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1.
Nat Commun ; 12(1): 599, 2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33500394

RESUMO

The ribosome represents a promising avenue for synthetic biology, but its complexity and essentiality have hindered significant engineering efforts. Heterologous ribosomes, comprising rRNAs and r-proteins derived from different microorganisms, may offer opportunities for novel translational functions. Such heterologous ribosomes have previously been evaluated in E. coli via complementation of a genomic ribosome deficiency, but this method fails to guide the engineering of refractory ribosomes. Here, we implement orthogonal ribosome binding site (RBS):antiRBS pairs, in which engineered ribosomes are directed to researcher-defined transcripts, to inform requirements for heterologous ribosome functionality. We discover that optimized rRNA processing and supplementation with cognate r-proteins enhances heterologous ribosome function for rRNAs derived from organisms with ≥76.1% 16S rRNA identity to E. coli. Additionally, some heterologous ribosomes undergo reduced subunit exchange with E. coli-derived subunits. Cumulatively, this work provides a general framework for heterologous ribosome engineering in living cells.


Assuntos
Escherichia coli/genética , Biossíntese de Proteínas/genética , Proteínas Ribossômicas/genética , Ribossomos/genética , Biologia Sintética/métodos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Filogenia , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , RNA Ribossômico 23S/genética , RNA Ribossômico 23S/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Óperon de RNAr/genética
2.
Medicine (Baltimore) ; 100(2): e24090, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33466172

RESUMO

OBJECTIVE: To understood the pathogen detection status and clinical characteristics of suspected pertussis in children and to observe the drug sensitivity and drug resistance genes of Bordetella pertussis (B. pertussis). METHODS: Three hundred fifty-one cases were collected and their nasopharyngeal swab samples were analyzed by culture and fluorescent quantitative polymerase chain reaction. The susceptibility to erythromycin, clindamycin, ampicillin, levofloxacin, and sulfamethoxazole-trimethoprim were tested by E-test for the positive strains, and the susceptibility to erythromycin was also tested for the KB disk diffusion method. The 23S rRNA gene of the positive strains was amplified and sequenced, and statistical analysis was performed in conjunction with clinical data. RESULTS: The positive rate of bacterial culture was 16.8% (59/351), and the positive rate of PCR was 62.4% (219/351). Two cases were positive about bacterial culture and negative for PCR. There were 221 confirmed cases of pertussis. The E-test results showed that the rate of the sensitivity of the 55 strains of pertussis to erythromycin and clindamycin was 50.9% (28/55), the minimum antibiotic concentration50 (MIC50) and MIC90 values were 0.094/>256 and 0.75/>256 mg/L, respectively, and the MIC50/MIC90 to ampicillin, levofloxacin, and sulfamethoxazole were 0.125/0.19, 0.38/0.5, and 0.125/0.25 mg/L, respectively. The KB disk diffusion method showed 27 of the 55 strains 49.1% (27/55) was resistant to erythromycin; all of the resistant strains had the 23S rRNA gene A2047G mutation, and their MIC of erythromycin was >256 mg/L. CONCLUSION: The diagnosis of pertussis by a fluorescent quantitative polymerase chain reaction method is more sensitive than that of bacterial culture. The resistance of B. pertussis to erythromycin was prominent. All of the strains of B. pertussis resistant to erythromycin in our center had the A2047G mutation of the 23S rRNA gene.


Assuntos
Bordetella pertussis/efeitos dos fármacos , Bordetella pertussis/genética , Farmacorresistência Bacteriana/genética , Testes de Sensibilidade Microbiana/métodos , Coqueluche/microbiologia , Antibacterianos/farmacologia , Criança , Pré-Escolar , China , Técnicas de Cultura , Eritromicina/farmacologia , Feminino , Humanos , Lactente , Masculino , Tipagem Molecular , Mutação , Reação em Cadeia da Polimerase , RNA Ribossômico 23S/genética , Coqueluche/diagnóstico
3.
Nucleic Acids Res ; 49(1): 547-567, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33330920

RESUMO

Genomic studies have indicated that certain bacterial lineages such as the Bacteroidetes lack Shine-Dalgarno (SD) sequences, and yet with few exceptions ribosomes of these organisms carry the canonical anti-SD (ASD) sequence. Here, we show that ribosomes purified from Flavobacterium johnsoniae, a representative of the Bacteroidetes, fail to recognize the SD sequence of mRNA in vitro. A cryo-electron microscopy structure of the complete 70S ribosome from F. johnsoniae at 2.8 Å resolution reveals that the ASD is sequestered by ribosomal proteins bS21, bS18 and bS6, explaining the basis of ASD inhibition. The structure also uncovers a novel ribosomal protein-bL38. Remarkably, in F. johnsoniae and many other Flavobacteriia, the gene encoding bS21 contains a strong SD, unlike virtually all other genes. A subset of Flavobacteriia have an alternative ASD, and in these organisms the fully complementary sequence lies upstream of the bS21 gene, indicative of natural covariation. In other Bacteroidetes classes, strong SDs are frequently found upstream of the genes for bS21 and/or bS18. We propose that these SDs are used as regulatory elements, enabling bS21 and bS18 to translationally control their own production.


Assuntos
Bacteroidetes/genética , Iniciação Traducional da Cadeia Peptídica , Sequências Reguladoras de Ácido Ribonucleico , Ribossomos/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Códon de Iniciação , Microscopia Crioeletrônica , Cristalografia por Raios X , Escherichia coli/genética , Flavobacterium/genética , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Puromicina/farmacologia , RNA Bacteriano/genética , RNA Mensageiro/genética , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , RNA Ribossômico 5S/genética , Ribossomos/ultraestrutura , Alinhamento de Sequência , Homologia de Sequência , Especificidade da Espécie
4.
BMC Infect Dis ; 20(1): 950, 2020 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-33308173

RESUMO

BACKGROUND: Antimicrobial resistance in M. genitalium is a growing clinical problem. We investigated the mutations associated with macrolide and fluoroquinolone resistance, two commonly used medical regimens for treatment in China. Our aim is to analyze the prevalence and diversity of mutations among M. genitalium-positive clinical specimens in Guangzhou, south China. METHODS: A total of 154 stored M. genitalium positive specimens from men and women attending a STI clinic were tested for macrolide and fluoroquinolone mutations. M. genitalium was detected via TaqMan MGB real-time PCR. Mutations associated with macrolide resistance were detected using primers targeting region V of the 23S rRNA gene. Fluoroquinolone resistant mutations were screened via primers targeting topoisomerase IV (parC) and DNA gyrase (gyrA). RESULTS: 98.7% (152/154), 95.5% (147/154) and 90.3% (139/154) of M. genitalium positive samples produced sufficient amplicon for detecting resistance mutations in 23S rRNA, gyrA and parC genes, respectively. 66.4% (101/152), 0.7% (1/147) and 77.7% (108/139) samples manifested mutations in 23S rRNA, gyrA and parC genes, respectively. A2072G (59/101, 58.4%) and S83I (79/108, 73.1%) were highly predominating in 23S rRNA and parC genes, respectively. Two samples had amino acid substitutions in gyrA (M95I and A96T, respectively). Two samples had two amino acid substitutions in parC (S83I + D87Y). 48.6% (67/138) of samples harbored both macrolide and fluoroquinolone resistance-associated mutations. The most common combination of mutations was A2072G (23S rRNA) and S83I (parC) (40/67, 59.7%). One sample had three amino acid changes in 23S rRNA, gyrA and parC genes (A2072G + A96T + S83I). CONCLUSIONS: The high antimicrobial resistance rate of M. genitalium in Guangzhou is a very worrying problem and suggests that antimicrobial resistance testing and the development of new antibiotic regimens are crucially needed.


Assuntos
Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana Múltipla/genética , Fluoroquinolonas/uso terapêutico , Macrolídeos/uso terapêutico , Mutação , Infecções por Mycoplasma/tratamento farmacológico , Mycoplasma genitalium/genética , Doenças Bacterianas Sexualmente Transmissíveis/tratamento farmacológico , China/epidemiologia , DNA Girase/genética , DNA Topoisomerase IV/genética , DNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Feminino , Humanos , Masculino , Infecções por Mycoplasma/epidemiologia , Infecções por Mycoplasma/microbiologia , Mycoplasma genitalium/isolamento & purificação , Prevalência , RNA Ribossômico 23S/genética , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos , Doenças Bacterianas Sexualmente Transmissíveis/epidemiologia , Doenças Bacterianas Sexualmente Transmissíveis/microbiologia
5.
BMC Bioinformatics ; 21(1): 492, 2020 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-33129268

RESUMO

BACKGROUND: The ability to compare samples or studies easily using metabarcoding so as to better interpret microbial ecology results is an upcoming challenge. A growing number of metabarcoding pipelines are available, each with its own benefits and limitations. However, very few have been developed to offer the opportunity to characterize various microbial communities (e.g., archaea, bacteria, fungi, photosynthetic microeukaryotes) with the same tool. RESULTS: BIOCOM-PIPE is a flexible and independent suite of tools for processing data from high-throughput sequencing technologies, Roche 454 and Illumina platforms, and focused on the diversity of archaeal, bacterial, fungal, and photosynthetic microeukaryote amplicons. Various original methods were implemented in BIOCOM-PIPE to (1) remove chimeras based on read abundance, (2) align sequences with structure-based alignments of RNA homologs using covariance models, and (3) a post-clustering tool (ReClustOR) to improve OTUs consistency based on a reference OTU database. The comparison with two other pipelines (FROGS and mothur) and Amplicon Sequence Variant definition highlighted that BIOCOM-PIPE was better at discriminating land use groups. CONCLUSIONS: The BIOCOM-PIPE pipeline makes it possible to analyze 16S, 18S and 23S rRNA genes in the same packaged tool. The new post-clustering approach defines a biological database from previously analyzed samples and performs post-clustering of reads with this reference database by using open-reference clustering. This makes it easier to compare projects from various sequencing runs, and increased the congruence among results. For all users, the pipeline was developed to allow for adding or modifying the components, the databases and the bioinformatics tools easily, giving high modularity for each analysis.


Assuntos
Archaea/genética , Bactérias/genética , Biodiversidade , Biologia Computacional/métodos , Código de Barras de DNA Taxonômico , Fungos/genética , Genes de RNAr , Software , Análise por Conglomerados , Simulação por Computador , Bases de Dados Genéticas , Microbiota/genética , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , Microbiologia do Solo
6.
Nat Commun ; 11(1): 5374, 2020 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-33097713

RESUMO

The emergence of resistance to azithromycin complicates treatment of Neisseria gonorrhoeae, the etiologic agent of gonorrhea. Substantial azithromycin resistance remains unexplained after accounting for known resistance mutations. Bacterial genome-wide association studies (GWAS) can identify novel resistance genes but must control for genetic confounders while maintaining power. Here, we show that compared to single-locus GWAS, conducting GWAS conditioned on known resistance mutations reduces the number of false positives and identifies a G70D mutation in the RplD 50S ribosomal protein L4 as significantly associated with increased azithromycin resistance (p-value = 1.08 × 10-11). We experimentally confirm our GWAS results and demonstrate that RplD G70D and other macrolide binding site mutations are prevalent (present in 5.42% of 4850 isolates) and widespread (identified in 21/65 countries across two decades). Overall, our findings demonstrate the utility of conditional associations for improving the performance of microbial GWAS and advance our understanding of the genetic basis of macrolide resistance.


Assuntos
Farmacorresistência Bacteriana/genética , Genoma Bacteriano , Estudo de Associação Genômica Ampla , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/genética , Antibacterianos/farmacologia , Azitromicina/farmacologia , Sítios de Ligação/genética , Gonorreia/tratamento farmacológico , Gonorreia/microbiologia , Humanos , Macrolídeos/farmacologia , Testes de Sensibilidade Microbiana , Mutação/efeitos dos fármacos , RNA Ribossômico 23S/genética
7.
Proc Natl Acad Sci U S A ; 117(33): 19879-19887, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32747536

RESUMO

The ribosome translates the genetic code into proteins in all domains of life. Its size and complexity demand long-range interactions that regulate ribosome function. These interactions are largely unknown. Here, we apply a global coevolution method, statistical coupling analysis (SCA), to identify coevolving residue networks (sectors) within the 23S ribosomal RNA (rRNA) of the large ribosomal subunit. As in proteins, SCA reveals a hierarchical organization of evolutionary constraints with near-independent groups of nucleotides forming physically contiguous networks within the three-dimensional structure. Using a quantitative, continuous-culture-with-deep-sequencing assay, we confirm that the top two SCA-predicted sectors contribute to ribosome function. These sectors map to distinct ribosome activities, and their origins trace to phylogenetic divergences across all domains of life. These findings provide a foundation to map ribosome allostery, explore ribosome biogenesis, and engineer ribosomes for new functions. Despite differences in chemical structure, protein and RNA enzymes appear to share a common internal logic of interaction and assembly.


Assuntos
Escherichia coli/genética , RNA Bacteriano/química , RNA Ribossômico 23S/química , Ribossomos/genética , Escherichia coli/química , Escherichia coli/metabolismo , Evolução Molecular , Conformação de Ácido Nucleico , Filogenia , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico 23S/genética , RNA Ribossômico 23S/metabolismo , Ribossomos/química , Ribossomos/metabolismo
8.
BMC Infect Dis ; 20(1): 615, 2020 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-32814558

RESUMO

BACKGROUND: The global prevalent ptxP3 strains varies from about 10% to about 50% of circulating B. pertussis population in different areas of China. METHODS: To investigate the difference of vaccination status between different genotypes in the circulating B. pertussis after 10 years of acellular pertussis vaccine (aPV) used in China. The nasopharyngeal swabs and isolates of B. pertussis from these patients were used to perform genotyping of antigen genes. We use antibiotic susceptibility test against erythromycin and sequencing methods for site 2047 of 23S rRNA to determine the resistance status. RESULTS: The ptxP1 allele with erythromycin resistant (ER) B. pertussis infection (total of 449 subjects) consisted of 84.70 to 96.70% from 2012 to 2016 in this study. Vaccinated with co-purified aPV was found in 133(133/403,33.0%), 1(1/9,11.1%) and 2(2/21,9.5%) in ptxP1/fhaB3-ER, ptxP1/fhaB2-ES and ptxP3/fhaB2-ES B. pertussis infected children each, which showed a significant difference (χ2 = 6.87, P = 0.032). CONCLUSIONS: The ptxP3-ES B. pertussis was rare while the ptxP1-ER B. pertussis was steadily increased in Xi'an, China from 2012 to 2016, where co-purified aPV was prevalent used. This pose a hypothesis that the co-purified aPV might protect against ptxP3 strains more efficient, which generated a rare chance for ptxP3 strains to be under the antibiotic pressure and further developed to be erythromycin resistance. A further cohort study and the mechanisms of the additional antigen proteins of co-purified aPV protected against B. pertussis should be consideration.


Assuntos
Bordetella pertussis/efeitos dos fármacos , Bordetella pertussis/genética , Toxina Pertussis/genética , Vacina contra Coqueluche/uso terapêutico , Coqueluche/epidemiologia , Alelos , Antibacterianos/farmacologia , Bordetella pertussis/isolamento & purificação , Pré-Escolar , China/epidemiologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Eritromicina/farmacologia , Genótipo , Humanos , Testes de Sensibilidade Microbiana , Nasofaringe/microbiologia , Vacina contra Coqueluche/imunologia , Prevalência , RNA Ribossômico 23S/genética , Estudos Retrospectivos , Vacinação , Coqueluche/microbiologia , Coqueluche/prevenção & controle
9.
J Med Microbiol ; 69(8): 1079-1088, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32729813

RESUMO

Introduction. Linezolid-resistant (LZR) Staphylococcus capitis has recently emerged in our hospital, and its potential resistance mechanisms are still not clear.Aim. This study aimed to investigate the epidemiology, clinical and genetic characteristics, resistance mechanisms and biofilm formation capacity of LZR S. capitis isolated from patients at Huashan Hospital, Shanghai, PR China between 2012 and 2018.Methodology. Strains were subjected to antimicrobial susceptibility testing (AST) with antibiotics using the broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. The presence of cfr, optrA and poxtA, as well as mutations in the 23S ribosomal (r)RNA and ribosomal proteins, was investigated using PCR and sequencing techniques. The genetic relationship between isolates was analysed using pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing (WGS). Biofilm biomasses were detected by using crystal violet staining.Results. Twenty-one LZR S. capitis strains displayed MICs of 32-512 µg ml-1. All LZR strains showed G2576T and C2104T mutations in the 23S rRNA V region. Besides G2576T and C2104T, no base mutations were detected in the V region. The cfr was detected in 12 strains, while optrA and poxtA were not amplified in 21 S. capitis strains. PFGE showed that the LZR S. capitis strains belonged to a single clone. The phylogenetic tree showed that 20 LZR S. capitis strains were highly similar to LNZR-1, isolated from Harbin (located in the north of China) in 2013, which showed resistance to linezolid.Conclusions. In this research, cfr-negative strains displayed linezolid MICs of 32 µg ml-1. In comparison, cfr-positive strains exhibited linezolid MICs of 128-512 µg ml-1, indicating that high levels of linezolid resistance appear to be related to the presence of cfr. The outbreak of LZR S. capitis in our hospital needs to be monitored closely.


Assuntos
Antibacterianos/farmacologia , Linezolida/farmacologia , Infecções Estafilocócicas/epidemiologia , Staphylococcus capitis/efeitos dos fármacos , Adulto , Idoso , Idoso de 80 Anos ou mais , Biofilmes/crescimento & desenvolvimento , China/epidemiologia , DNA Bacteriano/isolamento & purificação , Farmacorresistência Bacteriana/genética , Eletroforese em Gel de Campo Pulsado , Feminino , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mutação , Oxazolidinonas/farmacologia , RNA Ribossômico 23S/genética , Infecções Estafilocócicas/microbiologia , Staphylococcus capitis/genética , Staphylococcus capitis/fisiologia , Sequenciamento Completo do Genoma , Adulto Jovem
10.
Proc Natl Acad Sci U S A ; 117(28): 16333-16338, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601241

RESUMO

Bacterial transfer RNAs (tRNAs) contain evolutionarily conserved sequences and modifications that ensure uniform binding to the ribosome and optimal translational accuracy despite differences in their aminoacyl attachments and anticodon nucleotide sequences. In the tRNA anticodon stem-loop, the anticodon sequence is correlated with a base pair in the anticodon loop (nucleotides 32 and 38) to tune the binding of each tRNA to the decoding center in the ribosome. Disruption of this correlation renders the ribosome unable to distinguish correct from incorrect tRNAs. The molecular basis for how these two tRNA features combine to ensure accurate decoding is unclear. Here, we solved structures of the bacterial ribosome containing either wild-type [Formula: see text] or [Formula: see text] containing a reversed 32-38 pair on cognate and near-cognate codons. Structures of wild-type [Formula: see text] bound to the ribosome reveal 23S ribosomal RNA (rRNA) nucleotide A1913 positional changes that are dependent on whether the codon-anticodon interaction is cognate or near cognate. Further, the 32-38 pair is destabilized in the context of a near-cognate codon-anticodon pair. Reversal of the pairing in [Formula: see text] ablates A1913 movement regardless of whether the interaction is cognate or near cognate. These results demonstrate that disrupting 32-38 and anticodon sequences alters interactions with the ribosome that directly contribute to misreading.


Assuntos
Biossíntese de Proteínas/genética , RNA de Transferência/química , RNA de Transferência/genética , Anticódon/química , Anticódon/genética , Anticódon/metabolismo , Pareamento de Bases , Códon/genética , Códon/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Ribossômico 23S/química , RNA Ribossômico 23S/genética , RNA Ribossômico 23S/metabolismo , RNA de Transferência/metabolismo , Ribossomos/química , Ribossomos/metabolismo , Thermus thermophilus/genética , Thermus thermophilus/metabolismo
11.
BMC Infect Dis ; 20(1): 419, 2020 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-32546213

RESUMO

BACKGROUND: Four new variants of Chlamydia trachomatis (nvCTs), detected in several countries, cause false-negative or equivocal results using the Aptima Combo 2 assay (AC2; Hologic). We evaluated the clinical sensitivity and specificity, as well as the analytical inclusivity and exclusivity of the updated AC2 for the detection of CT and Neisseria gonorrhoeae (NG) on the automated Panther system (Hologic). METHODS: We examined 1004 clinical AC2 samples and 225 analytical samples spiked with phenotypically and/or genetically diverse NG and CT strains, and other potentially cross-reacting microbial species. The clinical AC2 samples included CT wild type (WT)-positive (n = 488), all four described AC2 diagnostic-escape nvCTs (n = 170), NG-positive (n = 214), and CT/NG-negative (n = 202) specimens. RESULTS: All nvCT-positive samples (100%) and 486 (99.6%) of the CT WT-positive samples were positive in the updated AC2. All NG-positive, CT/NG-negative, Trichomonas vaginalis (TV)-positive, bacterial vaginosis-positive, and Candida-positive AC2 specimens gave correct results. The clinical sensitivity and specificity of the updated AC2 for CT detection was 99.7 and 100%, respectively, and for NG detection was 100% for both. Examining spiked samples, the analytical inclusivity and exclusivity were 100%, i.e., in clinically relevant concentrations of spiked microbe. CONCLUSIONS: The updated AC2, including two CT targets and one NG target, showed a high sensitivity, specificity, inclusivity and exclusivity for the detection of CT WT, nvCTs, and NG. The updated AC2 on the fully automated Panther system offers a simple, rapid, high-throughput, sensitive, and specific diagnosis of CT and NG, which can easily be combined with detection of Mycoplasma genitalium and TV.


Assuntos
Infecções por Chlamydia/diagnóstico , Chlamydia trachomatis/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Análise de Sequência de RNA/métodos , Candida/genética , Candidíase/diagnóstico , Candidíase/microbiologia , Infecções por Chlamydia/microbiologia , Reações Cruzadas , Feminino , Gonorreia/diagnóstico , Gonorreia/microbiologia , Humanos , Neisseria gonorrhoeae/genética , RNA Bacteriano/genética , RNA Ribossômico 23S/genética , Sensibilidade e Especificidade , Tricomoníase/diagnóstico , Tricomoníase/parasitologia , Trichomonas vaginalis/genética
12.
Int J Syst Evol Microbiol ; 70(7): 4098-4104, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32539911

RESUMO

A new α-haemolytic streptococcal strain has been isolated from the dental plaque of a teenager with Down syndrome. Genetic and taxonomic analyses place this Streptococcus within the oralis group. It is a Gram-stain-positive, non-motile, non-spore-forming spherical alpha-haemolytic coccus arranged in chains, and it ferments a large number of monosaccharides and disaccharides, as well as polymeric carbohydrates. It differs biochemically from closely related species of Streptococcus due to its production of α-galactosidase, ß-galactosidase and N-acetyl-ß-d-glucosaminidase and by the absence of arginine dihydrolase deiminase and IgA1-protease. It grows in a temperature range of 25 to 40 °C (optimal growth temperature at 37 °C) and in a pH range of 4.5 to 8 (optimal pH at 7.0). A phylogenetic analysis based on its 16S and 23S rRNA gene sequences placed it close to Streptococcus dentisani CECT 7747T. The ANIb and ANIm values were 93.19 and 93.61 %, respectively, both below the accepted threshold to designate it as a new species of bacteria. A phylogenetic tree based on its core genome placed it close to Streptococcus oralis subsp. dentisani strain CECT 7747T with a distance in the expanded core phylogeny of 0.1298. The in silico DNA-DNA hybridization value was 52.2 % with respect to the closest species, S. oralis subsp. dentisani CECT 7747T. Based on these data, a new species of bacteria within the genus Streptococcus, family Streptococcaceae and order Lactobacillales is described, for which the name of Streptococcus downii sp. nov. is proposed (type strain CECT 9732T=CCUG 73139T).


Assuntos
Placa Dentária/microbiologia , Síndrome de Down , Boca/microbiologia , Filogenia , Streptococcus/classificação , Adolescente , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Humanos , Hibridização de Ácido Nucleico , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , Análise de Sequência de DNA , Streptococcus/isolamento & purificação
13.
PLoS One ; 15(5): e0233504, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32453777

RESUMO

One of the most pressing problems of enterococci infections is occurring resistance to linezolid, which is an antibiotic used in the treatment of infections caused by vancomycin-resistant strains (VRE). The main objective of our research was to investigate the relationship of 19 linezolid-resistant E. faecium isolates from 18 patients hospitalized at Clinical Hospital in Gdansk (Poland). One of the LZDREF was isolated in 2003 (K2003), and another 18 were collected from 2013 to 2017. Genotyping with PCR MP method indicated 14 main unrelated genetic profiles and no association with K2003 strain. Two isolates with the same genotype and genetically closely related two sub-types (2 isolates for each sub-type) were hospital-derived colonizations of patients. The other unrelated genotypes were discussed in the context of colonization, nosocomial infections, and commensal origin, taking into account prior exposure to linezolid. We determined the presence of a point mutation G2576T in six loci of 23S rDNA. There was also a significant correlation (p<0.0015) between the presence of MIC>32 value and the presence of G2576T point mutation on the sixth rrn. We also detected 5 virulence genes for all isolates: gelE, cylA, asa1, hyl, esp. Correlation (p≤0.0001) was observed between the presence of gelE gene encoding gelatinase and two other genes: cylA and asa1 encoding cytolysin and collagen binding protein responsible for aggregation of bacterial cells, respectively. Significant correlation was also observed between asa1 and cfr genes encoding 23S rRNA rybonuclease responsible for resistance to PhLOPSA antibiotics (p = 0.0004). The multidimensional analysis has also shown the correlation between cfr gene and GI-tract (p = 0, 0491), which suggests horizontal gene transfer inside the gut microbiota and the risk of colonization with linezolid-resistant strains without previously being treated with the antibiotic. The patient could have been colonized with LZDRVREF strains which in the absence of competitive microbiota quickly settle in ecological niches favourable for them and pose a risk for the patient.


Assuntos
Farmacorresistência Bacteriana , Enterococcus faecium/classificação , Infecções por Bactérias Gram-Positivas/microbiologia , Linezolida/farmacologia , Enterococcus faecium/genética , Enterococcus faecium/isolamento & purificação , Evolução Molecular , Feminino , Transferência Genética Horizontal , Técnicas de Genotipagem , Humanos , Masculino , Filogenia , Mutação Puntual , Polônia , RNA Ribossômico 23S/genética , Análise de Sequência de DNA , Simbiose , Fatores de Virulência/genética
14.
Int J Syst Evol Microbiol ; 70(5): 3413-3426, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32375955

RESUMO

Five cyanobacterial strains with Nostoc-like morphology from different localities of the Mazandaran province of Iran were characterized using a polyphasic approach. Three strains clustered within the Aliinostoc clade whereas one each of the remaining two strains clustered within the genera Desmonostoc and Desikacharya. The phylogenetic positioning of all the strains by the bayesian inference, neighbour joining and maximum parsimony methods inferred using 16S rRNA gene indicated them to represent novel species of the genera Aliinostoc, Desmonostoc and Desikacharya. The 16S-23S ITS secondary structure analysis revealed that all five strains under study represented novel species unknown to science. In accordance with the International Code of Nomenclature for algae, fungi and plants we describe three novel species of the genus Aliinostoc and one species each of the genera Desmonostoc and Desikacharya.


Assuntos
Cianobactérias/classificação , Filogenia , Microbiologia do Solo , Técnicas de Tipagem Bacteriana , Composição de Bases , Teorema de Bayes , Cianobactérias/isolamento & purificação , DNA Bacteriano/genética , Irã (Geográfico) , Conformação de Ácido Nucleico , Oryza , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , Análise de Sequência de DNA
15.
BMC Infect Dis ; 20(1): 314, 2020 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-32345231

RESUMO

BACKGROUND: Mycoplasma genitalium is an emerging sexually transmitted infection, with increasing rates of resistance to fluroquinolones and macrolides, the recommended treatments. Despite this, M. genitalium is not part of routine screening for Sexually Transmitted Infections (STIs) in many countries and the prevalence of infection and patterns of disease remain to be determined in many populations. Such data is of particular importance in light of the reported rise in antibiotic resistance in M. genitalium isolates. METHODS: Urine and urethral swab samples were collected from the primary public sexual health clinic in Singapore and tested for C. trachomatis (CT) or N. gonorrhoeae (NG) infection and for the presence of M. genitalium. Antibiotic resistance in M. genitalium strains detected was determined by screening for genomic mutations associated with macrolide and fluroquinolone resistance. RESULTS: We report the results of a study into M. genitalium prevalence at the national sexual health clinic in Singapore. M. genitalium was heavily associated with CT infection (8.1% of cases), but present in only of 2.4% in CT negative cases and not independently linked to NG infection. Furthermore, we found high rates of resistance mutations to both macrolides (25%) and fluoroquinolones (37.5%) with a majority of resistant strains being dual-resistant. Resistance mutations were only found in strains from patients with CT co-infection. CONCLUSIONS: Our results support targeted screening of CT positive patients for M. genitalium as a cost-effective strategy to reduce the incidence of M. genitalium in the absence of comprehensive routine screening. The high rate of dual resistance also highlights the need to ensure the availability of alternative antibiotics for the treatment of multi-drug resistant M. genitalium isolates.


Assuntos
Antibacterianos/farmacologia , Infecções por Chlamydia/diagnóstico , Chlamydia trachomatis/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla , Infecções por Mycoplasma/diagnóstico , Mycoplasma genitalium/efeitos dos fármacos , Instituições de Assistência Ambulatorial , Antibacterianos/uso terapêutico , Infecções por Chlamydia/complicações , Infecções por Chlamydia/tratamento farmacológico , Chlamydia trachomatis/genética , Chlamydia trachomatis/isolamento & purificação , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Farmacorresistência Bacteriana Múltipla/genética , Fluoroquinolonas/farmacologia , Fluoroquinolonas/uso terapêutico , Humanos , Macrolídeos/farmacologia , Macrolídeos/uso terapêutico , Infecções por Mycoplasma/complicações , Infecções por Mycoplasma/tratamento farmacológico , Infecções por Mycoplasma/epidemiologia , Mycoplasma genitalium/genética , Mycoplasma genitalium/isolamento & purificação , Prevalência , RNA Ribossômico 23S/química , RNA Ribossômico 23S/genética , RNA Ribossômico 23S/metabolismo , Análise de Sequência de DNA , Singapura/epidemiologia , Uretra/microbiologia
16.
Ann Vasc Surg ; 68: 569.e13-569.e20, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32339680

RESUMO

Melioidosis abdominal aortic aneurysm and splenic abscesses lead to poor prognosis and high mortality rate as high as 50% due to delayed/missed diagnosis. We describe an attempt to identify Burkholderia pseudomallei immediately, which was confirmed by polymerase chain reaction (PCR) and gene sequence analysis of 23S rRNA gene. PCR is not only an unambiguous identification of B. pseudomallei but also a rapid detection because B. pseudomallei may not be readily isolated. For patients of melioidosis abdominal aortic aneurysm with spleen abscess, prolonged antibiotic therapy, splenectomy and artificial vessel replacement provided an excellent result in our study. The progression, roentgenographic findings and histopathology character of melioidosis are similar to those of tuberculosis disease. PCR is useful to differentiate B. pseudomallei from Mycobacterium tuberculosis.


Assuntos
Aneurisma Infectado/microbiologia , Aneurisma da Aorta Abdominal/microbiologia , Burkholderia pseudomallei/genética , Melioidose/microbiologia , Reação em Cadeia da Polimerase , RNA Bacteriano/genética , RNA Ribossômico 23S/genética , Ribotipagem , Abscesso Abdominal/diagnóstico , Abscesso Abdominal/microbiologia , Aneurisma Infectado/diagnóstico por imagem , Aneurisma Infectado/cirurgia , Aneurisma da Aorta Abdominal/diagnóstico por imagem , Aneurisma da Aorta Abdominal/cirurgia , Implante de Prótese Vascular , Humanos , Masculino , Melioidose/diagnóstico , Melioidose/cirurgia , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Esplenopatias/diagnóstico , Esplenopatias/microbiologia
17.
Nat Commun ; 11(1): 1858, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32313034

RESUMO

Ribosome engineering is a powerful approach for expanding the catalytic potential of the protein synthesis apparatus. Due to the potential detriment the properties of the engineered ribosome may have on the cell, the designer ribosome needs to be functionally isolated from the translation machinery synthesizing cellular proteins. One solution to this problem was offered by Ribo-T, an engineered ribosome with tethered subunits which, while producing a desired protein, could be excluded from general translation. Here, we provide a conceptually different design of a cell with two orthogonal protein synthesis systems, where Ribo-T produces the proteome, while the dissociable ribosome is committed to the translation of a specific mRNA. The utility of this system is illustrated by generating a comprehensive collection of mutants with alterations at every rRNA nucleotide of the peptidyl transferase center and isolating gain-of-function variants that enable the ribosome to overcome the translation termination blockage imposed by an arrest peptide.


Assuntos
Bactérias/metabolismo , Engenharia de Proteínas/métodos , Ribossomos/química , Biologia Sintética/métodos , Alelos , Sistema Livre de Células , Cristalografia por Raios X , Modelos Moleculares , Modelos Teóricos , Conformação Molecular , Mutação , Peptídeos/química , Peptidil Transferases/química , Plasmídeos/genética , Biossíntese de Proteínas , Proteoma , RNA Mensageiro/genética , RNA Ribossômico/genética , RNA Ribossômico 23S/genética , Thermus thermophilus/química
18.
J Appl Microbiol ; 129(3): 738-752, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32155682

RESUMO

AIMS: To explore a prokaryotic species-specific DNA marker, 16S-23S rRNA gene internal transcribed spacer (ITS) sequence for identification and classification of Vibrio. METHODS AND RESULTS: Five hundred and seventy four ITS sequences from 60 Vibrio strains were collected, then the primary and secondary structures of ITS sequence were analysed. The ITS was divided into several subunits, and the species-specificity of these subunits were evaluated by blast. The variable subunit of ITS showed high species-specificity. A protocol to identify a Vibrio species based on ITS analysis was developed and verified. Both the specificity and sensitivity were 100%. The phylogeny analysis of Vibrio based on ITS showed that ITS devised a better classification than 16S rDNA. Finally, an identification method of Vibrio based on ITS sequencing in food samples was developed and evaluated. The results of ITS sequencing were (100%) consistent with the results identified by ISO standard. CONCLUSIONS: Vibrio could be accurately identified at the species level by using the ITS sequences. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study suggests that the ITS can be considered as a significant DNA marker for identification and classification of Vibrio species, and it posed a new path to screen the Vibrio in food sample.


Assuntos
DNA Espaçador Ribossômico/genética , Vibrio/classificação , Vibrio/isolamento & purificação , DNA Bacteriano/genética , Genes Bacterianos/genética , Filogenia , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , Especificidade da Espécie , Vibrio/genética
19.
Int J Infect Dis ; 96: 121-127, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32173573

RESUMO

OBJECTIVE: The aim of this study was to investigate the relationships between treatment outcomes of patients with urogenital Chlamydia trachomatis infections and minimum inhibitory concentrations (MICs) and drug resistance genes. METHODS: The clinical data of 92 patients diagnosed with Chlamydia trachomatis (C. trachomatis) infections were collected. Of these patients, 28 received regular treatment with azithromycin and 64 received minocycline. All patients underwent three monthly follow-ups after the completion of treatment. The microdilution method was used for the in vitro susceptibility tests. The acquisition of 23S rRNA mutations and presence of the tet(M) gene were detected by gene amplification and sequencing. RESULTS: The MICs of azithromycin, clarithromycin, erythromycin, tetracycline, doxycycline, and minocycline were comparable for isolates from the treatment failure and treatment success groups. Higher detection rates of 23S rRNA gene mutations and tet(M) were found in the treatment failure group (57.14% and 71.43%, respectively) than in the treatment success group (14.29% and 30.23%, respectively) (p < 0.05). The A2057G, C2452A, and T2611C gene mutations of 23S rRNA were detected in eight clinical isolates from the azithromycin treatment failure group, while the T2611C gene mutation was detected in one clinical strain from the treatment success group. CONCLUSIONS: The detection of resistance genes could better explain the high treatment failure rate than the MIC results in patients with urogenital C. trachomatis infections, highlighting the need for genetic antimicrobial resistance testing in infected patients.


Assuntos
Infecções por Chlamydia/tratamento farmacológico , Chlamydia trachomatis/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Adulto , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Azitromicina/farmacologia , Azitromicina/uso terapêutico , Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/genética , Chlamydia trachomatis/isolamento & purificação , Feminino , Doenças dos Genitais Femininos/tratamento farmacológico , Doenças dos Genitais Femininos/microbiologia , Doenças dos Genitais Masculinos/tratamento farmacológico , Doenças dos Genitais Masculinos/microbiologia , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Minociclina/farmacologia , Minociclina/uso terapêutico , RNA Ribossômico 23S/genética , Falha de Tratamento , Infecções Urinárias/tratamento farmacológico , Infecções Urinárias/microbiologia , Adulto Jovem
20.
Syst Appl Microbiol ; 43(3): 126074, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32169316

RESUMO

The Mediterranean world is the cradle for the diversification of a large number of plant species, including legumes belonging to the Tribe Genisteae. Nodule bacteria from three species of Genista legumes indigenous to northwestern Africa (G. ferox, G. numidica, G. tricuspidata) were sampled across a 150km region of Algeria in order to investigate symbiotic relationships. Partial 23S rRNA sequences from 107 isolates indicated that Bradyrhizobium was the predominant symbiont genus (96% of isolates), with the remainder belonging to Rhizobium or Mesorhizobium. A multilocus sequence analysis on 46 Bradyrhizobium strains using seven housekeeping (HK) genes showed that strains were differentiated into multiple clades with affinities to seven species: B. canariense (17 isolates), B. japonicum (2), B. ottawaense (2), B. cytisi/B. rifense (9), 'B. valentinum' (5), and B. algeriense (11). Extensive discordance between the HK gene phylogeny and a tree for four loci in the symbiosis island (SI) region implied that horizontal transfer of SI loci has been common. Cases of close symbiont relationship across pairs of legumes hosts were evident, with 33% of isolates having as their closest relative a strain sampled from a different Genista species. Nevertheless, tree permutation tests also showed that there was substantial host-related phylogenetic clustering. Thus, each of the three Genista hosts utilized a measurably different array of bacterial lineages.


Assuntos
Bradyrhizobium/classificação , Genista/microbiologia , Nódulos Radiculares de Plantas/microbiologia , Teorema de Bayes , Bradyrhizobium/genética , Bradyrhizobium/fisiologia , Biologia Computacional/métodos , Genes Essenciais , Genista/classificação , Haplótipos , Tipagem de Sequências Multilocus , Filogenia , Locos de Características Quantitativas , RNA Ribossômico 23S/genética , Microbiologia do Solo , Simbiose
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