RESUMO
Shigella spp. are a leading cause of human diarrheal disease worldwide, with Shigella flexneri being the most frequently isolated species in developing countries. This serogroup is presently classified into 19 serotypes worldwide. We report here a multicenter validation of a multiplex-PCR-based strategy previously developed by Q. Sun, R. Lan, Y. Wang, A. Zhao, et al. (J Clin Microbiol 49:3766-3770, 2011) for molecular serotyping of S. flexneri This study was performed by seven international laboratories, with a panel of 71 strains (researchers were blind to their identity) as well as 279 strains collected from each laboratory's own local culture collections. This collaborative work found a high extent of agreement among laboratories, calculated through interrater reliability (IRR) measures for the PCR test that proved its robustness. Agreement with the traditional method (serology) was also observed in all laboratories for 14 serotypes studied, while specific genetic events could be responsible for the discrepancies among methodologies in the other 5 serotypes, as determined by PCR product sequencing in most of the cases. This work provided an empirical framework that allowed the use of this molecular method to serotype S. flexneri and showed several advantages over the traditional method of serological typing. These advantages included overcoming the problem of availability of suitable antisera in testing laboratories as well as facilitating the analysis of multiple samples at the same time. The method is also less time-consuming for completion and easier to implement in routine laboratories. We recommend that this PCR be adopted, as it is a reliable diagnostic and characterization methodology that can be used globally for laboratory-based shigella surveillance.
Assuntos
Reação em Cadeia da Polimerase Multiplex/métodos , Sorotipagem/métodos , Shigella flexneri/classificação , Técnicas de Tipagem Bacteriana/métodos , Técnicas de Tipagem Bacteriana/normas , DNA Bacteriano/genética , Humanos , Internacionalidade , Reação em Cadeia da Polimerase Multiplex/normas , Sorogrupo , Shigella flexneri/imunologiaRESUMO
Klebsiella pneumoniae (K. pneumoniae), as an important hospital-acquired bacterium, is responsible for severe morbidity and mortality among the elderly, newborn and immune-compromised people. We established a rcsA gene-based label-free multiple cross displacement amplification (MCDA) assay for rapid, simple and sensitive detection of K. pneumoniae by using lateral flow biosensor (LFB). MCDA reaction was conducted at a fixed temperature (65⯰C) for only 30â¯min, and amplification results were directly indicated using LFB. The results showed that reaction products were detectable from as little as 100â¯fg and 4.8â¯CFU of pure K. pneumoniae templates, and from approximately 480â¯CFU in 1â¯mL of spiked clinical samples. All K. pneumoniae strains examined were positive for label-free MCDA-LFB analysis, and all non-K. pneumoniae strains used in the report were negative for label-free MCDA-LFB assay, indicating the high selectivity of the label free MCDA-LFB assay. Furthermore, to remove false-positive results, the label-free MCDA-LFB assay was supplemented with antarctic thermal sensitive uracil-DNA-glycosylase (AUDG) to eliminate the carryover contamination. Thus, label-free MCDA-LFB assay complemented with AUDG enzyme was a rapid, simple, sensitive and reliable technique for detection of target pathogen, which has the ability to effectively avoid carryover contamination, and can be a valuable tool for "on-site" detection, clinical diagnosis, and primary quarantine purposes.
Assuntos
Técnicas Biossensoriais/métodos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Nanopartículas/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Regiões Antárticas , Técnicas Bacteriológicas/métodos , Sequência de Bases , DNA Bacteriano/análise , Genes Bacterianos/genética , Infecções por Klebsiella/diagnóstico , Sensibilidade e Especificidade , Temperatura , Fatores de Tempo , Uracila-DNA GlicosidaseRESUMO
The authors report on a loop-mediated isothermal amplification (LAMP) scheme that uses antarctic thermally sensitive uracil-DNA-glycosylase (AUDG) for simultaneous detection of nucleic acids and elimination of carryover contamination. It was applied in a lateral flow assay (LFA) format. The assay has attractive features in that it does not require the use of labeled primers or probes, and can eliminate false-positive results generated by unwanted hybridization between two labeled primers or between a labeled primer and probe. LAMP amplification and AUDG digestion are conducted in a single pot, and the application of a closed-tube reaction prevents false-positives due to carryover contamination. The method was applied to the detection of the human pathogen Streptococcus pneumoniaein in pure cultures and spiked blood samples. This LFA can detect S. pneumoniae in pure cultures with a 25 fg.µL-1 detection limit and in spiked blood samples with a 470 cfu.mL-1 detection limit. Conceivably, this assay can be applied to the detection of various other targets if the specific LAMP primers are available. Graphical abstract á .
Assuntos
Técnicas Biossensoriais/métodos , DNA Bacteriano/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/isolamento & purificação , Temperatura , Uracila-DNA Glicosidase/metabolismo , DNA Bacteriano/genética , HumanosRESUMO
Vibrio cholerae is an important human pathogen that is responsible for cholera, a severe acute watery diarrhea. In the current study, a multiple cross displacement amplification (MCDA) coupled with amplicon detection by chromatographic lateral flow biosensor (LFB) method (MCDA-LFB) was successfully established and evaluated for the identification of V. cholerae. A set of 10 primers was designed specifically to recognize 10 different regions of the V. cholerae-specific gene ompW. The optimized time and temperature conditions for the MCDA were 30 min and 63°C, respectively. The MCDA-LFB assay correctly identified 31 strains of V. cholerae but did not detect 13 non-cholerae Vibrio strains and 30 non-Vibrio strains. The sensitivity of MCDA-LFB for target pathogen detection in pure culture was 10 fg per reaction. In the case of spiked shrimp samples without enrichment, the limit of detection was 4.1 CFUs per reaction or equivalent to 4.1 × 102 CFU g-1. The whole process, including shrimp homogenates processing (30 min), MCDA reaction (30 min) and results reporting (2 min), could be finished within 65 min. These results show that this assay is suitable for the rapid, sensitive and specific detection of V. cholerae in food, environmental and clinical samples.
Assuntos
Técnicas Biossensoriais/instrumentação , Nanopartículas/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Vibrio cholerae/genética , Animais , Proteínas da Membrana Bacteriana Externa/genética , Desenho de Equipamento , Microbiologia de Alimentos , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Penaeidae/microbiologiaRESUMO
Loop-mediated isothermal amplification (LAMP) is the most popular technique to amplify nucleic acid sequence without the use of temperature cycling. However, LAMP is often confounded by false-positive results, arising from interactions between (hetero-dimer) or within (self-dimerization) primers, off-target hybrids and carryover contaminants. Here, we devised a new LAMP technique that is self-avoiding molecular recognition system (SAMRS) components and antarctic thermal sensitive uracil-DNA-glycosylase (AUDG) enzyme-assisted, termed AUDG-SAMRS-LAMP. Incorporating SAMRS components into 3'-ends of LAMP primers can improve assay's specificity, which completely prevents the non-specific amplification yielding from off-target hybrids and undesired interactions between or within primers. Adding AUDG into reaction mixtures can effectively eliminate the false-positive results arising from carryover contamination, thus the genuine positive reactions are generated from the amplification of target templates. Furthermore, AUDG-SAMRS-LAMP results are confirmed using a new analysis strategy, which is developed for detecting LAMP amplicons by lateral flow biosensor (LFB). Only a single labeled primer is required in the analysis system, thus the false positive results arising from hybridization (the labeled primer and probe, or between two labeled primers) are avoided. Hence, the SAMRS components, AUDG and LFB convert traditional LAMP from a technique suited for the research laboratory into one that has practical value in the field of diagnosis. Human Tuberculosis (TB) is caused by infection with members of Mycobacterium tuberculosis complex (MTC), which are detected by the AUDG-SAMRS-LAMP technique to demonstrate the availability of target analysis. The proof-of-concept method can be reconfigured to detect various nucleic acids by redesigning the specific primers.
Assuntos
Mycobacterium tuberculosis/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico , Uracila-DNA Glicosidase/química , Técnicas Biossensoriais , DNA , Primers do DNA , Humanos , Ácidos Nucleicos , Sensibilidade e Especificidade , Tuberculose/diagnóstico , UracilaRESUMO
The taxonomic position of four phenotypically closely related strains isolated from faecal samples of yaks (Bos grunniens) collected from the Qinghai-Tibetan plateau, China, was determined by a polyphasic approach. The strains were non-spore-forming, non-motile Gram-stain-positive, ovoid cocci, occurring predominantly in pairs and short chains or in irregular clusters. The 16S rRNA gene of strain MN05T was related phylogenetically to those of Enterococcushaemoperoxidus, Enterococcusrotai, Enterococcusquebecensis, Enterococcusplantarum, Enterococcuscrotali, Enterococcusmoraviensis, Enterococcussilesiacus, Enterococcuscaccae, Enterococcustermitis, Enterococcusureasiticus and Enterococcusureilyticus, all belonging to the Enterococcusfaecalis species group. The sequence similarities of three selected genes of MN05T to those of the type strains of phylogenetically related species were measured, with values within the range of 99.2-99.5â% (16S rRNA gene), 90.0-97.3â% (rpoA) and 80.0-85.3â% (pheS), respectively. The genome of MN05T (3â842â361 bp) contained 4299 genes with a DNA G+C content of 37.5 mol%. A whole-genome phylogenetic tree based on 808 core genes confirmed that MN05T belongs to a distinct lineage, well separated from all recognized species of the Enterococcusfaecalis species group. DNA-DNA hybridization in silico showed that MN05T displayed less than 70â% DNA-DNA relatedness with the other 13 species of the Enterococcusfaecalis species group. Moreover, their phenotypic features distinguished the four strains from the other species of the Enterococcusfaecalis species group. Based upon these data obtained from the polyphasic characterization performed in the present study, a novel species of the genus Enterococcus, Enterococcus wangshanyuanii sp. nov., is proposed, with the type strain MN05T (=DSM 104047T=CGMCC 1.15942T).
Assuntos
Bovinos/microbiologia , Enterococcus/classificação , Fezes/microbiologia , Filogenia , Animais , Técnicas de Tipagem Bacteriana , Composição de Bases , China , DNA Bacteriano/genética , Enterococcus/genética , Enterococcus/isolamento & purificação , Hibridização de Ácido Nucleico , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
Two strains (pika_113T and pika_114) of a previously undescribed Actinomyces-like bacterium were recovered from the intestinal contents of plateau pika (Ochotona curzoniae) on the Tibet-Qinghai Plateau, China. Results from biochemical characterization indicated that the two strains were phenotypically homogeneous and distinct from other previously described species of the genus Actinomyces. Based on the comparison of 16S rRNA gene sequences and genome analysis, the bacteria were determined to be a hitherto unknown subline within the genus Actinomyces, being most closely related to type strains of Actinomyces denticolens and Actinomyces timonensis with a respective 97.2 and 97.1â% similarity in their 16S rRNA gene sequences. Phylogenetic analyses confirmed that pika_113T was well separated from any other recognized species of the genus Actinomyces and within the cluster with A. denticolens and A. timonensis. The genome of strain pika_113T displayed less than 42â% relatedness in DNA-DNA hybridization with all the available genomes of existing species of the genus Actinomyces in the NCBI database. Collectively, based on the phenotypic characteristics and phylogenetic analyses results, we propose the novel isolates as representatives of Actinomyces gaoshouyii sp. nov. The type strain of Actinomyces gaoshouyii is pika_113T (=CGMCC 4.7372T=DSM 104049T), with a genomic DNA G+C content of 71 mol%.
Assuntos
Actinomyces/classificação , Intestinos/microbiologia , Lagomorpha/microbiologia , Filogenia , Actinomyces/genética , Actinomyces/isolamento & purificação , Animais , Técnicas de Tipagem Bacteriana , DNA Bacteriano/genética , Hibridização de Ácido Nucleico , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , TibetRESUMO
Two strains (VUL4_1T and VUL4_2) of Gram-staining-positive, catalase-negative, non-spore-forming short rods were isolated from rectal swabs of Old World vultures (Gypaetus barbatus) in the Tibet-Qinghai Plateau, China. Analysis of morphological characteristics and biochemical tests indicated that the two strains closely resembled each other but were distinct from other species of the genus Actinomyces previously described. Based on the results of 16S rRNA gene sequence comparison and genome analysis, strains were determined to be members of the genus Actinomyces, closely related to the type strains of Actinomyces marimammalium (96.4â% 16S rRNA gene sequence similarity), Actinomyceshongkongensis (92.4â%), Actinomyceshordeovulneris (92.3â%) and Actinomycesnasicola (92.2â%), respectively. Optimal growth conditions were 37 °C, pH 6-7, with 1â% (w/v) NaCl. Strain VUL4_1T contained C18â:â1ω9c and C16â:â0 as the major cellular fatty acids and diphosphatidylglycerol as the major component of the polar lipids. The genomic DNA G+C content of VUL4_1T was 54.9 mol%. Strain VUL4_1T showed less than 70â% DNA-DNA relatedness with other species of the genus Actinomyces, further supporting strain VUL4_1T as a representative of a novel species. Based on the phenotypic data and phylogenetic inference, a novel species, Actinomyces liubingyangii sp. nov., is proposed with VUL4_1T (=CGMCC 4.7370T=DSM 104050T) as the type strain.
Assuntos
Falconiformes/microbiologia , Filogenia , Reto/microbiologia , Actinomyces/genética , Actinomyces/isolamento & purificação , Animais , Técnicas de Tipagem Bacteriana , DNA Bacteriano/genética , Ácidos Graxos/química , Hibridização de Ácido Nucleico , Fosfolipídeos/química , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Compostos de Espiro , TibetRESUMO
INTRODUCTION: Streptococcus suis serotype 2 is an important swine pathogen and emerging zoonotic agent causing meningitis and septicemia/septic shock. Strains are usually virulent (Eurasia) or of intermediate/low virulence (North America). Very few data regarding human and swine isolates from South America are available. CASE PRESENTATION: Seventeen new human S. suis cases in Argentina (16 serotype 2 strains and a serotype 5 strain) are reported. Alongside, 14 isolates from pigs are analyzed: 12 from systemic disease, one from lungs and one from tonsils of a healthy animal. All human serotype 2 strains and most swine isolates are sequence type (ST) 1, as determined by multilocus sequence typing and present a mrp+/epf+/sly+ genotype typical of virulent Eurasian ST1 strains. The remaining two strains (recovered from swine lungs and tonsils) are ST28 and possess a mrp+/epf - /sly- genotype typical of low virulence North American strains. Representative human ST1 strains as well as one swine ST28 strain were analyzed by whole-genome sequencing and compared with genomes from GenBank. ST1 strains clustered together with three strains from Vietnam and this cluster is close to another one composed of 11 strains from the United Kingdom. CONCLUSION: Close contact with pigs/pork products, a good surveillance system, and the presence of potentially virulent Eurasian-like serotype 2 strains in Argentina may be an important factor contributing to the higher number of human cases observed. In fact, Argentina is now fifth among Western countries regarding the number of reported human cases after the Netherlands, France, the UK and Poland.
RESUMO
JUSTIFICATIVA E OBJETIVOS: Os anestésicos locais são amplamente utilizados na prevenção ou na reversão de dor aguda e no tratamento de dor crônica. A reação de cardiotoxicidade induzida pelos anestésicos locais é um evento acidental sem terapia farmacológica, exceto a infusão de intralípides relatados recentemente cujo mecanismo de ação ainda não é bem compreendido. CONTEÚDO: A cardiolipina, um fosfolipídio aniônico, desempenha papel relevante na determinação de reação respiratória mitocondrial, metabolismo de ácidos graxos e apoptose celular. A disfunção do metabolismo energético mitocondrial é sugerida em associação com a cardiotoxicidade dos anestésicos locais, a partir de um estudo in vitro de que ela talvez se deva a fortes ligações eletrostáticas entre os anestésicos locais e a cardiolipina na membrana mitocondrial. Não há, contudo, evidência experimental. Portanto, levantamos a hipótese de que as interações anestésico-cardiolipina sejam o principal determinante associado à reação de cardiotoxicidade, o que pode ser estabelecido com a adoção de métodos teóricos e biológicos estruturais. Esse modelo de interação nos daria uma pista sobre o mecanismo da cardiotoxicidade dos anestésicos locais, visando a futuras pesquisas na área de desenvolvimento de fármacos de prevenção a esse evento na prática clínica. CONCLUSÕES: A interação entre a cardiolipina mitocondrial e os anestésicos locais pode ser a principal fonte de sua cardiotoxicidade, em função de seus efeitos sobre o metabolismo energético e o estado eletrostático.
BACKGROUND AND OBJECTIVES: Local anesthetics are used broadly to prevent or reverse acute pain and treat symptoms of chronic pain. Local anesthetic-induced cardiotoxic reaction has been considered the accidental event without currently effective therapeutic drugs except for recently reported intralipid infusion whose possible mechanism of action is not well known. CONTENTS: Cardiolipin, an anionic phospholipid, plays a key role in determining mitochondrial respiratory reaction, fatty acid metabolism and cellular apoptosis. Mitochondrial energy metabolism dysfunction is suggested as associated with local anesthetic cardiotoxicity, from an in vitro study report that the local anesthetic cardiotoxicity may be due to the strong electrostatic interaction of local anesthetics and cardiolipin in the mitochondria membrane, although there is a lack for experimental evidence. Herein we hypothesized that local anesthetic-cardiolipin interactions were the major determinant of local anesthetic-associated cardiotoxic reaction, established by means of theoretic and structural biological methods. This interacting model would give an insight on the underlying mechanism of local anesthetic cardiotoxicity and provide clues for further in depth research on designing preventive drugs for such inadvertent accidence in routine clinical practice. CONCLUSIONS: The interaction between local anesthetic and mitochondrial cardiolipin may be the underlying mechanism for cardiotoxicity affecting its energy metabolism and electrostatic status.
Assuntos
Humanos , Anestésicos Locais/farmacologia , Cardiolipinas/efeitos dos fármacos , Cardiopatias/induzido quimicamente , Mitocôndrias Cardíacas/efeitos dos fármacosRESUMO
BACKGROUND AND OBJECTIVES: Local anesthetics are used broadly to prevent or reverse acute pain and treat symptoms of chronic pain. Local anesthetic-induced cardiotoxic reaction has been considered the accidental event without currently effective therapeutic drugs except for recently reported intralipid infusion whose possible mechanism of action is not well known. CONTENTS: Cardiolipin, an anionic phospholipid, plays a key role in determining mitochondrial respiratory reaction, fatty acid metabolism and cellular apoptosis. Mitochondrial energy metabolism dysfunction is suggested as associated with local anesthetic cardiotoxicity, from an in vitro study report that the local anesthetic cardiotoxicity may be due to the strong electrostatic interaction of local anesthetics and cardiolipin in the mitochondria membrane, although there is a lack for experimental evidence. Herein we hypothesized that local anesthetic-cardiolipin interactions were the major determinant of local anesthetic-associated cardiotoxic reaction, established by means of theoretic and structural biological methods. This interacting model would give an insight on the underlying mechanism of local anesthetic cardiotoxicity and provide clues for further in depth research on designing preventive drugs for such inadvertent accidence in routine clinical practice. CONCLUSIONS: The interaction between local anesthetic and mitochondrial cardiolipin may be the underlying mechanism for cardiotoxicity affecting its energy metabolism and electrostatic status.
Assuntos
Anestésicos Locais/farmacologia , Cardiolipinas/efeitos dos fármacos , Cardiopatias/induzido quimicamente , Humanos , Mitocôndrias Cardíacas/efeitos dos fármacosRESUMO
Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are diarrheagenic pathogens that colonize the gut through the formation of attaching and effacing lesions, which depend on the translocation of effector proteins via a locus of enterocyte effacement-encoded type III secretion system. Recently, two effector proteins, EspJ and TccP, which are encoded by adjacent genes on prophage CP-933U in EHEC O157:H7, have been identified. TccP consists of a unique N-terminus region and several proline-rich domains. In this project we determined the distribution of tccP in O157:H7, in non-O157 EHEC, and in typical and atypical EPEC isolates. All the EHEC O157:H7 strains tested were tccP(+). Unexpectedly, tccP was also found in non-O157 EHEC, and in typical and atypical EPEC isolates, particularly in strains belonging to serogroups O26 (EHEC), O119 (typical EPEC), and O55 (atypical EPEC). We recorded some variation in the length of tccP, which reflects diversity in the number of the proline-rich repeats. These results show the existence of a class of "attaching and effacing" pathogens which express a combination of EPEC and EHEC virulence determinants.
Assuntos
Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Animais , Austrália , Brasil , China , Escherichia coli/química , Infecções por Escherichia coli/veterinária , Escherichia coli O157/química , Escherichia coli O157/genética , Microbiologia de Alimentos , Variação Genética , Humanos , Dados de Sequência Molecular , Reino UnidoRESUMO
Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are diarrheagenic pathogens that colonize the gut through the formation of attaching and effacing lesions, which depend on the translocation of effector proteins via a locus of enterocyte effacement-encoded type III secretion system. Recently, two effector proteins, EspJ and TccP, which are encoded by adjacent genes on prophage CP-933U in EHEC O157:H7, have been identified. TccP consists of a unique N-terminus region and several proline-rich domains. In this project we determined the distribution of tccP in O157:H7, in non-O157 EHEC, and in typical and atypical EPEC isolates. All the EHEC O157:H7 strains tested were tccP+. Unexpectedly, tccP was also found in non-O157 EHEC, and in typical and atypical EPEC isolates, particularly in strains belonging to serogroups O26 (EHEC), O119 (typical EPEC), and O55 (atypical EPEC). We recorded some variation in the length of tccP, which reflects diversity in the number of the proline-rich repeats. These results show the existence of a class of attaching and effacing pathogens which express a combination of EPEC and EHEC virulence determinants.