RESUMO
SARS-CoV-2, a severe respiratory disease primarily targeting the lungs, was the leading cause of death worldwide during the pandemic. Understanding the interplay between the oral microbiome and inflammatory cytokines during acute infection is crucial for elucidating host immune responses. This study aimed to explore the relationship between the oral microbiome and cytokines in COVID-19 patients, particularly those with and without sputum production. Saliva and blood samples from 50 COVID-19 patients were subjected to 16S ribosomal RNA gene sequencing for oral microbiome analysis, and 65 saliva and serum cytokines were assessed using Luminex multiplex analysis. The Mann-Whitney test was used to compare cytokine levels between individuals with and without sputum production. Logistic regression machine learning models were employed to evaluate the predictive capability of oral microbiome, salivary, and blood biomarkers for sputum production. Significant differences were observed in the membership (Jaccard dissimilarity: p = 0.016) and abundance (PhILR dissimilarity: p = 0.048; metagenomeSeq) of salivary microbial communities between patients with and without sputum production. Seven bacterial genera, including Prevotella, Streptococcus, Actinomyces, Atopobium, Filifactor, Leptotrichia, and Selenomonas, were more prevalent in patients with sputum production (p<0.05, Fisher's exact test). Nine genera, including Prevotella, Megasphaera, Stomatobaculum, Selenomonas, Leptotrichia, Veillonella, Actinomyces, Atopobium, and Corynebacteria, were significantly more abundant in the sputum-producing group, while Lachnoanaerobaculum was more prevalent in the non-sputum-producing group (p<0.05, ANCOM-BC). Positive correlations were found between salivary IFN-gamma and Eotaxin2/CCL24 with sputum production, while negative correlations were noted with serum MCP3/CCL7, MIG/CXCL9, IL1 beta, and SCF (p<0.05, Mann-Whitney test). The machine learning model using only oral bacteria input outperformed the model that included all data: blood and saliva biomarkers, as well as clinical and demographic variables, in predicting sputum production in COVID-19 subjects. The performance metrics were as follows, comparing the model with only bacteria input versus the model with all input variables: precision (95% vs. 75%), recall (100% vs. 50%), F1-score (98% vs. 60%), and accuracy (82% vs. 66%).
Assuntos
COVID-19 , Microbiota , Saliva , Escarro , Humanos , COVID-19/microbiologia , Escarro/microbiologia , Saliva/microbiologia , Feminino , Masculino , Pessoa de Meia-Idade , Adulto , Idoso , Citocinas/sangue , Citocinas/metabolismo , Pulmão/microbiologia , Pulmão/virologia , SARS-CoV-2/isolamento & purificação , RNA Ribossômico 16S/genética , Leptotrichia , Prevotella/isolamento & purificação , Streptococcus/isolamento & purificação , Actinomyces/isolamento & purificação , Boca/microbiologia , Boca/virologia , Aprendizado de Máquina , Biomarcadores/sangueRESUMO
OBJECTIVES: This study aimed to identify the oral microbiota factors contributing to low birth weight (LBW) in Chinese pregnant women and develop a prediction model using machine learning. METHODS: A nested case-control study was conducted in a prospective cohort of 580 Chinese pregnant women, with 23 LBW cases and 23 healthy delivery controls matched for age and smoking habit. Saliva samples were collected at early and late pregnancy, and microbiome profiles were analyzed through 16S rRNA gene sequencing. RESULTS: The relative abundance of Streptococcus was over-represented (median 0.259 vs. 0.116) and Saccharibacteria_TM7 was under-represented (median 0.033 vs. 0.068) in the LBW case group than in controls (p < 0.001, p = 0.015 respectively). Ten species were identified as microbiome biomarkers of LBW by LEfSe analysis, which included 7 species within the genus of Streptococcus or as part of 'nutritionally variant streptococci' (NVS), 2 species of opportunistic pathogen Leptotrichia buccalis and Gemella sanguinis (all LDA score>3.5) as risk biomarkers, and one species of Saccharibacteria TM7 as a beneficial biomarker (LDA= -4.5). The machine-learning model based on these 10 distinguished oral microbiota species could predict LBW, with an accuracy of 82 %, sensitivity of 91 %, and specificity of 73 % (AUC-ROC score 0.89, 95 % CI: 0.75-1.0). Results of α-diversity showed that mothers who delivered LBW infants had less stable salivary microbiota construction throughout pregnancy than the control group (measured by Shannon, p = 0.048; and Pielou's, p = 0.021), however the microbiome diversity did not improve the prediction accuracy of LBW. CONCLUSIONS: A machine-learning oral microbiome model shows promise in predicting low-birth-weight delivery. Even in cases where oral health is not significantly compromised, opportunistic pathogens or rarer taxa associated with adverse pregnancy outcomes can still be identified in the oral cavity. CLINICAL SIGNIFICANCE: This study highlights the potential complexity of the relationship between oral microbiome and pregnancy outcomes, indicating that mechanisms underlying the association between oral microbiota and adverse pregnancy outcomes may involve complex interactions between host factors, microbiota, and systemic conditions. Using machine learning to develop a predictive model based on specific oral microbiota biomarkers provides a potential for personalized medicine approaches. Future prediction models should incorporate clinical metadata to be clinically useful for improving maternal and child health.
Assuntos
Recém-Nascido de Baixo Peso , Aprendizado de Máquina , Microbiota , Boca , RNA Ribossômico 16S , Saliva , Streptococcus , Humanos , Feminino , Gravidez , Estudos de Casos e Controles , Recém-Nascido , Adulto , Saliva/microbiologia , Boca/microbiologia , Estudos Prospectivos , RNA Ribossômico 16S/análise , Streptococcus/isolamento & purificação , Biomarcadores/análise , China , Leptotrichia , Fatores de RiscoRESUMO
Type VI CRISPR-Cas systems are among the few CRISPR varieties that target exclusively RNA. The CRISPR RNA-guided, sequence-specific binding of target RNAs, such as phage transcripts, activates the type VI effector, Cas13. Once activated, Cas13 causes collateral RNA cleavage, which induces bacterial cell dormancy, thus protecting the host population from the phage spread. We show here that the principal form of collateral RNA degradation elicited by Leptotrichia shahii Cas13a expressed in Escherichia coli cells is the cleavage of anticodons in a subset of transfer RNAs (tRNAs) with uridine-rich anticodons. This tRNA cleavage is accompanied by inhibition of protein synthesis, thus providing defense from the phages. In addition, Cas13a-mediated tRNA cleavage indirectly activates the RNases of bacterial toxin-antitoxin modules cleaving messenger RNA, which could provide a backup defense. The mechanism of Cas13a-induced antiphage defense resembles that of bacterial anticodon nucleases, which is compatible with the hypothesis that type VI effectors evolved from an abortive infection module encompassing an anticodon nuclease.
Assuntos
Anticódon , Sistemas CRISPR-Cas , Escherichia coli , RNA de Transferência , RNA de Transferência/genética , RNA de Transferência/metabolismo , Anticódon/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Leptotrichia/genética , Leptotrichia/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Proteínas Associadas a CRISPR/genética , Bacteriófagos/genética , Clivagem do RNARESUMO
BACKGROUND: The aim of the present study was to evaluate the subgingival microbiome in patients with grade C molar-incisor pattern periodontitis (C-MIP) affecting the primary or permanent dentitions. METHODS: DNA was isolated from subgingival biofilm samples from diseased and healthy sites from 45 C-MIP patients and subjected to phylogenetic microarray analysis. C-MIP sites were compared between children affected in the primary to those affected in the permanent dentitions. Within-subject differences between C-MIP-affected sites and dentition-matched healthy sites were also evaluated. RESULTS: C-MIP sites of subjects affected in the primary dentition showed partially overlapping but distinct microbial communities from C-MIP permanent dentition sites (p < 0.05). Differences were due to increased levels in primary C-MIP sites of certain species of the genera Capnocytophaga and Leptotrichia, while C-MIP permanent dentition sites showed higher prevalence of Filifactor alocis. Aggregatibacter actinomycetemcomitans (Aa) was among species seen in high prevalence and levels in both primary and permanent C-MIP sites. Moreover, both permanent and primary C-MIP sites showed distinct microbial communities when compared to dentition-matched healthy sites in the same subject (p < 0.01). CONCLUSIONS: Primary and permanent teeth with C-MIP showed a dysbiotic microbiome, with children affected in the primary dentition showing a distinct profile from those affected in the permanent dentition. However, Aa was enriched in both primary and permanent diseased sites, confirming that this microorganism is implicated in C-MIP in both dentitions.
Assuntos
Aggregatibacter actinomycetemcomitans , Biofilmes , Dentição Permanente , Microbiota , Periodontite , Dente Decíduo , Humanos , Dente Decíduo/microbiologia , Masculino , Feminino , Criança , Periodontite/microbiologia , Aggregatibacter actinomycetemcomitans/isolamento & purificação , Adolescente , Capnocytophaga/isolamento & purificação , Leptotrichia , Gengiva/microbiologia , Estudos de Casos e Controles , DNA Bacteriano/análise , Pré-EscolarRESUMO
BACKGROUND: Leptotrichia spp. are fastidious facultative anaerobic, pencil-shaped, gramnegative rods that reside in the mouths, intestines, and female genital tracts of humans. Bacteremia and septic shock have been rarely reported in the immunocompromised host. We report a case of L. trevisanii bacteremia in a patient recently diagnosed with acute myeloid leukemia (AML) on chemotherapy. CASE PRESENTATION: A 75-year-old male with a history of diabetes, chronic kidney disease, and coronary artery disease status post-CABG presented with neutropenic fevers and signs of sepsis after the initiation of chemotherapy. Blood cultures were ordered and extensive gene sequencing helped identify Leptotrichia trevisanii as the causative pathogen. Subsequently, the patient was successfully treated with empiric cefepime. DISCUSSION: Opportunistic pathogens are involved in a variety of diseases and have been isolated from immunocompromised patients undergoing transplantation or in patients with comorbidities, like leukemia, lymphoma, or neutropenia. L. trevisanii has been reported as a cause of bloodstream infections in patients with hematologic malignancies receiving chemotherapy. CONCLUSION: This case highlights the key role that Leptotrichia trevisanii plays in the introduction of sepsis among immunocompromised patients, particularly with hematologic malignancies, like AML, on chemotherapy.
Assuntos
Bacteriemia , Infecções por Fusobacteriaceae , Neoplasias Hematológicas , Leucemia Mieloide Aguda , Masculino , Humanos , Feminino , Idoso , Leptotrichia/genética , Infecções por Fusobacteriaceae/complicações , Infecções por Fusobacteriaceae/diagnóstico , Infecções por Fusobacteriaceae/tratamento farmacológico , Leucemia Mieloide Aguda/complicações , Leucemia Mieloide Aguda/tratamento farmacológico , Bacteriemia/tratamento farmacológico , Neoplasias Hematológicas/complicaçõesRESUMO
PURPOSE: Antibiotics play an important role in treating periodontal diseases. Due to the effectiveness of antibiotic therapies, their usage in dentistry has significantly increased. The aim of this study focused on the in-vitro susceptibility of different gram-negative oral bacteria species - which are associated with periodontal diseases (Fusobacterium spp., Capnocytophaga spp. and Leptotrichia buccalis) and have different geographical origins (Asia and Europe) - against antimicrobials that are clinically relevant in dental therapy. MATERIALS AND METHODS: A total of 45 strains were tested (29 Fusobacterium spp., 13 Capnocytophaga spp. and 3 L. buccalis) that were either isolated from Chinese patients or were obtained from different strain collections. Their antimicrobial susceptibility to the antimicrobial agents benzylpenicillin, amoxicillin, amoxicillin-clavulanic acid, ciprofloxacin, moxifloxacin, clindamycin, doxycycline, tetracycline and metronidazole was tested using the E-Test. Strains with particular resistance to penicillin, clindamycin and metronidazole were further analysed for resistance genes. RESULTS: All tested bacterial isolates were sensitive to amoxicillin, amoxicillin-clavulanic acid, doxycycline and tetracycline, but showed variable sensitivity towards other antibiotics such as benzylpenicillin, ciprofloxacin, moxifloxacin, clindamycin and metronidazole. CONCLUSION: The results of the present study suggest that certain periodontal disease-related bacterial strains can be resistant towards antimicrobial agents commonly used in adjuvant periodontal therapy.
Assuntos
Anti-Infecciosos , Leptothrix , Doenças Periodontais , Humanos , Clindamicina , Metronidazol , Capnocytophaga , Doxiciclina , Fusobacterium , Combinação Amoxicilina e Clavulanato de Potássio/farmacologia , Combinação Amoxicilina e Clavulanato de Potássio/uso terapêutico , Moxifloxacina , Leptotrichia , Farmacorresistência Bacteriana , Testes de Sensibilidade Microbiana , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Amoxicilina/farmacologia , Amoxicilina/uso terapêutico , CiprofloxacinaRESUMO
Leptotrichia 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 Leptotrichia bacteremia. Patient risk factors, treatment approaches, and outcomes of a retrospective cohort of adult patients with Leptotrichia BSI at a tertiary medical center (Mayo Clinic Rochester [MCR]) were evaluated. Concurrently, species, temporal trends, and antimicrobial susceptibility testing (AST) results of Leptotrichia isolates submitted to a reference laboratory (Mayo Clinic Laboratories) over the past 10 years were examined. We identified 224 blood culture isolates of Leptotrichia species, with 26 isolates from patients treated at MCR. The most frequent species included L. trevisanii (49%), L. buccalis (24%), and L. wadei (16%). Leptotrichia 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. Leptotrichia 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.
Assuntos
Anti-Infecciosos , Bacteriemia , Sepse , Adulto , Feminino , Humanos , Pessoa de Meia-Idade , Metronidazol , Leptotrichia , Estudos Retrospectivos , Bacteriemia/microbiologia , Combinação Piperacilina e Tazobactam , Bactérias Gram-Negativas , Antibacterianos , Testes de Sensibilidade MicrobianaRESUMO
The misuse of antibiotics in our daily lives has led to the emergence of antimicrobial resistance. As a result, many antibiotics are becoming ineffective. This phenomenon is linked with high rates of mortality and morbidity. Therefore, new approaches are required to address this major health issue. Leptotrichia buccalis is a Gram-negative, rod-shaped bacterium which normally resides in the oral and vaginal cavities. It is an emerging bacterial pathogen which is developing new antibiotic-resistance mechanisms. No approved vaccine is available against this pathogen, which is a cause for growing concern. In this study, an in silico-based, multi-epitopes vaccine against this pathogen was designed by applying reverse vaccinology and immunoinformatic approaches. Of a total of 2193 predicted proteins, 294 were found to be redundant while 1899 were non-redundant. Among the non-redundant proteins, 6 were predicted to be present in the extracellular region, 12 in the periplasmic region and 23 in the outer-membrane region. Three proteins (trypsin-like peptidase domain-containing protein, sel1 repeat family protein and TrbI/VirB10 family protein) were predicted to be virulent and potential subunit vaccine targets. In the epitopes prediction phase, the three proteins were subjected to B- and T-cell epitope mapping; 19 epitopes were used for vaccine design. The vaccine construct was docked with MHC-I, MHC-II and TLR-4 immune receptors and only the top-ranked complex (based on global energy value) was selected in each case. The selected docked complexes were examined in a molecular dynamic simulation and binding free energies analysis in order to assess their intermolecular stability. It was observed that the vaccine binding mode with receptors was stable and that the system presented stable dynamics. The net binding free energy of complexes was in the range of -300 to -500 kcal/mol, indicating the formation of stable complexes. In conclusion, the data reported herein might help vaccinologists to formulate a chimeric vaccine against the aforementioned target pathogen.
Assuntos
Antibacterianos , Epitopos de Linfócito T , Biologia Computacional , Feminino , Humanos , Leptotrichia , Simulação de Acoplamento Molecular , Vacinas de Subunidades AntigênicasRESUMO
CRISPR systems are prokaryotic adaptive immune systems that use RNA-guided Cas nucleases to recognize and destroy foreign genetic elements. To overcome CRISPR immunity, bacteriophages have evolved diverse families of anti-CRISPR proteins (Acrs). Recently, Lin et al. (2020) described the discovery and characterization of 7 Acr families (AcrVIA1-7) that inhibit type VI-A CRISPR systems. We detail several inconsistencies that question the results reported in the Lin et al. (2020) study. These include inaccurate bioinformatics analyses and bacterial strains that are impossible to construct. Published strains were provided by the authors, but MS2 bacteriophage plaque assays did not support the published results. We also independently tested the Acr sequences described in the original report, in E. coli and mammalian cells, but did not observe anti-Cas13a activity. Taken together, our data and analyses prompt us to question the claim that AcrVIA1-7 reported in Lin et al. are type VI anti-CRISPR proteins.
Assuntos
Bacteriófagos , Proteínas Associadas a CRISPR , Animais , Bacteriófagos/genética , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Escherichia coli/genética , Escherichia coli/metabolismo , Leptotrichia/genética , Mamíferos/metabolismo , Prófagos/genética , Prófagos/metabolismo , Ribonucleases/metabolismoRESUMO
The aim of the study was to compare the microbiota composition and bacterial diversity of subgingival plaque in chronic periodontitis patients with and without gingival erosive oral lichen planus. The subgingival plaque samples of 20 chronic periodontitis patients with gingival erosive oral lichen planus (CP-OLP group) and 19 chronic periodontitis patients without gingival erosive oral lichen planus (CP group) were analyzed by 16S rRNA gene high-throughput sequencing. Compared with the CP group, the richness and diversity of subgingival plaque microflora in the CP-OLP group decreased significantly. There were some differences between the two groups in the composition of microflora on the levels of phylum and genus. Distributions of Prevotella and Leptotrichia in the CP-OLP group were significantly lower than those in the CP group. The dominant genera in CP-OLP group were Pseudomonas and Granulicatella. These results indicated that gingival erosive oral lichen planus may influence the structure and proportion of subgingival plaque microflora.
Assuntos
Periodontite Crônica/microbiologia , Genes de RNAr , Gengiva/microbiologia , Líquen Plano Bucal/genética , Microbiota , RNA Ribossômico 16S/genética , Adulto , Bactérias/genética , Biologia Computacional , Estudos Transversais , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Leptotrichia , Masculino , Pessoa de Meia-Idade , Prevotella , PseudomonasRESUMO
The CRISPR-Cas13 system based on a bacterial enzyme has been explored as a powerful new method for RNA manipulation. Due to the high efficiency and specificity of RNA editing/interference achieved by this system, it is currently being developed as a new therapeutic tool for the treatment of neurological and other diseases. However, the safety of this new generation of RNA therapies is still unclear. In this study, we constructed a vector expressing CRISPR-Cas13 under a constitutive neuron-specific promoter. CRISPR-Cas13 from Leptotrichia wadei was expressed in primary cultures of mouse cortical neurons. We found that the presence of CRISPR-Cas13 impedes the development of cultured neurons. These results show a neurotoxic action of Cas13 and call for more studies to test for and possibly mitigate the toxic effects of Cas13 enzymes in order to improve CRISPR-Cas13-based tools for RNA targeting.
Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Córtex Cerebral/enzimologia , Leptotrichia/enzimologia , Crescimento Neuronal , Neurônios/enzimologia , Animais , Proteínas Associadas a CRISPR/genética , Células Cultivadas , Córtex Cerebral/patologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Leptotrichia/genética , Camundongos , Neurônios/patologiaRESUMO
The in vitro antibacterial spectra and activities of five antimicrobial agents, including lascufloxacin (LSFX) and two quinolones, were investigated against 69 species of anaerobes in 31 genera and 188 strains in 9 genera, respectively. In this study, minimum inhibitory concentrations (MICs) of lascufloxacin against the reference strains associated with respiratory and head and neck infections. LSFX inhibited the growth of 33 gram-positive and gram-negative reference strains at ≤0.015-2 µg/mL, except for Leptotrichia buccalis. MICs ranges of LSFX against the clinical isolates of 44 Porphyromonas spp., 45 Prevotella spp., 25 Fusobacterium spp., 7 Leptotrichia spp., 25 Parvimonas micra, 25 other gram-positive anaerobic cocci, and 17 Veillonella spp., were ≤0.015-4, 0.125-4, 0.06-0.5, 2, 0.25-16, ≤0.015-2, ≤0.015-16 µg/mL, respectively. LSFX demonstrated potent antibacterial efficacy against a wide range of species isolated from specimens involved in respiratory as well as head and neck infections.
Assuntos
Fluoroquinolonas , Leptotrichia , Antibacterianos/farmacologia , Bactérias Anaeróbias , Firmicutes , Fluoroquinolonas/farmacologia , Humanos , Testes de Sensibilidade MicrobianaRESUMO
The objective of this pilot study was to describe the microbial profiles present in the plaque and saliva of children who continued to develop new carious lesions following treatment with silver diamine fluoride ("nonresponders") compared to caries active, caries-free, and children immediately receiving SDF treatment for untreated caries in order to identify potential microbial differences that may relate to a re-incidence of caries. Saliva and plaque samples from infected and contralateral sites were obtained from twenty children who were either caries free, had active carious lesions, were caries active and received SDF treatment immediately before sampling, or had previously received SDF treatment and developed new caries. In total, 8,057,899 Illumina-generated sequence reads from 60 samples were obtained. Reads were processed using the Quantitative Insights Into Microbial Ecology pipeline. Group differences were assessed using Analysis of Variance Models and Tukey Honest Significant Differences. To identify significant taxa between treatment groups, Linear discriminant analysis Effect Size (LefSe) and Analysis of Differential Abundance Taking Sample Variation Into Account were used. Differential abundant analysis indicated that members of the Lachnospiraceae family were significantly enriched in non-responders and the genus Tannerella and species Granulicatella adiances were also highly abundant in this group. LefSe analysis between non-responders and SDF-treated groups revealed that genera Leptotrichia and Granulicatella were enriched in non-responders. We observed the highest abundance of phosphotransferase system and lowest abundance of lipopolysaccharide synthesis in non-responders. The microbiome in dental biofilms is responsible for initiation and progression of dental caries. SDF has been shown to be effective in arresting the progression carious lesions, in part due to its antimicrobial properties. Findings suggest that the differential abundance of select microbiota and specific pathway functioning in individuals that present with recurrent decay after SDF treatment may contribute to a potential failure of silver diamine fluoride to arrest dental caries. However, the short duration of sample collection following SDF application and the small sample size emphasize the need for further data and additional analysis.
Assuntos
Cárie Dentária/tratamento farmacológico , Microbiota , Compostos de Amônio Quaternário/uso terapêutico , Compostos de Prata/uso terapêutico , Carnobacteriaceae/genética , Carnobacteriaceae/isolamento & purificação , Criança , Estudos Transversais , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Cárie Dentária/patologia , Placa Dentária/microbiologia , Análise Discriminante , Fluoretos Tópicos/uso terapêutico , Humanos , Leptotrichia/genética , Leptotrichia/isolamento & purificação , Projetos Piloto , Análise de Componente Principal , Saliva/microbiologia , Análise de Sequência de DNA , Falha de TratamentoRESUMO
Bacteria and archaea apply CRISPR-Cas surveillance complexes to defend against foreign invaders. These invading genetic elements are captured and integrated into the CRISPR array as spacer elements, guiding sequence-specific DNA/RNA targeting and cleavage. Recently, in vivo studies have shown that target RNAs with extended complementarity with repeat sequences flanking the target element (tag:anti-tag pairing) can dramatically reduce RNA cleavage by the type VI-A Cas13a system. Here, we report the cryo-EM structure of Leptotrichia shahii LshCas13acrRNA in complex with target RNA harboring tag:anti-tag pairing complementarity, with the observed conformational changes providing a molecular explanation for inactivation of the composite HEPN domain cleavage activity. These structural insights, together with in vitro biochemical and in vivo cell-based assays on key mutants, define the molecular principles underlying Cas13a's capacity to target and discriminate between self and non-self RNA targets. Our studies illuminate approaches to regulate Cas13a's cleavage activity, thereby influencing Cas13a-mediated biotechnological applications.
Assuntos
Proteínas de Bactérias/química , Proteínas Associadas a CRISPR/química , Sistemas CRISPR-Cas , Endodesoxirribonucleases/química , Leptotrichia/genética , RNA Guia de Cinetoplastídeos/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Clonagem Molecular , Microscopia Crioeletrônica , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Leptotrichia/metabolismo , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Clivagem do RNA , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
OBJECTIVES: the objective of the present exploratory study was to determine bacterial diversity and endotoxin levels in deep carious lesions of teeth presenting symptoms of reversible pulpitis. MATERIALS AND METHODS: Twenty patients with deep carious lesions, reporting clinical symptomatology compatible with reversible pulpitis (n = 10) or not reporting clinical symptomatology (n = 10), were selected. Carious dentin samples were obtained with the aid of sterile and pyrogen-free spoon excavators and harvested in two steps: before and after infected dentin removal. Samples were collected for checkerboard and for kinetic chromogenic LAL assay for determination of microbial profile and quantitation of endotoxin, respectively. Data were analyzed by Mann Whitney for bacteria and two-way ANOVA for endotoxins (5%). RESULTS: No difference on the studied bacteria was detected between the superficial and deep dentin layers. Symptomatic teeth showed greater presence of Lactobacillus species, Capnocytophaga sputigena, and Leptotrichia buccalis. For the endotoxins, symptomatic teeth resulted in greater quantity of endotoxins (p = 0.047), being 4.13 log10 EU/mL/µg dentin and 3.45 log10 EU/mL/µg dentin, for symptomatic and asymptomatic teeth, respectively. Dentin collected in different areas presented similar number of endotoxins (p = 0.139). CONCLUSION: The amount of the studied bacteria does not seem to be related to reported symptomatology of deep carious lesions, while endotoxins quantity is greater in symptomatic scenarios, regardless of the harvesting area. CLINICAL RELEVANCE: The understanding of bacterial amount in reversible pulpitis is important to establish a clinical protocol of treatment.
Assuntos
Cárie Dentária , Pulpite , Bactérias , Capnocytophaga , Dentina , Endotoxinas , Humanos , LeptotrichiaRESUMO
Although the prevalence of inflammatory bowel disease (IBD) has been increasing worldwide, the etiology remains elusive. Investigating oral microbiota dysbiosis is essential to understanding IBD pathogenesis. Our study evaluated variations in salivary microbiota and identified potential associations with IBD. The saliva microbiota of 22 IBD patients and 8 healthy controls (HCs) was determined using 16S ribosomal RNA (rRNA) gene sequencing and analyzed using QIIME2. A distinct saliva microbiota dysbiosis in IBD, characterized by alterations in microbiota biodiversity and composition, was identified. Saccharibacteria (TM7), Absconditabacteria (SR1), Leptotrichia, Prevotella, Bulleidia, and Atopobium, some of which are oral biofilm-forming bacteria, were significantly increased. Moreover, levels of inflammatory cytokines associated with IBD were elevated and positively correlated with TM7 and SR1. Functional variations include down-regulation of genetic information processing, while up-regulation of carbohydrate metabolism and protein processing in the endoplasmic reticulum in IBD. Our data implicate salivary microbiota dysbiosis involving in IBD pathogenesis.
Assuntos
Disbiose/microbiologia , Doenças Inflamatórias Intestinais/microbiologia , Metagenoma , Boca/microbiologia , Adulto , Disbiose/complicações , Disbiose/epidemiologia , Feminino , Microbioma Gastrointestinal , Humanos , Doenças Inflamatórias Intestinais/complicações , Leptotrichia/genética , Leptotrichia/patogenicidade , Masculino , Prevotella/genética , Prevotella/patogenicidadeRESUMO
OBJECTIVE: Microbiota comparisons between healthy and diseased dental tissues have accentuated the importance of cultivating and identifying bacterial species that play a role in the initiation and progression of dental caries. The objective of this study was to evaluate the bacterial community composition in caries-active and caries-free children. METHODS: Supragingival plaque samples were collected from 64 caries-active and 64 caries-free Middle Eastern children. The hypervariable V3-V4 of the bacterial 16S rRNA gene was sequenced with Human Oral Microbe Identification using Next Generation Sequencing. Microbial community structure and composition analyses were performed by processing operational taxonomic units. Bioinformatic analyses, including analysis of similarity, alpha and beta diversities, and principal coordinate analysis, were carried out. RESULTS: Diversity indices did not find differences between the caries-active and caries-free groups (p > 0.05). Similarity analysis demonstrated that the microbiota composition did not differ between the two groups. Comparative analysis at the species level revealed a significantly higher relative abundance of Leptotrichia shahii, Prevotella melaninogenica, Veillonella dispar, Leptotrichia HOT 498, and Streptococcus mutans in caries-active children (p < 0.05). Corynebacterium matruchotii, Lautropia mirabilis, Neisseria elongata, and Corynebacterium durum were relatively more abundant in the caries-free group (p < 0.05). Species belonging to the Leptotrichia, Prevotella, and Veillonella genera were significantly predominant in the caries-active subjects. CONCLUSION: In view of the lack of a clear association between Corynebacterium spp. and dental caries status in the literature, the predominance of these species in caries-free children warrants further research to understand their possible role in a health-associated microbial community. CLINICAL SIGNIFICANCE: Understanding the relationship between specific bacteria present in dental biofilms and health and disease is essential for preventing and combating dental caries. Using advanced next generation sequencing techniques, the present study demonstrated the complexity of the caries microbiome and identified species/genera whose virulence or protective properties should be further explored.
Assuntos
Cárie Dentária , Placa Dentária , Microbiota , Burkholderiaceae , Criança , Corynebacterium , Suscetibilidade à Cárie Dentária , Dentição , Humanos , Leptotrichia , Microbiota/genética , RNA Ribossômico 16S/genética , VeillonellaRESUMO
The oral aerotolerant anaerobe Leptotrichia goodfellowii is an unusual cause of endocarditis and is amenable to treatment with ß-lactam antibiotics. Because this organism is difficult to identify by conventional methods, molecular detection is a key diagnostic modality. Broad-range 16S rDNA PCR followed by Sanger sequencing constitute the first-line molecular approach, yet poor DNA quality, contaminating DNA, or low template quantity make identification challenging. Here we report a case of culture-negative, aortic and mitral valve endocarditis in a 66-yr-old woman with a history of cardiomyopathy, atrial fibrillation with intracardiac pacer, poor dentition, and recent tooth infection. In this case, 16S rDNA amplicon Sanger sequencing was not sufficient for pathogen identification because of interfering DNA, but deconvolution of the clinical sample using reflexive next-generation amplicon sequencing enabled confident identification of a single pathogenic organism, L. goodfellowii The patient developed a sigmoid colon perforation and died despite additional surgical treatment. Most Leptotrichia endocarditis cases have been subacute and have been successfully treated with antibiotics, with or without valve replacement. This case highlights both an unusual etiologic agent of endocarditis, as well as the rational utilization of advanced molecular diagnostics tools for characterizing serious infections.
Assuntos
Endocardite Bacteriana/diagnóstico , Endocardite/diagnóstico , Infecções por Fusobacteriaceae/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Leptotrichia/genética , Leptotrichia/isolamento & purificação , Idoso , DNA Ribossômico/genética , Feminino , Humanos , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
Nucleic acid detection by isothermal amplification and the collateral cleavage of reporter molecules by CRISPR-associated enzymes is a promising alternative to quantitative PCR. Here, we report the clinical validation of the specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) assay using the enzyme Cas13a from Leptotrichia wadei for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the virus that causes coronavirus disease 2019 (COVID-19)-in 154 nasopharyngeal and throat swab samples collected at Siriraj Hospital, Thailand. Within a detection limit of 42 RNA copies per reaction, SHERLOCK was 100% specific and 100% sensitive with a fluorescence readout, and 100% specific and 97% sensitive with a lateral-flow readout. For the full range of viral load in the clinical samples, the fluorescence readout was 100% specific and 96% sensitive. For 380 SARS-CoV-2-negative pre-operative samples from patients undergoing surgery, SHERLOCK was in 100% agreement with quantitative PCR with reverse transcription. The assay, which we show is amenable to multiplexed detection in a single lateral-flow strip incorporating an internal control for ribonuclease contamination, should facilitate SARS-CoV-2 detection in settings with limited resources.