Gut microbiome in hemodialysis patients treated with calcium acetate or treated with sucroferric oxyhydroxide: a pilot study

Purpose It has been proved that the gut microbiome is altered in patients with chronic kidney disease. This contributes to chronic inflammation and increases cardiovascular risk and mortality, especially in those undergoing hemodialysis. Phosphate binders may potentially induce changes in their microbiome. This trial aimed to compare the changes in the gut microbiome of hemodialysis patients treated with calcium acetate to those treated with sucroferric oxyhydroxide. Methods Twelve hemodialysis patients were distributed to receive calcium acetate or sucroferric oxyhydroxide for 5 months. Blood samples (for biochemical analysis) and stool samples (for microbiome analysis) were collected at baseline, 4, 12, and 20 weeks after treatment initiation. Fecal DNA was extracted and a 16S rRNA sequencing library was constructed targeting the V3 and V4 hypervariable regions. Results Regarding clinical variables and laboratory parameters, no statistically significant differences were observed between calcium acetate or sucroferric oxyhydroxide groups. When analyzing stool samples, we found that all patients were different (p = 0.001) among themselves and these differences were kept along the 20 weeks of treatment. The clustering analysis in microbial profiles grouped the samples of the same patient independently of the treatment followed and the stage of the treatment. Conclusion These results suggest that a 5-month treatment with either calcium acetate or sucroferric oxyhydroxide did not modify baseline diversity or baseline bacterial composition in hemodialysis patients, also about the high-variability profiles of the gut microbiome found among these patients.

Distribution of ermB, ermF, tet(W), and tet(M) Resistance Genes in the Vaginal Ecosystem of Women during Pregnancy and Puerperium

The inhabitants of the vaginal ecosystem can harbor genetic determinants conferring antimicrobial resistance. However, detailed data about the distribution of resistance genes in the vaginal microbiome of pregnant women are still lacking. Therefore, we assessed the presence of macrolide (i.e., erm genes) and tetracycline (i.e., tet genes) resistance markers in the vaginal environment of Caucasian women at different gestational ages. Furthermore, the detection of resistance genes was related to the composition of the vaginal microbiota. A total of 228 vaginal samples, collected at different trimesters of pregnancy or during the puerperium, were tested for the presence of ermB, ermF, tet(W), and tet(M) by in-house end-point PCR assays. The composition of the vaginal microbiota was assessed through a microscopic evaluation (i.e., Nugent score) and by means of sequencing V3–V4 hypervariable regions of the bacterial 16 rRNA gene. Overall, the most detected resistance gene was tet(M) (76.7%), followed by ermB (55.2%). In 17% of women, mainly with a ‘normal’ vaginal microbiota, no resistance genes were found. Except for tet(W), a significant correlation between the positivity of resistance genes and a dysbiotic vaginal status (i.e., bacterial vaginosis (BV)) was noticed. Indeed, samples positive for at least one resistance determinant were characterized by a decrease in Lactobacillus spp. and an increase of BV-related genera (Prevotella, Gardnerella, Atopobium, Sneathia). A high predominance of vaginal Lactobacillus spp. (>85%) was associated with a lower risk of tet(W) gene detection, whereas the presence of Megasphaera (>1%) increased the risk of positivity for all analyzed genes. Different types of vaginal microbiota are associated with peculiar resistance profiles, being a lactobacilli-dominated ecosystem poor in or free of resistance genes. These data could open new perspectives for promoting maternal and neonatal health.

The Microbiota Profile Analysis of Combined Periodontal-Endodontic Lesions Using 16S rRNA Next-Generation Sequencing

Objective. The primary aim of this investigation was to analyze the microbiome in patients with combined periodontal-endodontic lesions. Method. Patients with loose and/or painful teeth referred for treatment from March 2020 to December 2020 in the First People’s Hospital of Jinzhong were recruited. Samples were collected from teeth diagnosed as chronic periodontics (PE), ulcerative pulpitis (PU), and retrograde pulpitis (RE). Genomic DNA was extracted. The quantitative polymerase chain reaction, targeting the 16S ribosomal RNA (rRNA), was adopted for the quantification of bacteria. Then, the V3-V4 hypervariable regions of the 16S rRNA gene were amplified and subjected to next-generation sequencing. The statistical analysis was performed by R software (V3.5.1). Results. A total of 57 qualified samples were collected from 48 patients and analyzed (7 PE, 21 PU, and 19 RE). By linear discriminant analysis effect size, Kingella and Barnesiella were significantly increased in the periodontal pocket of retrograde pulpitis (RE-PE), compared with PE. The relative abundance of Clostridiales Incertae Sedis XI, Fusobacteriaceae, Fusobacterium, Parvimonas, Micrococcaceae, and Rothia was significantly increased in the pulp of retrograde pulpitis (RE-PU) than PU and RE-PE. Prevotella, Leptotrichia, Porphyromonas, Streptococcus, and Fusobacterium are consistently at a high abundance, across PU, RE-PE, and RE-PU. Conclusion. The current study highlighted the evidence that a specific microbial community is associated with the occurrence of retrograde pulpitis. The microenvironment of the root canal and pulp chamber will select microbiota. This study offered insights into the pathogenesis of retrograde pulpitis.

Bacterial Endophytes As Indicators of Susceptibility To Cercospora Leaf Spot (CLS) Disease In Beta vulgaris L.

Background: The fungus Cercospora beticola causes Cercospora Leaf Spot (CLS) of sugar beet (Beta vulgaris L.). Despite the global importance of this disease, durable resistance to CLS has still not been obtained. Therefore, the development of tolerant hybrids is still a major goal for sugar beet breeding. Although recent studies have suggested that the leaf microbiome composition can offer useful predictors to assist plant breeders, this is an untapped resource in sugar beet breeding efforts. Methods: Using Ion GeneStudio S5 technology to sequence amplicons from seven 16S rRNA hypervariable regions, the most recurring endophytes discriminating CLS-symptomatic and symptomless sea beets (Beta vulgaris L.ssp. maritima) were identified. This allowed the design of taxon-specific primer pairs to quantify the abundance of the most representative endophytic species in large naturally occurring populations of sea beet and subsequently in sugar beet breeding genotypes under either CLS symptomless or infection stages using qPCR. Results: Among the screened bacterial genera, Methylobacterium and Mucilaginibacter were found to be significantly (p<0.05) more abundant in symptomatic sea beets with respect to symptomless. In cultivated sugar beet material under CLS infection, the comparison between resistant and susceptible genotypes confirmed that the susceptible genotypes hosted higher contents of the above-mentioned bacterial genera. Conclusions: These results suggest that the abundance of these species can be correlated with increased sensitivity to CLS disease. This evidence can further prompt novel protocols to assist plant breeding of sugar beet in the pursuit of improved pathogen resistance.

Diversity of Site-Specific Microbes of Occlusal and Proximal Lesions in Severe- Early Childhood Caries (S-ECC)

Background: Severe-early childhood caries (S-ECC) is a global problem of significant concern, commonly manifest as a dentinal lesion on the occlusal and proximal surfaces of the affected deciduous dentition. Although there are major ecological differences between these two niches, it is unclear whether these are reflected in the composition of their dysbiotic cariogenic microbiome. Therefore, we compared the compositional differences in the microbiota of occlusal and proximal caries lesions in S-ECC. Methods: Deep-dentine caries samples (19-occlusal and 19-proximal) from asymptomatic primary molars of children with S-ECC (n=19) belonging to caries-code 5/6, according to ICDAS classification, were evaluated. Employing two primer pools, we amplified and compared the bacterial 16S rRNA gene sequences of the seven hypervariable regions (V2 to V4 and V6 to V9) using a next-generation sequencing based assay.Results: Bray-Curtis dissimilarity data indicated that occlusal lesions had a more homogeneous microbial community structure than the proximal lesions with significant compositional differences at species level (p=0.01; R-value of 0.513). Together, the occlusal and proximal niches harbored 263 species, of which 202 (76.8%) species were common to both locales, while 49 (18.6%) and 12 (4.6%) disparate species were exclusively isolated from the proximal and occlusal niches, respectively. The most commonly found genera at both locales included Streptococcus, Prevotella, and Lactobacillus, with 33, 27, and 22 species each, respectively. In addition, Streptococcus mutans predominated in the proximal cavities (p≤0.05), as opposed to Atopobium parvulum (p=0.01) in the occlusal niches, while Vellonella alcalescens was present in similar proportions in both habitats (p≥0.05). Conclusions: Distinct differences between the caries microbiota of occlusal and proximal caries in S-ECC exist. The former niche appears to provide a habitat for a more homogeneous growth of communal microbiota than the latter. This may be due to the conditions prevalent in relatively quiescent inter-proximal regions, as opposed to the occlusal regions exposed to the ebb and flow of salivary and masticatory forces, and/or the anatomical and structural differences in the two locales. The clinical implications of these findings in terms of the rate and severity of caries progression remain to be determined.

Haplogroup Distribution of 309 Thais from Admixed Populations across the Country by HVI and HVII Sanger-Type Sequencing

The mitochondrial DNA (mtDNA) control region sequences for the hypervariable regions I (HVI) and II (HVII) of 309 Thai citizens were investigated using Sanger-type sequencing to generate an mtDNA reference dataset for forensic casework, and the haplogroup distribution within geographically proximal Asian populations was analyzed. The population sample set contained 264 distinct haplotypes and showed high haplotype diversity, low matching probability, and high powers of discrimination, at 0.9985, 0.4744%, and 0.9953, respectively, compared with previous reports. Subhaplogroup F1a showed the highest frequency in the Thai population, similar to Southeast Asian populations. The haplotype frequencies in the northern, northeastern, and southern populations of Thailand illustrate the relevance of social, religious, and historical factors in the biogeographical origin of the admixed Thai population as a whole. The HVI and HVII reference datasets will be useful for forensic casework applications, with improved genetic information content and discriminatory power compared to currently available techniques.

Long-read metagenomics of multiple displacement amplified DNA of low-biomass human gut phageomes by SACRA preprocessing chimeric reads

The human gut bacteriophage community (phageome) plays an important role in the host’s health and disease; however, the entire structure is poorly understood, partly owing to the generation of many incomplete genomes in conventional short-read metagenomics. Here, we show long-read metagenomics of amplified DNA of low-biomass phageomes with multiple displacement amplification (MDA), involving the development of a novel bioinformatics tool, SACRA, that efficiently preprocessed numerous chimeric reads generated through MDA. Using five samples, SACRA markedly reduced the average chimera ratio from 72% to 1.5% in PacBio reads with an average length of 1.8-kb. De novo assembly of chimera-less PacBio long reads reconstructed contigs of ≥ 5-kb with an average proportion of 27%, which was 1% in contigs from MiSeq short reads, thereby dramatically improving contig length and genome completeness. Comparison of PacBio and MiSeq contigs found MiSeq contig fragmentations frequently near local repeats and hypervariable regions in the phage genomes, and those caused by multiple homologous phage genomes coexisting in the community. We also developed a reference-independent method to assess the completeness of the linear phage genomes. Overall, we established a SACRA-coupled long-read metagenomics robust to highly diverse gut phageomes, identifying high-quality circular and linear phage genomes with adequate sequence quantity.

Vaginal microbiome topic modeling of laboring Ugandan women with and without fever

The composition of the maternal vaginal microbiome influences the duration of pregnancy, onset of labor, and even neonatal outcomes. Maternal microbiome research in sub-Saharan Africa has focused on non-pregnant and postpartum composition of the vaginal microbiome. Here we aimed to illustrate the relationship between the vaginal microbiome of 99 laboring Ugandan women and intrapartum fever using routine microbiology and 16S ribosomal RNA gene sequencing from two hypervariable regions (V1–V2 and V3–V4). To describe the vaginal microbes associated with vaginal microbial communities, we pursued two approaches: hierarchical clustering methods and a novel Grades of Membership (GoM) modeling approach for vaginal microbiome characterization. Leveraging GoM models, we created a basis composed of a preassigned number of microbial topics whose linear combination optimally represents each patient yielding more comprehensive associations and characterization between maternal clinical features and the microbial communities. Using a random forest model, we showed that by including microbial topic models we improved upon clinical variables to predict maternal fever. Overall, we found a higher prevalence of Granulicatella, Streptococcus, Fusobacterium, Anaerococcus, Sneathia, Clostridium, Gemella, Mobiluncus, and Veillonella genera in febrile mothers, and higher prevalence of Lactobacillus genera (in particular L. crispatus and L. jensenii), Acinobacter, Aerococcus, and Prevotella species in afebrile mothers. By including clinical variables with microbial topics in this model, we observed young maternal age, fever reported earlier in the pregnancy, longer labor duration, and microbial communities with reduced Lactobacillus diversity were associated with intrapartum fever. These results better defined relationships between the presence or absence of intrapartum fever, demographics, peripartum course, and vaginal microbial topics, and expanded our understanding of the impact of the microbiome on maternal and potentially neonatal outcome risk.

Molecular Characterization of a Natural Mutant of Feline Calicivirus in China

During epidemiological surveillance of Feline calicivirus (FCV) isolates in Shanghai, China, a natural mutant of FCV, designated SH1909, was successfully isolated from a stray cat. The complete genome sequence of SH1909 was determined in this study. Sequence comparison and analysis showed that thirteen unique aa residues substitutions and single-aa insertion of N or Y were observed in SH1909, which indicated that SH1909 was a novel and natural mutant of FCV. Interestingly, phylogenetic analysis based on LC-VP1 showed SH1909 could be clustered into an independent evolutionary branch with some Chinese isolates and was more distantly related to vaccine strains, indicating its potential to escape from vaccine-elicited immunity. According to the predicted B-cell epitopes of LC-VP1, amino acid mutation sites and positive selective sites, peptide C (aa sites: 445–460) and, peptide D (aa sites: 425–440) located in the hypervariable regions of LC-VP1, may result in the decreased immunological protection. Moreover, amino acid sites 439 and 449 may be responsible for the potential immune escape of SH1909. This study provides an important insight into genetic variations of FCV and vaccine development.

HyperEx: A Tool to Extract Hypervariable Regions from 16S rRNA Sequencing Data

The 16S ribosomal RNA gene is one of the most studied genes in biology. This 16S ribosomal RNA importance is due to its wide application in phylogenetics and taxonomic elucidation of bacteria and archaea. Indeed, 16S ribosomal RNA is present in almost all bacteria and archaea and has, among many other useful characteristics, a low mutation rate. The 16S ribosomal RNA is composed of nine hypervariable regions which are commonly targeted by high throughput sequencing technologies in identification or community studies like metabarcoding studies. Unfortunately, the hypervariable regions do not have the same taxonomic resolution among all bacteria taxa. This requires a preliminary in silico analysis to determine the best hypervariable regions to target in a particular study. Nevertheless, to the best of our knowledge, no automated primer-based open-source tool exists to extract hypervariable regions from complete or near-complete 16S rRNA sequencing data. Here we present HyperEx which efficiently extracts the hypervariable region of interest based on embedded primers or user-given primers. HyperEx implements the Myers algorithm for the exact pairwise sequence alignment. HyperEx is freely available under the MIT license as an operating system independent Rust command-line tool at https://github.com/Ebedthan/hyperex and https://crates.io.