucleatum was significantly higher in metastatic non-excised CRC, compared to controls (p < 0.001), non-metastatic excised CRC (p = 0.023), and metastatic excised CRC (p = 0.023). This effect was mainly attributed to the presence of the primary tumor (p = 0.006) but not the presence of distant metastases (p = 0.217). The association of cbDNA with other clinical parameters or co-morbidities was also evaluated, revealing a higher detection of E. coli in CRC patients with diabetes (p = 0.004). These results highlighted the importance of bacterial translocation in CRC patients and the potential role of F. nucleatum as an intratumoral oncomicrobe in CRC.

Seasonal patterns of microbial diversity across the world oceans

AbstractUnderstanding the patterns of marine microbial diversity (Bacteria + Archaea) is essential, as variations in their alpha‐ and beta‐diversities can affect ecological processes. Investigations of microbial diversity from global oceanographic expeditions and basin‐wide transects show positive correlations between microbial diversity and either temperature or productivity, but these studies rarely captured seasonality, especially in polar regions. Here, using multiannual alpha‐diversity data from eight time series in the northern and southern hemispheres, we show that marine microbial community richness and evenness generally correlate more strongly with daylength than with temperature or chlorophyll a (a proxy for photosynthetic biomass). This pattern is observable across time series found in the northern and southern hemispheres regardless of collection method, DNA extraction protocols, targeted 16S rRNA hypervariable region, sequencing technology, or bioinformatics pipeline.

The impacts of film mulching and ridging on N2O emissions, relevant functional genes, and microbial communities in rain-fed potato fields

Rain-fed potato (Solanum tuberosum) fields in drylands significantly contribute to nitrous oxide (N2O) emissions, making them an important focus of agricultural greenhouse gas research. Film mulching and ridging are key agricultural methods in potato cultivation. Investigating the impact of these methods on N2O emissions, nitrifying/denitrifying functional genes, and microbial communities can provide a theoretical basis for soil emission reduction and more sustainable dryland agriculture. We examine the effects of flat tillage with mulching, ridge tillage with mulching, flat tillage without mulching, and ridge tillage without mulching, on potato fields under natural rainfall conditions in Wuchuan County, China. N2O emission fluxes were monitored using a static (dark) chamber and gas chromatography. Real-time quantitative PCR (q-PCR) was used to quantify abundances of nitrifying and denitrifying bacteria related to N2O emissions at various potato-growth stages. Illumina high-throughput sequencing was used to investigate microbial community structure by targeting 16S rRNA genes; related soil elements (soil temperatures and moisture) are analyzed. Mulching and ridging indirectly influence N2O emissions, nitrifying/denitrifying functional gene copy numbers, and microbial community structure by altering soil temperature and moisture. Cumulative N2O emissions and emission intensity were both consistently higher in ridge tillage with mulching during the potato-growing period. Ammonia-oxidizing archaea are the main microorganisms that control N2O emissions, with nitrification-coupled denitrification also being an important mechanism contributing to high N2O emissions during soil dry–wet cycles. Increased soil temperature and moisture elevated N2O emissions and functional gene copy numbers. The combination of mulching and ridging effectively uses the characteristics of both practices, making Nitrospira the dominant genus, and significantly increases N2O emissions.

Clostridium perfringens in central Colombia: frequency, toxin genes, and risk factors

Clostridium perfringens is an opportunistic bacterium that causes intestinal diseases in both humans and animals. This study aimed to assess the frequency of C. perfringens and the presence of toxin-encoding genes in fecal samples from individuals with or without gastrointestinal symptoms in the Department of Boyacá, Colombia. Additionally, risk factors associated with carriage and disease development were analyzed. A total of 114 stool samples were analyzed using a molecular test based on specific polymerase chain reaction (PCR) targeting 16S-rRNA and alpha toxin (cpa) genes. For individuals with a positive result for the PCR test, stool samples were cultured on Tryptose Sulfite Cycloserine (TSC) agar. Two to five colonies forming units were selected based on phenotypic characteristics, resulting in 56 bacterial isolates. These isolates were then analyzed for toxin-coding genes associated with gastrointestinal diseases. In addition, sociodemographic and clinical data from 77 individuals were also analyzed. The overall frequency of C. perfringens was 19.3% (n = 22/114). The detection frequency in 77 individuals with clinical data was 16.6% (n = 5/30) among symptomatic individuals and 21.2% (n = 10/47) among asymptomatic individuals. All 56 isolates obtained carried the cpa gene, while cpb2 was present in 10.7% (n = 6/56); cpe and cpb genes were not detected. Notably, diabetes and autoimmune diseases are significantly associated with an increased risk of C. perfringens detection (adjusted OR 8.41: 95% CI 1.32–35.89). This study highlights an elevated frequency of C. perfringens and the presence of the cpb2 gene in asymptomatic individuals compared with their symptomatic counterparts. These findings offer insights into the distribution and virulence factors of C. perfringens at a micro-geographical level. This information supports the need for developing tailored prevention strategies based on local characteristics to promote active surveillance programs based on molecular epidemiology.

Environmental DNA reveals fine‐scale spatial and temporal variation of marine mammals and their prey species in a Scottish marine protected area

AbstractMarine mammal foraging grounds are popular focal points for marine protected area (MPA) implementation, despite being temporally dynamic, requiring continuous monitoring to infer prey availability and abundance. Marine mammal distributions are assumed to be driven by their prey in foraging areas, but limited understanding of prey distributions often prevents us from exploring how shifting prey availability impacts both seasonal and long‐term marine mammal distributions. Environmental DNA (eDNA) metabarcoding could enhance the understanding of marine mammal habitat use in relation to their prey through simultaneous monitoring of both. However, eDNA applications focused on marine mammals or predator–prey dynamics have been limited to date. In this study, we assess spatiotemporal changes in the distribution and abundance of cetaceans, minke whales (Balaenoptera acutorostrata), bottlenose dolphins (Tursiops truncatus) and harbor porpoises (Phocoena phocoena) in relation to key prey species in a newly established MPA, employing eDNA metabarcoding. We recovered 105 molecular operational taxonomic units (OTUs) from marine vertebrates using two primer sets targeting 12S and 16S genes, along with 112 OTUs from a broader eukaryotic primer set targeting 18S rRNA. Overall, key forage fish prey species, sandeels and clupeids, were the most abundant teleost fishes detected, although their availability varied temporally and with distance from shore. We also found clear spatial partitioning between coastal bottlenose dolphins and the more pelagic minke whales and harbor porpoises, paralleling availability of their main prey species. Other species of conservation interest were also detected including the critically endangered European eel (Anguilla anguilla), Atlantic bluefin tuna (Thunnus thynnus), and the invasive pink salmon (Oncorhynchus gorbuscha). This study demonstrates the application of eDNA to detect spatiotemporal trends in the occurrence and abundance of cetacean predators and their prey, furthering our understanding of fine‐scale habitat use within MPAs. Future long‐term monitoring of predator–prey dynamics with eDNA could improve our ability to predict climate‐induced shifts in foraging grounds and enhance rapid responses with appropriate management actions.

Prevalence and predictors of NTM in presumed/confirmed drug-resistant TB.

Non-tuberculous mycobacteria (NTM) are increasingly isolated in individuals with presumed/confirmed pulmonary TB. We aimed to estimate the prevalence and species distribution of NTM among presumed/confirmed drug-resistant TB (DR-TB) individuals and determine NTM isolation predictors. Sputum samples collected for DR-TB diagnosis and follow-up from 2012 to 2021 in Ghana were retrospectively analysed. Samples were subjected to sputum smear microscopy (SSM) and mycobacterial culture. The MPT64 assay was performed on positive cultures to distinguish between Mycobacterium tuberculosis complex MTBc and NTM. NTM isolates were re-cultured for species identification using GenoType® Mycobacterium CM/AS line-probe assay, polymerase chain reaction, and Sanger sequencing targeting 16S rRNA and rpoB genes. MTBc isolates identified by GenoType underwent spoligotyping. A logistic regression model was used to identify the predictors of NTM isolation. Of the 2,492 samples, 839 (33.7%) tested culture-positive for mycobacteria, with 257 (30.6%) presumed to be NTM. Of these, 53 (23.6%) were identified at the species level, with a predominance of M. intracellulare (66.0%). MPT64 testing missed 18 (3%) MTBc isolates. Logistic regression showed increased odds of NTM isolation in follow-up samples (aOR 2.41, 95% CI 1.46-3.99). NTM species were isolated from 46 patients, with four classified as NTM pulmonary disease. Enhancing our understanding of local NTM epidemiology and improving local diagnostic capabilities can optimise patient management strategies and outcomes.