Fluorescence in situ hybridization (FISH) is a highly specific technique for pathogenic bacteria detection that requires no culturing and provides simultaneous information on pathogenic bacteria abundance, morphology, and spatial localization. However, the limited sensitivity and poor multiplexing capacity of conventional FISH have hindered its broader application. Herein, we proposed a fingerprinting FISH (FinFISH) strategy driven by DNA self-assembly for multiplexed pathogenic bacteria detection. Using respiratory pathogens as representative models, FinFISH employs three distinct fluorophores in combinatorial labeling to generate identifiable fluorescent fingerprints for each species. In this work, FAM, Cy3, and Cy5 were selected as the fluorescent reporters because they represent well-established fluorophore combinations for multicolor imaging and combinatorial encoding, with minimal spectral overlap under standard fluorescence microscopy conditions. This strategy enables pathogen detection far beyond the limitations imposed by fluorescence channel numbers, effectively overcoming the throughput bottleneck of conventional imaging systems while offering high scalability for further expansion. Additionally, a custom-designed enclosed chip featuring multichannel reaction chambers improves parallel sample processing and simplifies experimental operation. Experimental results demonstrated that FinFISH not only performed well in identifying pathogenic bacteria within simulated sputum and urine samples but also proved applicable to clinical samples. Moreover, FinFISH provides additional semiquantitative insights into mixed infections. With future integration of expanded probe design and artificial intelligence (AI)-assisted analysis, FinFISH has the potential to advance clinical pathogenic bacteria diagnostics, microbial colocalization studies, and spatial analysis of intratumoral bacteria.

The aim of this study was to compare, during milking, the bacteriomes of goat milk from farms in Mexico representing traditional and semi-intensive production systems. Metagenomic DNA was isolated from pooled milk samples collected at different milking stages, and following 16S rRNA-targeted sequencing, alpha (Shannon H’ and Simpson D) and beta (Bray–Curtis) diversity indices were calculated. Within the semi-intensive system, fore-stripping showed lower diversity (H’ = 1.5 vs. H’ = 4.0) but greater evenness (D = 0.5 vs. D = 0.8) than the milking stage. In contrast, no differences between stages in the traditional system were observed. The Bray–Curtis index revealed that the use of the semi-intensive system explained 99.4% of the variability, while the traditional system accounted for only 0.5%. In the semi-intensive system, fore-stripping was dominated by Mesoplasma (51.9%) and Staphylococcus (42.1%), whereas Enterococcus (27.2%) and Lactococcus (18.5%) prevailed during milking. Meanwhile, in the traditional system, Pseudomonas (46.9% and 22.7) and Lactococcus (22.7% and 29.2%) predominated in both stages. Management practices strongly influence the microbiological profile of milk, leading to changes in not only the diversity and abundance of pathogenic bacteria but also in the presence of beneficial lactic acid bacteria and, hence, the overall expected milk quality.

PFOA and a dash of aluminum: The perfect recipe for growing drug-resistant biofilms in urban water supply.

The vaginal microbiota of a healthy woman is a unique microecosystem comprising more than 300 bacterial species. The main vaginal bacteria of a healthy woman are Lactobacillus spp. (90–95%), with the most common being L. crispatus, L. iners, L. jensenii, and L. gasseri. Based on the dominant Lactobacillus species, five types of communities are distinguished: CST I, II, III, IV, and V. Lactobacilli ensure a normal vaginal pH (3.8–4.4) and inhibit the growth of other microorganisms, particularly Escherichia coli, Trichomonas vaginalis, Gardnerella vaginalis, Prevotella bivia, etc. Different lactobacilli species produce different levels of reactive oxygen species. Many studies demonstrated a close relationship between vaginal microbiota disturbance and the development of malignancies, particularly cervical, vagina, and vulvar cancer. This review analyzes published studies, which showed a positive correlation between a low content of lactobacilli, an increase in the abundance of pathogenic bacteria, and cancer of the anogenital region; the association between some bacteria, the persistence of the human papillomavirus, and the development of dysplasia of the lower reproductive system was demonstrated. Timely diagnosis and treatment of vulvovaginal infections and vaginal dysbiosis would reduce the risk of human papillomavirus persistence and, consequently, the development of dysplasia and cancers of the lower genital tract.

Background: Diarrhea-predominant irritable bowel syndrome (D-IBS) is a clinically common functional intestinal disease, classified into “diarrhea,” “abdominal pain,” and “depression syndrome” categories according to traditional Chinese medicine (TCM). The exact pathogenesis of D-IBS is still not fully understood. Gut microbiota regulates gastrointestinal nerve, endocrine, and immune functions and maintains gastrointestinal homeostasis through interaction with the brain–gut axis. In this study, we assessed the changes in gut microbiota in a D-IBS rat model with liver depression, spleen deficiency, and liver depression and spleen deficiency syndrome. We also discussed the biological basis of liver depression and spleen deficiency syndrome and the associations among the three syndromes from the perspective of gut microbiota.Methods: Ninety rats were divided into nine groups randomly: normal group (ZC), spleen deficiency syndrome groups (four PX groups), liver depression syndrome groups (two GY groups), and liver depression and spleen deficiency syndrome groups (two GYPX groups). The abdominal wall withdrawal reflex (AWR) test detected visceral sensitivity, while changes in gut microbiota were analyzed using 16S rRNA sequencing.Results: The visceral sensitivity of rats in the model group was significantly higher than that in the ZC group, and the visceral sensitivity of the GYPX groups was significantly higher compared to the PX and GY groups. 16S rRNA sequencing analysis showed that the D-IBS model gut microbiota's species number, alpha diversity, and beta diversity were changed; the Bacteroidota increased, and the Firmicutes decreased in the model group. The abundance of pathogenic bacteria, such as Bacteroidales, significantly increased in the GYPX groups compared to other groups.Conclusion: Oral administration of senna combined with restraint stress had different effects on visceral hypersensitivity, gut microbiota composition, and metabolic pathways in rats with D-IBS liver depression and spleen deficiency syndrome, and the liver depression factors play an important role in the pathogenesis of liver depression and spleen deficiency syndrome in D-IBS.

High viscosity of alginate means a relatively low substrate concentration, which limits the efficiency of hydrolysis, resulting in one of the main challenges for the large-scale production of alginate oligosaccharides (AOS). In this study, a pilot-scale degradation product (PSDP) of the recombinant enzyme 102C300C-Vgb was produced for the first time at a substrate concentration of up to 20% sodium alginate. The optimal conditions for SA digestion were: enzyme dosage of 25 U/g, enzymatic temperature of 45 °C, enzymatic pH of 7.0, and enzymatic time of 24 h. Under these conditions, the yield of enzymatic hydrolysis was consistently in the range of 69% to 70%. The average molecular weight (Mw) of PSDP was 1496.36 Da, mainly containing oligosaccharides with degrees of polymerization ranging from 2 to 4. The low-Mw PSDP was subsequently applied in the diet of sea cucumber Stichopus japonicus. The results showed that the body wall weight of S. japonicus increased significantly after 40 days of feeding with a 0.09% PSDP-supplemented diet. Furthermore, PSDP-supplemented diet significantly increased the thickness of the serosal and submucosal layers and the width folds of mucosa of the sea cucumber gut. The abundance of pathogenic bacteria was reduced effectively, and that of beneficial bacteria increased significantly after being fed with PSDP. The results demonstrated that PSDP can serve as a digestive health enhancer for sea cucumbers, promoting their healthy growth.

The gut microbiome’s role in the pathogenesis of hematologic malignancies is actively being explored; yet studies on chronic lymphocytic leukemia (CLL) are limited. Using the Eμ-TCL1 murine model of CLL, we identify a unique and dysbiotic disease-associated gut microbiome that develops in mice over time. Leukemic mice show an increase in abundance of pathogenic bacteria, specifically members of the Proteobacteria phylum. We found that this dysbiotic microenvironment is associated with CLL involvement within the intestinal tract and high levels of markers indicative of altered tight junction permeability (e.g., claudin-2, soluble CD14, and zonulin). Moreover, utilizing the syngeneic model of CLL in tandem with an antibiotic-mediated microflora ablation approach, we found that leukemic mice receiving microflora-ablating antibiotics show marked changes to the gut microbiome and a delayed disease onset compared with mice receiving antibiotic-free water. Immunophenotyping of murine spleens showed that this delay in disease was accompanied by more tumor-reactive CD8+ T cells that coexpressed fewer inhibitory receptors (e.g., PD-1, LAG-3, and TIM-3). These findings confirm that a dysbiotic gut microbiome develops during CLL disease and demonstrate unique intestinal involvement and potential immune dysregulation occurring during CLL progression that may be influencing the overall microbial signature.Significance:There is a growing appreciation for the gut microbiome’s role in hematologic malignancies. Despite this, its role in CLL remains obscure. This study demonstrates a dysbiotic microbiome within CLL that may contribute to further intestinal and immune dysregulation present during CLL progression.

BackgroundNeonatal mortality remains high in many low- and middle-income countries (LMICs), with neonatal sepsis and antimicrobial resistance (AMR) posing significant threats to newborns, particularly in sub-Saharan Africa (SSA). Tanzania is among the countries with the highest neonatal mortality rates, with sepsis being a major contributor. Gut dysbiosis has been identified as a risk factor for neonatal sepsis in high-income countries, due to factors like abundance of pathogenic bacteria, decrease in microbiome diversity, intestinal barrier defects and bacterial translocation. Understanding gut dysbiosis in the local setting and its role in sepsis development may offer new prevention strategies, such as probiotics for high-risk preterm infants.ObjectivesThis prospective neonatal cohort, established at Muhimbili National Hospital (MNH) in Dar es Salaam, Tanzania, aims to analyze the gut microbiome of preterm infants and explore associations with neonatal late-onset sepsis (LOS). Additionally, data on bacterial pathogens of bloodstream infections and AMR prevalence will be identified. Secondary endpoints include clinical LOS, sepsis-related death, death from any cause, and hospital discharge outcomes.MethodsEligible preterm neonates (28 + 0 to <34 weeks of gestational age, birth weight ≥ 1000g) will be recruited with maternal consent. Socio-demographic and clinical data, microbiological details of blood pathogens, and a set of fresh frozen fecal samples during the 28 days observation period will be collected. The study targets a sample size of 1350 participants and we expect 72–135 culture-proven LOS during a study period of 18 months. Fecal samples will undergo next-generation sequencing (NGS) to analyze microbial community functions in comparison to matched controls.DiscussionThis collaborative study between universities in Tanzania and Germany, aims to analyze the neonatal microbiome in relation to sepsis development and AMR of blood culture isolates to enhance neonatal sepsis care, improve diagnostics and treatment. The project will offer insights into potential therapeutic strategies for the future, promote academic exchange, capacity building and research on African microbiomes.

The SARS-CoV-2 virus and its rapid spread have made it a global health concern. The aim of this was to investigate the microbial and metabolic faecal profiles of paediatric patients hospitalised for COVID-19 to try to identify biomarkers of predisposition to severity. The study included 16 patients (aged 4-14 years old) from six different Spanish hospitals and 20 age-matched healthy controls. The gut microbiota was characterised by sequencing of 16S rDNA amplicons and internal transcribed space amplicons, while the metabolic profile was analysed by liquid chromatography high resolution mass spectrometry. A different microbial profile was observed between patients and controls, with a significantly higher abundance of sequences belonging to the phyla Bacteroidota and Pseudomonadota in patients. A different metabolic profile was observed between the two groups. Non-infected children had higher faecal levels of vitamins such as niacin, thiamine, and vitamin D3 derivatives, which were negatively correlated with the abundance of pathogenic bacteria, such as members of Enterobacteriaceae. Hospitalisation due to SARS-CoV-2 infection in children was associated with changes in the gut microbiota and an altered metabolomic profile. For the first time, several relevant biological compounds were found to be reduced in the faeces of children hospitalised with COVID-19 compared to healthy controls.

Partial RAG deficiency (pRD) can manifest with systemic and tissue-specific immune dysregulation, with inflammatory bowel disease (IBD) in 15% of the patients. We aimed at identifying the immunopathological and microbial signatures associated with IBD in patients with pRD and in a mouse model of pRD (Rag1w/w) with spontaneous development of colitis. pRD patients with IBD and Rag1w/w mice showed a systemic and colonic Th1/Th17 inflammatory signature. Restriction of fecal microbial diversity, abundance of pathogenic bacteria, and depletion of microbial species producing short-chain fatty acid were observed, which were associated with impaired induction of lamina propria peripheral Treg cells in Rag1w/w mice. The use of vedolizumab in Rag1w/w mice and of ustekinumab in a pRD patient were ineffective. Antibiotics ameliorated gut inflammation in Rag1w/w mice, but only bone marrow transplantation (BMT) rescued the immunopathological and microbial signatures. Our findings shed new light in the pathophysiology of gut inflammation in pRD and establish a curative role for BMT to resolve the disease phenotype.

Selenium-enriched Lactiplantibacillus plantarum alleviates alkalinity stress-induced selective hepatic insulin resistance in common carp.

Endophthalmitis is a serious eye infection that can occur after intravitreal injections. Topical antibiotics are frequently used as a preventative measure, but their impact on the conjunctival microbiome is not fully understood. Conjunctival swabs were collected from 33 eyes of 33 patients undergoing intravitreal injections, both before and after a 3-day course of prophylactic topical levofloxacin 0.5%. Conjunctival microbiome analysis was conducted using 16S rRNA sequencing on the Illumina MiSeq platform. Bioinformatics processing identified unique amplicon sequence variants (ASVs) to evaluate microbial diversity and community composition. Alpha and beta diversity indices were analyzed. Topical levofloxacin treatment resulted in no significant change in alpha diversity indices, including Shannon index, Chao1, Shannon, PD whole tree, and observed ASVs, indicating stable microbial richness and evenness. In contrast, beta diversity analysis, assessed through Bray-Curtis dissimilarity, revealed significant differences in microbial composition between pre- and post-treatment samples. These changes included a decrease in the abundance of Staphylococcus and Bacillus species and an increase in the abundance of Streptococcus, Haemophilus, and Neisseria. Although prophylactic topical levofloxacin was found to alter the conjunctival microbiome and showed inconsistent effects on the abundance of pathogenic bacteria, its clinical effectiveness as a preventative measure against endophthalmitis remains inconclusive. Further studies are needed to clarify its role in infection prevention.

The vaginal microbiota plays an important role in reproductive health, especially in the process of artificial insemination. The imbalance of microbiota may affect pregnancy outcomes. Therefore, this study aims to explore the composition of vaginal microbiota and its impact on artificial insemination pregnancy outcomes, combined with microscopic images and PCR fluorescence methods, in order to provide scientific basis for improving the pregnancy outcomes of husband sperm artificial insemination. This study included female patients who underwent artificial insemination with husband's sperm and collected reproductive tract samples. By observing the microbial community morphology in the sample under a microscope and analyzing microbial DNA using PCR fluorescence method, the microbial ecological status is evaluated. Among women with successful pregnancy, the proportion of beneficial bacteria (such as lactic acid bacteria) is higher, while the abundance of pathogenic bacteria is significantly reduced. Specific microbial markers detected by PCR fluorescence method are positively correlated with pregnancy rate. Therefore, the microecological state of the female reproductive tract has a significant impact on the pregnancy outcome of artificial insemination. Maintaining a good microecological balance, especially increasing the proportion of beneficial bacteria, can help improve the success rate of artificial insemination.

Deep sequencing technologies can be used to evaluate pathogens in environmental samples. The objective of this study was to use this technology to evaluate Sargassum samples that were characterized by different stranding times, one classified as short-term stranded (STS) and another classified as long-term stranded (LTS) Sargassum. Nine replicates of the STS Sargassum showed a range in Shannon diversity between 3.04 and 3.38, whereas 11 replicates of LTS showed a range between 1.17 and 1.22. Nonmetric multidimensional scaling analysis showed distinct differences between STS and LTS by about 0.5 coordinate units, while variations within replicates ranged by 0.1 coordinate units. Comparison between the two Sargassum samples showed a greater abundance of Vibrio species in STS Sargassum when compared to LTS Sargassum, with major pathogenic forms observed for Vibrio alginolyticus (11%), Vibrio parahaemolyticus (1.5%), and Vibrio vulnificus (0.29%). Additional known human pathogens were observed, including Listeria monocytogenes, Legionella pneumophila, and Staphylococcus aureus, as well as the presence of gut commensals and fecal coliforms. Overall results show that deep sequencing analysis of these environmental samples was reproducible. Given the abundance of pathogenic bacteria, more research is needed to evaluate the risk of disease transmission as Sargassum strands and decomposes on coastal beaches.

Introduction. Alcohol dependence (AD) and sleep disturbance (SD) independently affect gut microbiota, potentially disrupting the circadian rhythm of the microbiota and the host. However, the impact of SD on the composition and rhythmicity of gut flora in AD patients remains poorly understood.Gap Statement. Characteristics of gut flora and diurnal oscillations in AD patients experiencing SD are unknown.Aim. This study aims to explore alterations in gut flora and diurnal oscillations in AD patients experiencing SD.Methodology. Thirty-two AD patients and 20 healthy subjects participated, providing faecal samples at 7 : 00 AM, 11 : 00 AM, 3 : 00 PM and 7 : 00 PM for gut microbiota analysis using 16S rDNA sequencing. AD patients were further categorized into those with poor sleep (ADwPS) and those with good sleep (ADwGS) for further analyses.Results. The ADwPS group demonstrated elevated levels of anxiety, depression and withdrawal severity compared to the ADwGS group (all P<0.05). The β-diversity of gut microbiota in the ADwPS group differed from that in the ADwGS group (P<0.05). Bacterial abundances at various taxonomic levels, including Cyanobacteria and Pseudomonadales, differed between the ADwPS and ADwGS groups (all P<0.05). Utilizing unweighted UniFrac analysis, the β-diversity of gut microbiota in the ADwPS group demonstrated robust diurnal oscillation (P<0.05), whereas this pattern was statistically insignificant in the ADwGS group. Notably, the abundance of pathogenic bacteria like Pseudomonadales and Pseudomonadaceae exhibited marked diurnal fluctuation in the ADwPS group (all P<0.05).Conclusion. SD in AD patients extends beyond alcohol-induced alterations, impacting gut microbiota composition, function and diurnal oscillation patterns. This highlights its add-on influence, supplementing AD-related changes.

Identifying changes in vaginal fluid using SERS: Advancing diagnosis of vulvovaginal candidiasis

Metagenomic analysis of pathogenic bacteria and virulence factor genes in coastal sediments from highly urbanized cities of India

Antibiotic resistance genes (ARGs) are an emerging issue which are receiving increasing concerns in drinking water safety. However, the factors (e.g. treatment processes and water quality) affecting the removal efficiency of ARGs in the drinking water treatment plants (DWTPs) is still unclear. This work investigated the ARG profiles in each treatment process of two DWTPs located in a northwest Chinese city. The results showed that tetracycline and sulfonamide resistance genes were predominant among the 14 targeted ARGs. After the treatment, the Z water treatment plant which demonstrated a higher removal rate of ARGs (ranging from 50 to 80%), compared to the S plant (50-75%). And the average removal rate of tetracycline resistance genes (tetA, tetG, tetQ, tetX) was about 49.18% (S plant) and 67.50% (Z plant), as well as the removal rate of 64.2% and 72.9% for sulfonamide resistance (sul1 and sul2) at S and Z water plants, respectively. It was found that the relative abundance of main microbial communities (such as Bacteroidota, Actinobacteria, Verrucomicrobiota, Roseomonas), α-diversity index, as well as the abundance of pathogenic bacteria were all significantly reduced after different treatment processes. Network co-occurrence analysis revealed that Methylocystis possibly was the potential host for most ARGs, and sul1 was found across a broad spectrum of microorganisms in the drinking water environment. Adonis analysis showed that heavy metals and microbial communities explain solely 44.1% and 35.7% of variances of ARGs within DWTPs. This study provides insights into the contamination status and removal efficiencies of ARGs in DWTPs, offering valuable references for future studies on ARG removal, propagation, and diffusion patterns in drinking water treatment.

High temperature aggravated hypoxia-induced intestine toxicity on juvenile Chinese mitten crab (Eriocheir sinensis)

Land use/cover drive functional patterns of bacterial communities in sediments of a subtropical river, China