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1.
BMC Microbiol ; 21(1): 266, 2021 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-34607564

RESUMO

BACKGROUND: The anoxic redox control binary system plays an important role in the response to oxygen as a signal in the environment. In particular, phosphorylated ArcA, as a global transcription factor, binds to the promoter regions of its target genes to regulate the expression of aerobic and anaerobic metabolism genes. However, the function of ArcA in Plesiomonas shigelloides is unknown. RESULTS: In the present study, P. shigelloides was used as the research object. The differences in growth, motility, biofilm formation, and virulence between the WT strain and the ΔarcA isogenic deletion mutant strain were compared. The data showed that the absence of arcA not only caused growth retardation of P. shigelloides in the log phase, but also greatly reduced the glucose utilization in M9 medium before the stationary phase. The motility of the ΔarcA mutant strain was either greatly reduced when grown in swim agar, or basically lost when grown in swarm agar. The electrophoretic mobility shift assay results showed that ArcA bound to the promoter regions of the flaK, rpoN, and cheV genes, indicating that ArcA directly regulates the expression of these three motility-related genes in P. shigelloides. Meanwhile, the ability of the ΔarcA strain to infect Caco-2 cells was reduced by 40%; on the contrary, its biofilm formation was enhanced. Furthermore, the complementation of the WT arcA gene from pBAD33-arcA+ was constructed and all of the above features of the pBAD33-arcA+ complemented strain were restored to the WT level. CONCLUSIONS: We showed the effect of ArcA on the growth, motility, biofilm formation, and virulence of Plesiomonas shigelloides, and demonstrated that ArcA functions as a positive regulator controls the motility of P. shigelloides by directly regulating the expression of flaK, rpoN and cheV genes.

2.
Breast Cancer Res Treat ; 189(2): 533-539, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34196900

RESUMO

PURPOSE: Mutations in hereditary breast cancer genes play an important role in the risk for cancer. METHODS: Cancer susceptibility genes were sequenced in 664 unselected breast cancer cases from Guatemala. Variants were annotated with ClinVar and VarSome. RESULTS: A total of 73 out of 664 subjects (11%) had a pathogenic variant in a high or moderate penetrance gene. The most frequently mutated genes were BRCA1 (37/664, 5.6%) followed by BRCA2 (15/664, 2.3%), PALB2 (5/664, 0.8%), and TP53 (5/664, 0.8%). Pathogenic variants were also detected in the moderate penetrance genes ATM, BARD1, CHEK2, and MSH6. The high ratio of BRCA1/BRCA2 mutations is due to two potential founder mutations: BRCA1 c.212 + 1G > A splice mutation (15 cases) and BRCA1 c.799delT (9 cases). Cases with pathogenic mutations had a significantly earlier age at diagnosis (45 vs 51 years, P < 0.001), are more likely to have had diagnosis before menopause, and a higher percentage had a relative with any cancer (51% vs 37%, P = 0.038) or breast cancer (33% vs 15%, P < 0.001). CONCLUSIONS: Hereditary breast cancer mutations were observed among Guatemalan women, and these women are more likely to have early age at diagnosis and family history of cancer. These data suggest the use of genetic testing in breast cancer patients and those at high risk as part of a strategy to reduce breast cancer mortality in Guatemala.


Assuntos
Neoplasias da Mama , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/epidemiologia , Neoplasias da Mama/genética , Feminino , Genes BRCA2 , Células Germinativas , Guatemala , Humanos
3.
J Microbiol Biotechnol ; 31(4): 520-528, 2021 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-33746188

RESUMO

Plesiomonas shigelloides, a member of the family Vibrionaceae, is a gram-negative, rod-shaped, facultative anaerobic bacterium with flagella. P. shigelloides has been isolated from such sources as freshwater, surface water, and many wild and domestic animals. P. shigelloides contains 102 Oantigens and 51 H-antigens. The diversity of O-antigen gene clusters is relatively poorly understood. In addition to O1 and O17 reported by other laboratories, and the 12 O serogroups (O2, O10, O12, O23, O25, O26, O32, O33, O34, O66, O75, and O76) reported previously by us, in the present study, nine new P. shigelloides serogroups (O8, O17, O18, O37, O38, O39, O44, O45, and O61) were sequenced and annotated. The genes for the O-antigens of these nine groups are clustered together in the chromosome between rep and aqpZ. Only O38 possesses the wzm and wzt genes for the synthesis and translocation of O-antigens via the ATP-binding cassette (ABC) transporter pathway; the other eight use the Wzx/Wzy pathway. Phylogenetic analysis using wzx and wzy showed that both genes are diversified. Among the nine new P. shigelloides serogroups, eight use wzx/wzy genes as targets. In addition, we developed an O-antigen-specific PCR assay to detect these nine distinct serogroups with no cross reactions among them.


Assuntos
Família Multigênica , Antígenos O/genética , Plesiomonas/classificação , Sorotipagem , Filogenia , Plesiomonas/genética , Reação em Cadeia da Polimerase , Análise de Sequência de DNA
4.
J Med Genet ; 2021 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397747

RESUMO

High-quality interpretation of BRCA1/2 variants plays a critical role in the clinical practice of precision medicine. However, a comprehensive system to evaluate the quality and accuracy of variant interpretation has yet to be established. This study investigates the performance of an interpretation system in evaluating the capacities of BRCA1/2 interpretation among distinct laboratories in China. The evaluation system is based on a reference database that contains 750 different variants in BRCA1/2 Evaluation was performed among 41 laboratories in China. We classified their performance into five levels. Only level A was considered qualified. This level allows for a 0.3% error rate for clinical decision-related misinterpretation; 26 of 41 laboratories (63%) met the qualified standard, while 7 laboratories were at levels D and E, which indicated egregious mistakes and systemic problems in variant interpretation. Due to strict quality demands, the interpretation of several variants was amended, which largely influenced the quality rate. The number of qualified laboratories would decrease from 26 to 17 if those incorrect recommended interpretations were not corrected. This evaluation system provides a potential approach for standardisation of variant interpretation and lowers the discordance of variant interpretation between different laboratories. A well-designed interpretation ability evaluation is essential to evaluate the interpretation level of laboratories before they provide service in real-world clinical settings.

5.
Microb Pathog ; 147: 104443, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32777352

RESUMO

Vibrio cholerae is a natural inhabitant of aquatic environments and causes the epidemic diarrheal disease known as cholera. Fatty acid metabolism is closely related to the pathogenicity of V. cholerae. The TetR family transcriptional repressor PsrA regulates the ß-oxidation pathway in Pseudomonas aeruginosa; however, little is known about its regulation in V. cholerae. In this study, qRT-PCR revealed that the expression of vc1741 (psrA) increased 40-fold in the small intestines of infant mice compared with that grown in LB medium. The Δvc1741 mutant showed a significant defected in the ability to colonize the small intestines of infant mice with a competitive index (CI) of 0.53. EMSAs indicated that VC1741 could directly bind to the promoter regions of vc1741-fadE1, fadBA, and fadIJ operons, and these bindings were reversed upon addition of the long-chain fatty acid (LCFA), oleic acid. The expression levels of the fadB, fadA, fadI, and fadJ genes were all elevated by approximately 2-fold in the Δvc1741 mutant strain compared with that in the wild-type strain in LB medium, indicating that VC1741 is a repressor for these genes involved in fatty acid degradation. Moreover, ΔfadBA, ΔfadB, and ΔfadA isogenic mutants showed defective abilities to colonize the small intestines of infant mice, with CI values of 0.64, 0.73, and 0.74, respectively. These data provided a mechanistic model in which LCFAs affect the expression of VC1741 to control fatty acid degradation and virulence in V. cholerae.


Assuntos
Vibrio cholerae , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ácidos Graxos , Regulação Bacteriana da Expressão Gênica , Intestinos , Camundongos , Ácido Oleico , Vibrio cholerae/genética , Vibrio cholerae/metabolismo
6.
Microb Pathog ; 144: 104197, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32283260

RESUMO

Vibrio cholerae, the agent of severe diarrheal disease cholera, is known to form biofilm to persist in the environmental and the host,s intestines. The bacteria execute a complex regulatory pathway producing virulence factors that allow colonization and cause disease in response to environmental signals in the intestine, including low oxygen-limited condition. VpsR and VpsT are primary regulators of the biofilm formation-regulatory network. In this study, we determined that anaerobic induction enhanced biofilm formation via the two component system, ArcB/A, which functions as a positive regulator of toxT expression. The biofilm formation has reduced approximately 2.4-fold in the ΔarcA mutant compared to the wild type in anaerobic condition. Chip-qPCR and EMSA assays confirmed that ArcA can bind directly to the vpsT promoter and then activates the expression of biofilm formation related genes, vpsA-K and vpsL-Q. Meanwhile, the ΔarcA mutant decreased the ability of colonization in intestine with CI (competition index) of 0.27 compared to wild type strain. These results suggest that ArcA links the expression of virulence and biofilm synthesis genes during anaerobic condition, and contributes to understand the complex relationship between biofilm formation and the intestinal signals during infection.


Assuntos
Anaerobiose , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Proteínas Repressoras/genética , Vibrio cholerae/genética , Vibrio cholerae/metabolismo , Fatores de Virulência/genética , Cólera/microbiologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Oxigênio/farmacologia , Regiões Promotoras Genéticas , Virulência/genética , Fatores de Virulência/metabolismo
7.
Environ Microbiol ; 22(10): 4231-4243, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-31868254

RESUMO

Vibrio cholerae is a waterborne bacterium responsible for worldwide outbreaks of acute and fatal cholera. Recently, small regulatory RNAs (sRNAs) have become increasingly recognized as important regulators of virulence gene expression in response to environmental signals. In this study, we determined that two-component system EnvZ/OmpR was required for intestinal colonization in V. cholerae O1 EI Tor strain E12382. Analysis of the characteristics of OmpR revealed a potential binding site in the intergenic region between vc1470 and vc1471, and qRT-PCR showed that expression of the intergenic region increased 5.3-fold in the small intestine compared to LB medium. Race and northern blot assays were performed and demonstrated a new sRNA, coaR (cholerae osmolarity and acidity related regulatory RNA). A ΔcoaR mutant showed a deficient colonization ability in small intestine with CI of 0.15. We identified a target of coaR, tcpI, a negative regulator of the major pilin subunit of TcpA. The ΔtcpI mutant has an increased colonization with CI of 3.16. The expression of coaR increased 2.8-fold and 3.3-fold under relative acidic and hypertonic condition. In summary, coaR was induced under the condition of high osmolarity and acid stress via EnvZ/OmpR and explained that tcpI relieves pH-mediated repression of toxin co-regulated pilus synthesis.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Fímbrias/metabolismo , Intestinos/microbiologia , RNA Bacteriano/genética , Transativadores/metabolismo , Vibrio cholerae/patogenicidade , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Cólera/microbiologia , Cólera/patologia , Toxina da Cólera/genética , Proteínas de Fímbrias/biossíntese , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Humanos , Camundongos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transativadores/genética , Vibrio cholerae/genética , Vibrio cholerae/metabolismo , Virulência/genética
8.
Front Microbiol ; 10: 741, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31024508

RESUMO

Plesiomonas shigelloides is a Gram-negative, flagellated, rod-shaped, ubiquitous, and facultative anaerobic bacterium. It has been isolated from various sources, such as freshwater, surface water, and many wild and domestic animals. P. shigelloides is associated with diarrheal diseases of acute secretory gastroenteritis, an invasive shigellosis-like disease, and a cholera-like illness in humans. At present, 102 somatic antigens and 51 flagellar antigens of P. shigelloides have been recognized; however, very little is known about variations of O-antigens among P. shigelloides species. In this study, 12 O-antigen gene clusters of P. shigelloides, O2H1a1c (G5877), O10H41 (G5892), O12H35 (G5890), O23H1a1c (G5263), O25H3 (G5879), O26H1a1c (G5889), O32H37 (G5880), O33H38 (G5881), O34H34 (G5882), O66H3 (G5270), O75H34 (G5885), and O76H39 (G5886), were sequenced and analyzed. The genes that control O-antigen synthesis are present as chromosomal gene clusters that maps between rep and aqpZ, and most of the synthesis and translocation of OPS (O-specific polysaccharide) belongs to Wzx/Wzy pathway with the exception of O12, O25, and O66, which use the ATP-binding cassette (ABC) transporter pathway. Phylogenetic analysis of wzx and wzy show that the wzx and wzy genes are specific to individual O-antigens and can be used as targets in molecular typing. Based on the sequence data, an O-antigen specific suspension array that detects 12 distinct OPS' has been developed. This is the first report to catalog the genetic features of P. shigelloides O-antigen variations and develop a suspension array for the molecular typing. The method has several advantages over traditional bacteriophage and serum agglutination methods and lays the foundation for easier identification and detection of additional O-antigen in the future.

9.
J Mol Diagn ; 21(4): 677-686, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31026599

RESUMO

The absence of interpretation guidelines and limited data on BRCA1/2 mutations in the Chinese population have impeded the detection of BRCA variants based on next-generation sequencing (NGS) in China. This study was performed to establish a reference system for performance evaluation of BRCA genetic testing and variant interpretation, which includes interpretation rules, reference materials (RMs), and a reference database (RD). BRCA1/2 mutations identified in cell lines and clinical cases were selected to establish RMs. All mutations were detected by NGS and validated by Sanger sequencing. Variant call format files and standard variant data sets were collected and annotated to build the RD. Participant laboratories were invited to validate this reference system. Interpretation rules for BRCA variants in the Chinese population were generated as a standard for BRCA variant interpretation. Mutational analysis demonstrated that BRCA2 mutations (55%) were more common than BRCA1 mutations (45%) in Chinese patients. Eliminating duplicates from 19,886 variants, the RD contained 750 unique BRCA mutations. Most BRCA1/2 mutations in the reference system were pathogenic or likely pathogenic (RMs, 77.5%; RD, 57%). In total, 91 novel pathogenic/likely pathogenic variants were identified in the RD. The reference system can contribute to NGS performance and high-quality interpretation to facilitate clinical decision making. It could also accelerate the development and application of BRCA mutation detection technologies in China.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Análise Mutacional de DNA , Genética Populacional , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Grupo com Ancestrais do Continente Asiático/genética , China , Bases de Dados Genéticas , Humanos , Navegador
10.
J Microbiol Methods ; 159: 75-80, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30817946

RESUMO

Plesiomonas shigelloides is widely associated with human diarrheal disease. Research on this pathogen has been hampered by the absence of an effective genetic manipulation system. In the present study, an efficient and precise conjugation transfer procedure, mediated by suicide vector pRE112 was used to overcome this limitation. The efficiency of generating double recombinants was average 74.3%, and the conjugation protocol may be applied to other P. shigelloides strains. We also identified that the SipD protein of P. shigelloides G5884 (serotype O45) is 65% similar to the SipD in Salmonella pathogenicity island 1 (SPI-1), which is a key element of the type III secretion system related to Salmonella invasion. A P. shigelloides sipD null mutant was generated via the conjugation system, using the suicide vector pRE112. The isogenic mutant strain lacking sipD showed a 50% reduction in its capacity to invade Caco-2 cells.


Assuntos
Proteínas de Bactérias/genética , Técnicas de Transferência de Genes , Plesiomonas/genética , Conjugação Genética , Mutação
11.
Am J Physiol Gastrointest Liver Physiol ; 316(4): G527-G538, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30789748

RESUMO

Hepatic steatosis is the beginning phase of nonalcoholic fatty liver disease, and hyperhomocysteinemia (HHcy) is a significant risk factor. Soluble epoxide hydrolase (sEH) hydrolyzes epoxyeicosatrienoic acids (EETs) and other epoxy fatty acids, attenuating their cardiovascular protective effects. However, the involvement of sEH in HHcy-induced hepatic steatosis is unknown. The current study aimed to explore the role of sEH in HHcy-induced lipid disorder. We fed 6-wk-old male mice a chow diet or 2% (wt/wt) high-metnionine diet for 8 wk to establish the HHcy model. A high level of homocysteine induced lipid accumulation in vivo and in vitro, which was concomitant with the increased activity and expression of sEH. Treatment with a highly selective specific sEH inhibitor (0.8 mg·kg-1·day-1 for the animal model and 1 µM for cells) prevented HHcy-induced lipid accumulation in vivo and in vitro. Inhibition of sEH activated the peroxisome proliferator-activated receptor-α (PPAR-α), as evidenced by elevated ß-oxidation of fatty acids and the expression of PPAR-α target genes in HHcy-induced hepatic steatosis. In primary cultured hepatocytes, the effect of sEH inhibition on PPAR-α activation was further confirmed by a marked increase in PPAR-response element luciferase activity, which was reversed by knock down of PPAR-α. Of note, 11,12-EET ligand dependently activated PPAR-α. Thus increased sEH activity is a key determinant in the pathogenesis of HHcy-induced hepatic steatosis, and sEH inhibition could be an effective treatment for HHcy-induced hepatic steatosis. NEW & NOTEWORTHY In the current study, we demonstrated that upregulation of soluble epoxide hydrolase (sEH) is involved in the hyperhomocysteinemia (HHcy)-caused hepatic steatosis in an HHcy mouse model and in murine primary hepatocytes. Improving hepatic steatosis in HHcy mice by pharmacological inhibition of sEH to activate peroxisome proliferator-activated receptor-α was ligand dependent, and sEH could be a potential therapeutic target for the treatment of nonalcoholic fatty liver disease.


Assuntos
Inibidores Enzimáticos/farmacocinética , Epóxido Hidrolases , Ácidos Graxos/metabolismo , Fígado Gorduroso , Hiper-Homocisteinemia , PPAR alfa/metabolismo , Animais , Modelos Animais de Doenças , Descoberta de Drogas , Epóxido Hidrolases/antagonistas & inibidores , Epóxido Hidrolases/metabolismo , Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/enzimologia , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Hiper-Homocisteinemia/complicações , Hiper-Homocisteinemia/metabolismo , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Regulação para Cima
12.
Cell Biol Toxicol ; 35(1): 59-66, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30430365

RESUMO

Circulating tumor cells (CTCs) have important application prospects in the early diagnosis, treatment evaluation, and prognostic prediction of tumors. In this study, we enrolled a total of 65 patients with different stages and molecular subtypes of breast cancer and isolated and enriched for CTCs from peripheral blood using the ClearCell FX1 platform, which is based on a label-free spiral microfluidic method. The ClearCell platform can successfully isolate CTCs from peripheral blood with different detection rates in breast cancer patients. We also compared the difference between the ClearCell and CellSearch platforms for isolating CTCs. To further determine the genetic information of CTCs, we performed single-cell whole-exome sequencing (WES) in three CTCs isolated from one patient. The sequencing results indicated the presence of a few hundreds of single-nucleotide variants (SNVs) in each CTC, with only 16 SNVs being shared by all three CTCs. These shared SNVs may have a crucial impact on the development of breast cancer. Here, we report, for the first time, the complete process and results of performing single-cell WES on CTCs isolated by the ClearCell FX1 platform.


Assuntos
Neoplasias da Mama/patologia , Microfluídica/métodos , Células Neoplásicas Circulantes/patologia , Neoplasias da Mama/genética , Feminino , Humanos , Sequenciamento Completo do Exoma
13.
Can J Microbiol ; 64(4): 231-241, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29357266

RESUMO

Capsular polysaccharides (CPSs), or K-antigens, are the major surface antigens of Escherichia coli. More than 80 serologically unique K-antigens are classified into 4 groups (Groups 1-4) of capsules. Groups 1 and 4 contain the Wzy-dependent polymerization pathway and the gene clusters are in the order galF to gnd; Groups 2 and 3 contain the ABC-transporter-dependent pathway and the gene clusters consist of 3 regions, regions 1, 2 and 3. Little is known about the variations among the gene clusters. In this study, 9 serotypes of K-antigen gene clusters (K2ab, K11, K20, K24, K38, K84, K92, K96, and K102) were sequenced and correlated with their CPS chemical structures. On the basis of sequence data, a K-antigen-specific suspension array that detects 10 distinct CPSs, including the above 9 CPSs plus K30, was developed. This is the first report to catalog the genetic features of E. coli K-antigen variations and to develop a suspension array for their molecular typing. The method has a number of advantages over traditional bacteriophage and serum agglutination methods and lays the foundation for straightforward identification and detection of additional K-antigens in the future.


Assuntos
Antígenos de Bactérias/genética , Antígenos de Superfície/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Variação Genética , Tipagem Molecular , Família Multigênica , DNA Bacteriano/genética , Antígenos O/genética , Reação em Cadeia da Polimerase , Polissacarídeos
14.
Antonie Van Leeuwenhoek ; 110(12): 1515-1525, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28695408

RESUMO

Among the 50 species and 70 serogroups of Legionella identified, Legionella pneumophila, comprising three subsp. (subsp. pneumophila, subsp. fraseri, and subsp. pasculleii), is recognized as the major cause of epidemic legionellosis. Rapid and reliable assays to identify pathogenic Legionella spp., and the three L. pneumophila subsp. in particular, are in great demand. In this study, we analyzed the gyrB genes of eleven Legionella spp. and subsp., comprising L. anisa, L. bozemanii, L. dumoffii, L. feeleii, L. gormanii, L. longbeachae, L. micdadei, L. waltersii, L. pneumophila subsp. pneumophila, L. pneumophila subsp. fraseri, and L. pneumophila subsp. pasculleii. We developed a rapid oligonucleotide microarray detection technique to identify accurately these common pathogenic Legionella spp. and L. pneumophila subsp. To detect multiple Legionella species with high specificity, 31 reproducible probes were designed in the array. Sixty-one strains were analyzed in total, including 37 target pathogens and 24 non-target bacterial species used to validate the microarray. The sensitivity of the detection was 1.0 ng using genomic DNA of three Legionella spp., L. anisa, L. dumoffii, and L. waltersii, or 13 CFU/100 mL using the cultured L. pneumophila subsp. pneumophila. Eight isolated strains were tested using the microarray with 100% accuracy. The data indicated that the technique is an efficient method to diagnose and detect Legionella spp. and subsp. in basic microbiology, clinical diagnosis, epidemiological surveillance, and food safety applications. In addition, a phylogenetic study based on the gyrB gene revealed the genetic relationship among the different Legionella spp. and subsp.


Assuntos
DNA Girase/genética , Legionella pneumophila/classificação , Legionella pneumophila/genética , Legionella/classificação , Legionella/genética , Análise de Sequência com Séries de Oligonucleotídeos , Microbiologia Ambiental , Humanos , Legionelose/diagnóstico , Legionelose/microbiologia , Doença dos Legionários/diagnóstico , Doença dos Legionários/microbiologia , Filogenia , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade
15.
Proc Natl Acad Sci U S A ; 113(48): E7730-E7739, 2016 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-27849586

RESUMO

Vibrio cholerae has caused seven cholera pandemics since 1817, imposing terror on much of the world, but bacterial strains are currently only available for the sixth and seventh pandemics. The El Tor biotype seventh pandemic began in 1961 in Indonesia, but did not originate directly from the classical biotype sixth-pandemic strain. Previous studies focused mainly on the spread of the seventh pandemic after 1970. Here, we analyze in unprecedented detail the origin, evolution, and transition to pandemicity of the seventh-pandemic strain. We used high-resolution comparative genomic analysis of strains collected from 1930 to 1964, covering the evolution from the first available El Tor biotype strain to the start of the seventh pandemic. We define six stages leading to the pandemic strain and reveal all key events. The seventh pandemic originated from a nonpathogenic strain in the Middle East, first observed in 1897. It subsequently underwent explosive diversification, including the spawning of the pandemic lineage. This rapid diversification suggests that, when first observed, the strain had only recently arrived in the Middle East, possibly from the Asian homeland of cholera. The lineage migrated to Makassar, Indonesia, where it gained the important virulence-associated elements Vibrio seventh pandemic island I (VSP-I), VSP-II, and El Tor type cholera toxin prophage by 1954, and it then became pandemic in 1961 after only 12 additional mutations. Our data indicate that specific niches in the Middle East and Makassar were important in generating the pandemic strain by providing gene sources and the driving forces for genetic events.


Assuntos
Cólera/epidemiologia , Pandemias , Vibrio cholerae/genética , Cólera/microbiologia , Evolução Molecular , Genoma Bacteriano , Humanos , Anotação de Sequência Molecular , Filogenia , Polimorfismo de Nucleotídeo Único
16.
PLoS One ; 11(5): e0155115, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27171009

RESUMO

Hafnia alvei is a facultative and rod-shaped gram-negative bacterium that belongs to the Enterobacteriaceae family. Although it has been more than 50 years since the genus was identified, very little is known about variations among Hafnia species. Diversity in O-antigens (O-polysaccharide, OPS) is thought to be a major factor in bacterial adaptation to different hosts and situations and variability in the environment. Antigenic variation is also an important factor in pathogenicity that has been used to define clones within a number of species. The genes that are required to synthesize OPS are always clustered within the bacterial chromosome. A serotyping scheme including 39 O-serotypes has been proposed for H. alvei, but it has not been correlated with known OPS structures, and no previous report has described the genetic features of OPS. In this study, we obtained the genome sequences of 21 H. alvei strains (as defined by previous immunochemical studies) with different lipopolysaccharides. This is the first study to show that the O-antigen gene cluster in H. alvei is located between mpo and gnd in the chromosome. All 21 of the OPS gene clusters contain both the wzx gene and the wzy gene and display a large number of polymorphisms. We developed an O serotype-specific wzy-based suspension array to detect all 21 of the distinct OPS forms we identified in H. alvei. To the best of our knowledge, this is the first report to identify the genetic features of H. alvei antigenic variation and to develop a molecular technique to identify and classify different serotypes.


Assuntos
Variação Genética , Hafnia alvei/classificação , Hafnia alvei/genética , Antígenos O/genética , Sorotipagem/métodos , Vias Biossintéticas , DNA Bacteriano/genética , Genoma Bacteriano , Hafnia alvei/imunologia , Família Multigênica , Antígenos O/química , Filogenia , Reação em Cadeia da Polimerase , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
17.
Antonie Van Leeuwenhoek ; 108(6): 1405-1423, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26415652

RESUMO

The Legionella pneumophila serogroups O1, O4, O6, O7, O10 and O13 are pathogenic strains associated with pneumonia. The surface O-antigen gene clusters of L. pneumophila serogroups O4, O6, O7, O10 and O13 were sequenced and analyzed, with the function annotated on the basis of homology to that of the genes of L. pneumophila serogroup O1 (L. pneumophila subsp. pneumophila str. Philadelphia 1). The gene locus of the six L. pneumophila serogroups contains genes of yvfE, neuABCD, pseA-like for nucleotide sugar biosynthesis, wecA for sugar transfer, and wzm as well as wzt for O-antigen processing. The detection of O-antigen genes allows the fine differentiation at species and serogroup level without the neccessity of nucleotide sequencing. The O-antigen-processing genes wzm and wzt, which were found to be distinctive for different for different serogroups, have been used as the target genes for the detection and identification of L. pneumophila strains of different O serogroups. In this report, a multiplex PCR assay based on wzm or wzt that diferentiates all the six serogroups by amplicon size was developed with the newly designed specific primer pairs for O1 and O7, and the specific primer pairs for O4, O6, O10, and O13 reported previously. The array was validated by analysis of 34 strains including 15 L. pneumophila O-standard reference strains, eight reference strains of other Legionella non-pneumophila species, six other bacterial species, and five L. pneumophila environmental isolates. The detection sensitivity was one ng genomic DNA. The accurate and sensitive assay is suitable for the identification and detection of strains of these serogroups in environmental and clinical samples.


Assuntos
Legionella pneumophila/classificação , Legionella pneumophila/genética , Técnicas de Diagnóstico Molecular/métodos , Tipagem Molecular/métodos , Família Multigênica , Reação em Cadeia da Polimerase Multiplex/métodos , Antígenos O/genética , Primers do DNA/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Sensibilidade e Especificidade , Análise de Sequência de DNA , Sorogrupo
18.
PLoS One ; 10(7): e0133927, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26208181

RESUMO

This study established a multiplex PCR-based microarray to detect simultaneously a diverse panel of 17 sexually transmitted diseases (STDs)-associated pathogens including Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma, Herpes simplex virus (HSV) types 1 and 2, and Human papillomavirus (HPV) types 6, 11, 16, 18, 31, 33, 35, 39, 54 and 58. The target genes are 16S rRNA gene for N. gonorrhoeae, M. genitalium, M. hominism, and Ureaplasma, the major outer membrane protein gene (ompA) for C. trachomatis, the glycoprotein B gene (gB) for HSV; and the L1 gene for HPV. A total of 34 probes were selected for the microarray including 31 specific probes, one as positive control, one as negative control, and one as positional control probe for printing reference. The microarray is specific as the commensal and pathogenic microbes (and closely related organisms) in the genitourinary tract did not cross-react with the microarray probes. The microarray is 10 times more sensitive than that of the multiplex PCR. Among the 158 suspected HPV specimens examined, the microarray showed that 49 samples contained HPV, 21 samples contained Ureaplasma, 15 contained M. hominis, four contained C. trachomatis, and one contained N. gonorrhoeae. This work reports the development of the first high through-put detection system that identifies common pathogens associated with STDs from clinical samples, and paves the way for establishing a time-saving, accurate and high-throughput diagnostic tool for STDs.


Assuntos
Análise de Sequência com Séries de Oligonucleotídeos/métodos , Doenças Sexualmente Transmissíveis/diagnóstico , Doenças Sexualmente Transmissíveis/etiologia , Humanos , Reação em Cadeia da Polimerase Multiplex , RNA Ribossômico 16S/genética , Sensibilidade e Especificidade
19.
PLoS One ; 9(12): e113863, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25469776

RESUMO

Legionella pneumophila has been recognized as the major cause of legionellosis since the discovery of the deadly disease. Legionella spp. other than L. pneumophila were later found to be responsible to many non-pneumophila infections. The non-L. pneumophila infections are likely under-detected because of a lack of effective diagnosis. In this report, we have sequenced the 16S-23S rRNA gene internal transcribed spacer (ITS) of 10 Legionella species and subspecies, including L. anisa, L. bozemanii, L. dumoffii, L. fairfieldensis, L. gormanii, L. jordanis, L. maceachernii, L. micdadei, L. pneumophila subspp. fraseri and L. pneumophila subspp. pasculleii, and developed a rapid oligonucleotide microarray detection technique accordingly to identify 12 most common Legionella spp., which consist of 11 pathogenic species of L. anisa, L. bozemanii, L. dumoffii, L. gormanii, L. jordanis, L. longbeachae, L. maceachernii, L. micdadei, and L. pneumophila (including subspp. pneumophila, subspp. fraseri, and subspp. pasculleii) and one non-pathogenic species, L. fairfieldensis. Twenty-nine probes that reproducibly detected multiple Legionella species with high specificity were included in the array. A total of 52 strains, including 30 target pathogens and 22 non-target bacteria, were used to verify the oligonucleotide microarray assay. The sensitivity of the detection was at 1.0 ng with genomic DNA or 13 CFU/100 mL with Legionella cultures. The microarray detected seven samples of air conditioner-condensed water with 100% accuracy, validating the technique as a promising method for applications in basic microbiology, clinical diagnosis, food safety, and epidemiological surveillance. The phylogenetic study based on the ITS has also revealed that the non-pathogenic L. fairfieldensis is the closest to L. pneumophila than the nine other pathogenic Legionella spp.


Assuntos
DNA Bacteriano/genética , DNA Espaçador Ribossômico/genética , Legionella/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , DNA Bacteriano/química , DNA Espaçador Ribossômico/química , Humanos , Legionella/classificação , Legionelose/microbiologia , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , Análise de Sequência de DNA , Especificidade da Espécie
20.
J Microbiol Biotechnol ; 24(10): 1445-54, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24950883

RESUMO

Severe acute respiratory syndrome (SARS), a disease that spread widely in the world during late 2002 to 2004, severely threatened public health. Although there have been no reported infections since 2004, the extremely pathogenic SARS coronavirus (SARS-CoV), as the causative agent of SARS, has recently been identified in animals, showing the potential for the re-emergence of this disease. Previous studies showed that 27 single nucleotide polymorphism (SNP) mutations among the spike (S) gene of this virus are correlated closely with the SARS pathogenicity and epidemicity. We have developed a SNP DNA microarray in order to detect and genotype these SNPs, and to obtain related information on the pathogenicity and epidemicity of a given strain. The microarray was hybridized with PCR products amplified from cDNAs obtained from different SARS-CoV strains. We were able to detect 24 SNPs and determine the type of a given strain. The hybridization profile showed that 19 samples were detected and genotyped correctly by using our microarray, with 100% accuracy. Our microarray provides a novel method for the detection and epidemiological surveillance of SARS-CoV.


Assuntos
Técnicas de Genotipagem/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Polimorfismo de Nucleotídeo Único , Vírus da SARS/classificação , Vírus da SARS/genética , Virologia/métodos , Animais , Humanos , Epidemiologia Molecular/métodos
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