The bladder microbiome, metabolome, cytokines, and phenotypes in patients with systemic lupus erythematosus

Background and aims: Emerging studies reveal a unique bacterial community in the human bladder, with alteration of composition associated to disease states. Systemic lupus erythematosus (SLE) is a complex autoimmune disease that is characterized by frequent impairment of the kidney. Here, we explored the bladder microbiome, metabolome, and cytokine profiles in SLE patients, as well as correlations between microbiome and metabolome, cytokines, and disease profiles. Methods and materials: We recruited a cohort of 50 SLE patients and 50 individually matched asymptomatic controls. We used transurethral catheterization to collect urine samples, 16S rRNA gene sequencing to profile bladder microbiomes, and LC-MS/MS to perform untargeted metabolomic profiling. Results: Compared to controls, SLE patients possessed a unique bladder microbial community and increased alpha diversity. These differences were accompanied by differences in urinary metabolomes, cytokines, and patients’ disease profiles. The SLE-enriched genera, including Bacteroides, were positively correlated with several SLE-enriched metabolites, including olopatadine. The SLE-depleted genera, such as Pseudomonas, were negatively correlated to SLE-depleted cytokines, including IL-8. Alteration of the bladder microbiome was associated with disease profile. For example, the genera Megamonas and Phocaeicola were negatively correlated with serum complement C3, and Streptococcus was positively correlated with IgG. Conclusions: Our present study reveals associations between the bladder microbiome and the urinary metabolome, cytokines, and disease phenotypes. Our results could help identify biomarkers for SLE.

Viral dysbiosis in children with new-onset celiac disease

Viruses are common components of the intestinal microbiome, modulating host bacterial metabolism and interacting with the immune system, with a possible role in the pathogenesis of immune-mediated diseases such as celiac disease (CeD). The objective of this study was to characterize the virome profile in children with new-onset CeD. We used metagenomic analysis of viral DNA in mucosal and fecal samples from children with CeD and controls and performed sequencing using the Nextera XT library preparation kit. Abundance log2 fold changes were calculated using differential expression and linear discriminant effect size. Shannon alpha and Bray–Curtis beta diversity were determined. A total of 40 children with CeD and 39 controls were included. We found viral dysbiosis in both fecal and mucosal samples. Examples of significantly more abundant species in fecal samples of children with CeD included Human polyomavirus 2, Enterobacteria phage mEpX1, and Enterobacteria phage mEpX2; whereas less abundant species included Lactococcus phages ul36 and Streptococcus phage Abc2. In mucosal samples however, no species were significantly associated with CeD. Shannon alpha diversity was not significantly different between CeD and non-CeD groups and Bray–Curtis beta diversity showed no significant separation between CeD and non-CeD samples in either mucosal or stool samples, whereas separation was clear in all samples. We identified significant viral dysbiosis in children with CeD, suggesting a potential role in the pathogenesis of CeD indicating the need for further studies.

Comparative analysis of DNA extraction and PCR product purification methods for cervicovaginal microbiome analysis using cpn60 microbial profiling

Background The microbiota of the lower female genital tract plays an important role in women’s health. Microbial profiling using the chaperonin60 (cpn60) universal target (UT) improves resolution of vaginal species associated with negative health outcomes compared to the more commonly used 16S ribosomal DNA target. However, the choice of DNA extraction and PCR product purification methods may bias sequencing-based microbial studies and should be optimized for the sample type and molecular target used. In this study, we compared two commercial DNA extraction kits and two commercial PCR product purification kits for the microbial profiling of cervicovaginal samples using the cpn60 UT. Methods DNA from cervicovaginal secretions and vaginal lavage samples as well as mock community standards were extracted using either the specialized QIAamp DNA Microbiome Kit, or the standard DNeasy Blood & Tissue kit with enzymatic pre-treatment for enhanced lysis of gram-positive bacteria. Extracts were PCR amplified using well-established cpn60 primer sets and conditions. Products were then purified using a column-based method (QIAquick PCR Purification Kit) or a gel-based PCR clean-up method using the QIAEX II Gel Extraction Kit. Purified amplicons were sequenced with the MiSeq platform using standard procedures. The overall quality of each method was evaluated by measuring DNA yield, alpha diversity, and microbial composition. Results DNA extracted from cervicovaginal samples using the DNeasy Blood and Tissue kit, pre-treated with lysozyme and mutanolysin, resulted in increased DNA yield, bacterial diversity, and species representation compared to the QIAamp DNA Microbiome kit. The column-based PCR product purification approach also resulted in greater average DNA yield and wider species representation compared to a gel-based clean-up method. In conclusion, this study presents a fast, effective sample preparation method for high resolution cpn60 based microbial profiling of cervicovaginal samples.

Enumeration of Citrus endophytic bacterial communities based on illumine metagenomics technique

Citrus is a valuable crop in Pakistan because it is rich in vitamin C and antioxidants. Huanglongbing (HLB) has an influence on citrus production around the world caused by a bacterium “ Candidatus liberibacter asiaticus ” (CLas), africanus and americanus. The structure and diversity of bacterial species in various ecosystems can be quickly examined using NGS. This approach is considerably quicker and more precise than outdated methods. Healthy or citrus greening infected leaf samples of Grapefruit, Citrus aurantifolia , and Citrus reticulata Blanco was used for diversity analysis. In this study high throughput, NGS technique was used to access the population of both cultivable and non-cultivable bacterial endophytes from citrus leaves, by using PCR amplicons of 16S rDNA sequences (V5–V7 regions) with Illumina Hi seq. As a result, a total number of 68,722 sequences were produced from the test samples. According to the NGS-based diversity classification, the most common genera of exploited bacterial endophytes were Proteobacteria, Firmicutes, Bacteroides, Cyanobacteria, and Actinobacteria. Citrus aurantifolia and Citrus paradisi showed almost equal diversity, whereas Citrus reticulata Blanco had a higher proportion of Proteobacteria and Cyanobacteria in their leaves. To determine alpha diversity (AD), additional data was analyzed using statistical indices such as Shannon, Chao1, and Simpson. According to the inverse Simpson diversity index, the abundance of the microbial population in six different citrus samples was 0.48, 0.567, and 0.163, respectively. The metagenomics of microbiota in plant tissues was successfully recorded by NGS technology, which can help us learn more about the interactions between plants and microbes. This research is the first step toward a better understanding of 16SrRNA-based metagenomics from citrus in Pakistan using Illumina (Hi seq) Technology.

Sub-Chronic Difenoconazole Exposure Induced Gut Microbiota Dysbiosis in Mice

Difenoconazole (DIF) is a widely separated triazole fungicide in many countries. The excessive usage of DIF increases the high volume of residues in agriculture production and water bodies. Some previous studies demonstrated the toxic effects of DIF on non-target animals, however, there were still some gaps in the knowledge of the potential hazards of DIF to mammals and human health. Herein, 7-week-old male mice were exposed to 30 and 100 mg/kg/day DIF for 14 and 56 days. We observed that 56 days of DIF exposure decreased the colonic mucus expression of alcin blue-periodic acid-schiff (AB-PAS) stain and the immunochemical stain of muc2 protein. The transcript levels of mucin protein (muc1, muc2 and muc3) decreased significantly in the gut of mice followed 56 days of 100 mg/kg/day DIF exposure. In addition, the gut microbiota composition was also affected after 14 or 56 days of DIF exposure. Although the mucus expression after 14 days of DIF exposure only decreased slightly, the gut microbiota composition compared with the control group was changed significantly. Moreover, the DIF-30 and DIF-100 caused respectively different changes on the gut microbiota. The relative abundance of Bacteroidetes decreased significantly after 14 days and 56 days of DIF exposure. After 14 days of DIF exposure, there were 35 and 18 differential genera in the DIF-30 and DIF-100 group, respectively. There were 25 and 32 differential genera in the DIF-30 and DIF-100 group after 56 days of exposure, respectively. Meanwhile, the alpha diversity indexes, including observed species, Shannon, Simpson, Chao1 and ACE, in gut microbiota decreased significantly after 56 days of DIF exposure. Interestingly, the relative abundance of Akkermansia increased significantly after 56 days of 100 mg/kg/d DIF exposure. Although Akkermansia was considered as one probiotic, the phenomenon of dramatic Akkermansia increase with the decrease in gut microbiota diversity needed further discussion. These results provided some new insights on how DIF exposure impacts the mucus barrier and induces gut microbiota dysbiosis.

Microbial Responses to the Reduction of Chemical Fertilizers in the Rhizosphere Soil of Flue-Cured Tobacco

The overuse of chemical fertilizers has resulted in the degradation of the physicochemical properties and negative changes in the microbial profiles of agricultural soil. These changes have disequilibrated the balance in agricultural ecology, which has resulted in overloaded land with low fertility and planting obstacles. To protect the agricultural soil from the effects of unsustainable fertilization strategies, experiments of the reduction of nitrogen fertilization at 10, 20, and 30% were implemented. In this study, the bacterial responses to the reduction of nitrogen fertilizer were investigated. The bacterial communities of the fertilizer-reducing treatments (D10F, D20F, and D30F) were different from those of the control group (CK). The alpha diversity was significantly increased in D20F compared to that of the CK. The analysis of beta diversity revealed variation of the bacterial communities between fertilizer-reducing treatments and CK, when the clusters of D10F, D20F, and D30F were separated. Chemical fertilizers played dominant roles in changing the bacterial community of D20F. Meanwhile, pH, soil organic matter, and six enzymes (soil sucrase, catalase, polyphenol oxidase, urease, acid phosphatase, and nitrite reductase) were responsible for the variation of the bacterial communities in fertilizer-reducing treatments. Moreover, four of the top 20 genera (unidentified JG30-KF-AS9, JG30-KF-CM45, Streptomyces, and Elsterales) were considered as key bacteria, which contributed to the variation of bacterial communities between fertilizer-reducing treatments and CK. These findings provide a theoretical basis for a fertilizer-reducing strategy in sustainable agriculture, and potentially contribute to the utilization of agricultural resources through screening plant beneficial bacteria from native low-fertility soil.

Consistent changes in the intestinal microbiota of Atlantic salmon fed insect meal diets

Background Being part of fish's natural diets, insects have become a practical alternative feed ingredient for aquaculture. While nutritional values of insects have been extensively studied in various fish species, their impact on the fish microbiota remains to be fully explored. In an 8-week freshwater feeding trial, Atlantic salmon (Salmo salar) were fed either a commercially relevant reference diet or an insect meal diet wherein black soldier fly (Hermetia illucens) larvae meal comprised 60% of total ingredients. Microbiota of digesta and mucosa origin from the proximal and distal intestine were collected and profiled along with feed and water samples. Results The insect meal diet markedly modulated the salmon intestinal microbiota. Salmon fed the insect meal diet showed similar or lower alpha-diversity indices in the digesta but higher alpha-diversity indices in the mucosa. A group of bacterial genera, dominated by members of the Bacillaceae family, was enriched in salmon fed the insect meal diet, which confirms our previous findings in a seawater feeding trial. We also found that microbiota in the intestine closely resembled that of the feeds but was distinct from the water microbiota. Notably, bacterial genera associated with the diet effects were also present in the feeds. Conclusions We conclude that salmon fed the insect meal diets show consistent changes in the intestinal microbiota. The next challenge is to evaluate the extent to which these alterations are attributable to feed microbiota and dietary nutrients, and what these changes mean for fish physiology and health.

Reduced calorie diet combined with NNMT inhibition establishes a distinct microbiome in DIO mice

Treatment with a nicotinamide N-methyltransferase inhibitor (NNMTi; 5-amino-1-methylquinolinium) combined with low-fat diet (LD) promoted dramatic whole-body adiposity and weight loss in diet-induced obese (DIO) mice, rapidly normalizing these measures to age-matched lean animals, while LD switch alone was unable to restore these measures to age-matched controls in the same time frame. Since mouse microbiome profiles often highly correlate with body weight and fat composition, this study was designed to test whether the cecal microbiomes of DIO mice treated with NNMTi and LD were comparable to the microbiomes of age-matched lean counterparts and distinct from microbiomes of DIO mice maintained on a high-fat Western diet (WD) or subjected to LD switch alone. There were minimal microbiome differences between lean and obese controls, suggesting that diet composition and adiposity had limited effects. However, DIO mice switched from an obesity-promoting WD to an LD (regardless of treatment status) displayed several genera and phyla differences compared to obese and lean controls. While alpha diversity measures did not significantly differ between groups, beta diversity principal coordinates analyses suggested that mice from the same treatment group were the most similar. K-means clustering analysis of amplicon sequence variants by animal demonstrated that NNMTi-treated DIO mice switched to LD had a distinct microbiome pattern that was highlighted by decreased Erysipelatoclostridium and increased Lactobacillus relative abundances compared to vehicle counterparts; these genera are tied to body weight and metabolic regulation. Additionally, Parasutterella relative abundance, which was increased in both the vehicle- and NNMTi-treated LD-switched groups relative to the controls, significantly correlated with several adipose tissue metabolites’ abundances. Collectively, these results provide a novel foundation for future investigations.

Age and injury size influence the magnitude of fecal dysbiosis in adult burn patients

Clinical studies have demonstrated that age ≥ 50 years old is an independent risk factor associated with poor prognosis after burn injury, the second leading cause of traumatic injuries in the aged population. While mechanisms driving age-dependent post-burn mortality are perplexing, changes in the intestinal microbiome however may contribute to the heightened, dysregulated systemic response seen in aging burn patients. The fecal microbiome from 22 patients admitted to a verified burn center from July 2018 to February 2019 were stratified based on age of 50 years and total burn surface area (TBSA) size of ≥10%. Significant differences (P = 0.014) in overall microbiota community composition (i.e., beta diversity) were measured across the four patient groups, young <10% TBSA, young ≥10% TBSA, older <10% TBSA, and older ≥10% TBSA. Differences in beta diversity were driven by %TBSA (P = 0.013) and trended with age (P = 0.087). Alpha diversity components, richness, evenness, and Shannon diversity were measured. We observed significant differences in bacterial species evenness (P = 0.0023) and Shannon diversity (P = 0.0033) between the groups. There were significant correlations between individual bacterial species and levels of SCFA. Specifically, levels of fecal butyrate correlated with the presence of Enterobacteriaceae, an opportunistic gut pathogen, when elevated in burn patients lead to worsen outcomes. Overall, our findings reveal that age-specific changes in the fecal microbiome following burn injuries may contribute to immune system dysregulation in patients with varying TBSA burns and potentially lead to worsen clinical outcomes with heightened morbidity and mortality.

Comparative Microbiomes of the Respiratory Tract and Joints of Feedlot Cattle Mortalities

A comparative study of microbiota of the respiratory tract and joints of bovine respiratory disease (BRD) cattle mortalities was undertaken. Nasopharynx, trachea, lung and joint samples were collected from 32 cattle that died of BRD, “cases”, and 8 that died of other causes, “controls”. Bacterial diversity was lower (p < 0.05) in the nasopharynx, trachea and lungs of cases as compared to controls. In cases, alpha-diversity (p < 0.05) was lower in the lungs and joints than the nasopharynx. Proteobacteria, Tenericutes, Bacteroidetes, Firmicutes and Actinobacteria were the most abundant phyla in all samples. Relative abundances of Mycoplasma spp. in the lung, Pasteurella spp. in the trachea and lung, and Histophilus spp. in the lung, trachea and nasopharynx of cases were higher (p < 0.001) than controls. Mycoplasma spp. comprised 20.5% of bacterial flora in the joint, 36.0% in the lung, 22.4% in the trachea and 8.8% in the nasopharynx. Mannheimia spp. (21.8%) and Histophilus spp. (10.4%) were more abundant in lungs. Cattle that died of BRD possessed less diverse respiratory microbiomes with a higher abundance of respiratory pathogens. Mycoplasma spp. were prominent members of pneumonic lungs and joints displaying septic arthritis.