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Nature Protocols Oct 2019Rapid detection of nucleic acids is integral to applications in clinical diagnostics and biotechnology. We have recently established a CRISPR-based diagnostic platform...
Rapid detection of nucleic acids is integral to applications in clinical diagnostics and biotechnology. We have recently established a CRISPR-based diagnostic platform that combines nucleic acid pre-amplification with CRISPR-Cas enzymology for specific recognition of desired DNA or RNA sequences. This platform, termed specific high-sensitivity enzymatic reporter unlocking (SHERLOCK), allows multiplexed, portable, and ultra-sensitive detection of RNA or DNA from clinically relevant samples. Here, we provide step-by-step instructions for setting up SHERLOCK assays with recombinase-mediated polymerase pre-amplification of DNA or RNA and subsequent Cas13- or Cas12-mediated detection via fluorescence and colorimetric readouts that provide results in <1 h with a setup time of less than 15 min. We also include guidelines for designing efficient CRISPR RNA (crRNA) and isothermal amplification primers, as well as discuss important considerations for multiplex and quantitative SHERLOCK detection assays.
Topics: CRISPR-Cas Systems; DNA Primers; Endonucleases; Humans; Leptotrichia; Nucleic Acid Amplification Techniques; Nucleic Acids; Protein Engineering; RNA, Guide, CRISPR-Cas Systems; Recombinant Proteins; Ribonucleases; Workflow; Zika Virus; Zika Virus Infection
PubMed: 31548639
DOI: 10.1038/s41596-019-0210-2 -
Microbiology Spectrum Oct 2022As the fourth most common gynecological cancer, cervical cancer has resulted in more than 300,000 deaths worldwide in 2020. The expression of the human papillomavirus...
As the fourth most common gynecological cancer, cervical cancer has resulted in more than 300,000 deaths worldwide in 2020. The expression of the human papillomavirus (HPV) oncogenes E6 and E7 is significantly involved in the initiation and progression of cervical neoplasia. Additionally, the composition of the vaginal microbiome was also closely associated with the ability of HPV to induce cervical cancer. However, the relationship between the expression of HPV E6/E7 oncogene and the composition of the vaginal microbiome has not been clearly explored. In our present study, to investigate the relationship between the HPV E6/E7 oncogene and vaginal microbiome, cervical swabs from 115 patients were collected, and their vaginal microbiomes were analyzed by using metagenomics sequencing. Along with the progression of cervical lesions, the diversity of cervical flora increased gradually, and the abundance of decreased. Compared with the HPV group, the prevalence of HPV E6/E7 and oncogene expression level were significantly upregulated in cervical intraepithelial neoplasia (CIN) and cervical cancer (CC) groups. Additionally, a positive correlation between the expression of the HPV oncogene and the genera , Salmonella, , Pseudomonas, and in the HPV group was observed. In the CIN group, the enrichment of the genera and was weakly linked with HPV oncogene overexpression. In the CC group, a strong association between the overabundance of the genera and and the high expression of HPV oncogene was also found. The increased diversity of the vaginal microbiota and the decreased abundance were significantly associated with the severity of cervical disease, and the expression of the HPV oncogene could also be regulated by certain pathogens in different stages of cervical lesions. The main findings of this study were that we clarified the associations of the different bacterial species with the expression of human papillomavirus (HPV) oncogenes at different stages of cervical cancer. Along with the severity of cervical lesions, the abundance of the genus and species of obviously declined, while the aerobic and anaerobic bacteria, as well as the prevalence and expression of HPV E6/E7 oncogene, were increased dramatically.
Topics: Female; Humans; Papillomaviridae; Uterine Cervical Neoplasms; Papillomavirus Infections; Alphapapillomavirus; Oncogene Proteins, Viral; Dysbiosis; Papillomavirus E7 Proteins; Oncogenes; Uterine Cervical Dysplasia
PubMed: 36036584
DOI: 10.1128/spectrum.00151-22 -
Frontiers in Immunology 2022() eradication has been reported to cause short-term disruption of gut microbiota. It is acknowledged that probiotics supplementation mitigates side effects induced by... (Randomized Controlled Trial)
Randomized Controlled Trial
BACKGROUND
() eradication has been reported to cause short-term disruption of gut microbiota. It is acknowledged that probiotics supplementation mitigates side effects induced by eradication, yet its role on alleviating dysbiosis of microbiota is obscure.
OBJECTIVES
To evaluate the impact of probiotics on gastrointestinal microbiota after eradication therapy.
METHODS
This was a multicenter, double-blinded, randomized trial done at seven centers in China. A total of 276 treatment-naïve -positive patients were randomly assigned to receive 14-day bismuth-containing quadruple therapy (esomeprazole, bismuth, amoxicillin, furazolidone) combined with probiotics (Bifidobacterium Tetragenous viable Bacteria Tablets) (n=140) or placebo (n=136) for 28 days. Saliva, gastric mucosa and fecal samples were collected before and after therapy for 16S rRNA gene sequencing.
RESULTS
The incidence of gastrointestinal adverse events was lower in probiotics group compared to placebo group (23.6% vs 37.7%, p=0.016), while there was no significant difference in eradication rate. We found dramatic perturbations of gut microbiota immediately following eradication, with the predominance of Proteobacteria in replacement of commensal Firmicutes and Bacteroidetes, and gradually restored after two weeks. The reduction of gut Bacteroidetes caused by eradication drugs was neutralized with probiotics supplementation. The gastric microbiota was completely reconstituted with depleted and other taxa flourished. Of note, patients treated with probiotics showed smaller fluctuations of gastric microbiota compared to those with placebo. We also observed changes of saliva microbiota after eradication, illustrated by the overgrowth of and depletion of . The expansion of some pathogenic genera, including , , in the mouth was suppressed by probiotics.
CONCLUSION
This study not only demonstrated the beneficial effect of probiotics implementation on side events during eradication but also provided a comprehensive profile of microbiome alterations along gastrointestinal tract that modulated by probiotics.
Topics: Humans; Helicobacter pylori; Gastrointestinal Microbiome; Helicobacter Infections; Bismuth; RNA, Ribosomal, 16S; Anti-Bacterial Agents; Probiotics; Bacteroidetes
PubMed: 36426355
DOI: 10.3389/fimmu.2022.1033063 -
Nature Microbiology Dec 2022CRISPR-Cas13 proteins are RNA-guided RNA nucleases that defend against incoming RNA and DNA phages by binding to complementary target phage transcripts followed by...
CRISPR-Cas13 proteins are RNA-guided RNA nucleases that defend against incoming RNA and DNA phages by binding to complementary target phage transcripts followed by general, non-specific RNA degradation. Here we analysed the defensive capabilities of LbuCas13a from Leptotrichia buccalis and found it to have robust antiviral activity unaffected by target phage gene essentiality, gene expression timing or target sequence location. Furthermore, we find LbuCas13a antiviral activity to be broadly effective against a wide range of phages by challenging LbuCas13a against nine E. coli phages from diverse phylogenetic groups. Leveraging the versatility and potency enabled by LbuCas13a targeting, we applied LbuCas13a towards broad-spectrum phage editing. Using a two-step phage-editing and enrichment method, we achieved seven markerless genome edits in three diverse phages with 100% efficiency, including edits as large as multi-gene deletions and as small as replacing a single codon. Cas13a can be applied as a generalizable tool for editing the most abundant and diverse biological entities on Earth.
Topics: Gene Editing; Bacteriophages; CRISPR-Cas Systems; Escherichia coli; Phylogeny; RNA; Antiviral Agents
PubMed: 36316451
DOI: 10.1038/s41564-022-01258-x -
Journal of Clinical Microbiology Feb 2023species are anaerobic, Gram-negative bacilli increasingly recognized as pathogens capable of causing invasive infections such as bloodstream infection (BSI),... (Review)
Review
species are anaerobic, Gram-negative bacilli increasingly recognized as pathogens capable of causing invasive infections such as bloodstream infection (BSI), particularly among immunocompromised patients. However, there is a paucity of data regarding epidemiology, antimicrobial susceptibility, optimal treatment, and clinical outcomes among patients with bacteremia. Patient risk factors, treatment approaches, and outcomes of a retrospective cohort of adult patients with BSI at a tertiary medical center (Mayo Clinic Rochester [MCR]) were evaluated. Concurrently, species, temporal trends, and antimicrobial susceptibility testing (AST) results of isolates submitted to a reference laboratory (Mayo Clinic Laboratories) over the past 10 years were examined. We identified 224 blood culture isolates of species, with 26 isolates from patients treated at MCR. The most frequent species included L. trevisanii (49%), L. buccalis (24%), and L. wadei (16%). species demonstrated >90% susceptibility to penicillin, metronidazole, ertapenem, and piperacillin-tazobactam. However, 96% (74/77) of isolates were resistant to moxifloxacin. For patients treated at MCR, the mean patient age was 55 years (standard deviation [SD], 17), with 9 females (35%), and all were neutropenic at the time of BSI. The primary sources of infection were gastrointestinal (58%), intravascular catheter (35%), and odontogenic (15%). Patients were treated with metronidazole (42%), piperacillin-tazobactam (27%), or carbapenems (19%). The mean duration of treatment was 11 days (SD, 4.5), with a 60-day all-cause mortality of 19% and no microbiologic relapse. species are rare but important causes of BSI in neutropenic patients. Due to evolving antimicrobial susceptibility profiles, a review of AST results is necessary when selecting optimal antimicrobial therapy.
Topics: Adult; Female; Humans; Middle Aged; Metronidazole; Leptotrichia; Retrospective Studies; Bacteremia; Anti-Infective Agents; Piperacillin, Tazobactam Drug Combination; Gram-Negative Bacteria; Sepsis; Anti-Bacterial Agents; Microbial Sensitivity Tests
PubMed: 36715514
DOI: 10.1128/jcm.01733-22 -
Dentistry Journal Aug 2022To characterize the microflora profile of supragingival biofilm in patients with and without full-crown prostheses. Plaque samples of full-crown prostheses and teeth...
To characterize the microflora profile of supragingival biofilm in patients with and without full-crown prostheses. Plaque samples of full-crown prostheses and teeth in patients with porcelain-fused-to-metal crowns, all-ceramic crowns, and no prostheses were collected (three patients per group), using 16S rRNA high-throughput sequencing technology to conduct DNA sequencing on the samples and using Qiime, R, and PICRUSt2 software to perform bioinformatics analyses and functional analyses on sequencing data. In total, 110,209 valid sequences were obtained in the experiment, corresponding to 11 phyla and 120 genera. The predominant species shared by the three groups were phyla , , , , and and genera , , , , , , , , and . The species-difference analysis showed that genus significantly increased after the patient wore the dental prosthesis. Compared with the no-prosthesis samples, the functional analysis showed that cell motility increased in the samples from full-crown prostheses, while replication and repair, and translation decreased. This study reveals the changes in the oral microbial community of patients with full-crown prostheses, which could provide insights regarding the safety of materials for long-term use in the oral cavity.
PubMed: 36005250
DOI: 10.3390/dj10080152 -
Microorganisms Jan 2023The oral microbiome is an emerging field that has been a topic of discussion since the development of next generation sequencing and the implementation of the human... (Review)
Review
The oral microbiome is an emerging field that has been a topic of discussion since the development of next generation sequencing and the implementation of the human microbiome project. This article reviews the current literature surrounding the oral microbiome, briefly highlighting most recent methods of microbiome characterization including cutting edge omics, databases for the microbiome, and areas with current gaps in knowledge. This article also describes reports on microorganisms contained in the oral microbiome which include viruses, archaea, fungi, and bacteria, and provides an in-depth analysis of their significant roles in tissue homeostasis. Finally, we detail key bacteria involved in oral disease, including oral cancer, and the current research surrounding their role in stimulation of inflammatory cytokines, the role of gingival crevicular fluid in periodontal disease, the creation of a network of interactions between microorganisms, the influence of the planktonic microbiome and cospecies biofilms, and the implications of antibiotic resistance. This paper provides a comprehensive literature analysis while also identifying gaps in knowledge to enable future studies to be conducted.
PubMed: 36838283
DOI: 10.3390/microorganisms11020318 -
Journal of Dental Research Jul 2023Dental caries lesions are a clinical manifestation of disease, preceded by microbial dysbiosis, which is poorly characterized and thought to be associated with...
Dental caries lesions are a clinical manifestation of disease, preceded by microbial dysbiosis, which is poorly characterized and thought to be associated with saccharolytic taxa. Here, we assessed the associations between the oral microbiome of children and various caries risk factors such as demographics and behavioral and clinical data across early childhood and characterized over time the salivary and dental plaque microbiome of children before clinical diagnosis of caries lesions. Children ( = 266) were examined clinically at ~1, 2.5, 4, and 6.5 y of age. The microbiome samples were collected at 1, 2.5, and 4 y. Caries groups consisted of children who remained caries free (International Caries Detection and Assessment System [ICDAS] = 0) at all time points (CFAT) ( = 50); children diagnosed with caries (ICDAS ≥ 1) at 6.5 y (C6.5), 4 y (C4), or 2.5 y of age (C2.5); and children with early caries or advanced caries lesions at specific time points. Microbial community analyses were performed on zero-radius operational taxonomic units (zOTUs) obtained from V4 of 16S ribosomal RNA gene amplicon sequences. The oral microbiome of the children was affected by various factors, including antibiotic use, demographics, and dietary habits of the children and their caregivers. At all time points, various risk factors explained more of the variation in the dental plaque microbiome than in saliva. At 1 y, composition of saliva of the C4 group differed from that of the CFAT group, while at 2.5 y, this difference was observed only in plaque. At 4 y, multiple salivary and plaque zOTUs of genera and were significantly higher in samples of the C6.5 group than those of the CFAT group. In conclusion, up to 3 y prior to clinical caries detection, the oral microbial communities were already in a state of dysbiosis that was dominated by proteolytic taxa. Plaque discriminated dysbiotic oral ecosystems from healthy ones better than saliva.
Topics: Child; Humans; Child, Preschool; Dental Caries; Dental Plaque; Dysbiosis; Saliva; Microbiota; RNA, Ribosomal, 16S
PubMed: 37042041
DOI: 10.1177/00220345231160756 -
World Journal of Gastroenterology Dec 2020Microbiota profiles differ between patients with pancreatic cancer and healthy people, and understanding these differences may help in early detection of pancreatic...
BACKGROUND
Microbiota profiles differ between patients with pancreatic cancer and healthy people, and understanding these differences may help in early detection of pancreatic cancer. Saliva sampling is an easy and cost-effective way to determine microbiota profiles compared to fecal and tissue sample collection.
AIM
To investigate the saliva microbiome distribution in patients with pancreatic adenocarcinoma (PDAC) and the role of oral microbiota profiles in detection and risk prediction of pancreatic cancer.
METHODS
We conducted a prospective study of patients with pancreatic cancer ( = 41) and healthy individuals ( = 69). Bacterial taxa were identified by 16S ribosomal ribonucleic acid gene sequencing, and a linear discriminant analysis effect size algorithm was used to identify differences in taxa. Operational taxonomic unit values of all selected taxa were converted into a normalized Z-score, and logistic regressions were used to calculate risk prediction of pancreatic cancer.
RESULTS
Compared with the healthy control group, carriage of and (z-score) was associated with a higher risk of PDAC [odds ratio (OR) = 5.344, 95% confidence interval (CI): 1.282-22.282, = 0.021 and OR = 6.886, 95%CI: 1.423-33.337, = 0.016, respectively]. and (z-score) were considered a protective microbe that decreased the risk of PDAC (OR = 0.187, 95%CI: 0.055-0.631, = 0.007 and OR = 0.309, 95%CI: 0.100-0.952, = 0.041, respectively). Among the patients with PDAC, patients reporting bloating have a higher abundance of ( = 0.039), ( = 0.024), and ( = 0.041); while patients reporting jaundice had a higher amount of ( = 0.008); patients reporting dark brown urine had a higher amount of ( = 0.035). Patients reporting diarrhea had a lower amount of and ( = 0.024 and = 0.034), and patients reporting vomiting had decreased ( = 0.036).
CONCLUSION
Saliva microbiome was able to distinguish patients with pancreatic cancer and healthy individuals. may be specific for patients living in Sichuan Province, southwest China. Symptomatic patients had different bacteria profiles than asymptomatic patients. Combined symptom and microbiome evaluation may help in the early detection of pancreatic cancer.
Topics: Adenocarcinoma; China; Humans; Microbiota; Pancreatic Neoplasms; Prospective Studies; RNA, Ribosomal, 16S; Saliva
PubMed: 33505144
DOI: 10.3748/wjg.v26.i48.7679 -
World Journal of Oncology Jun 2023Reports have shown increased positive correlations with the salivary microbiota and pancreatic carcinogenesis. A European study showed that high levels of were... (Review)
Review
Reports have shown increased positive correlations with the salivary microbiota and pancreatic carcinogenesis. A European study showed that high levels of were correlated with periodontium damage and were associated with a risk of pancreatic cancer (two-fold). A recent study, using oral mouthwash samples (n = 361 with pancreatic adenocarcinoma), determined that the presence of and along with and were a risk factor for pancreatic cancer. The link between pancreatic cancer and periodontitis has been documented. Interestingly, periodontitis presents with inflammation and microbial dysbiosis, both of which have been characterized in pancreatic cancer. This review highlights multiple roles in which oral anaerobic bacteria can spread to the pancreas and contribute to pancreatic cancer.
PubMed: 37350809
DOI: 10.14740/wjon1596