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1.
Methods Mol Biol ; 2278: 141-148, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33649954

RESUMO

Bifidobacteria are commensal bacteria, which naturally colonize the gastrointestinal tract of a large number of animals, including humans, contributing to their health and well-being. An important taxonomic marker for the identification of members of the bifidobacterial group is the presence of the fructose-6-phosphate phosphoketolase (F6PPK) activity. The F6PPK enzyme is involved in the bifidus shunt based on the ability of F6PPK to split fructose-6-phosphate into erythrose-4-phosphate and acetyl phosphate. Here, we describe the two main methods utilized to detect the presence of F6PPK activity, that is, the enzymatic assay and the presence of the D-xylulose-5-phosphate/fructose-6-phosphate phosphoketolase bifidobacterial gene.


Assuntos
Aldeído Liases/metabolismo , Proteínas de Bactérias/metabolismo , Bifidobacterium/metabolismo , Ensaios Enzimáticos/métodos , Aldeído Liases/genética , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana/métodos , Bifidobacterium/genética , Bifidobacterium/isolamento & purificação , Técnicas de Cultura de Células/métodos , Genes Bacterianos , Reação em Cadeia da Polimerase/métodos
2.
Artigo em Inglês | MEDLINE | ID: mdl-33507857

RESUMO

Cockle mortality events have been reported in northern France since 2012. In the present study, we describe and investigate the implication of a potential bacterial causative agent in cockle mortality. Bacteria isolated from five different cockle mortality events were characterized and studied. Using phenotypic analysis combined with DNA-DNA hybridization (DDH) and whole genome sequencing, the isolates were shown to belong to Vibrio aestuarianus, a species regularly detected in France during oyster mortality events. Comparison of the strains from cockles with strains from French oysters and the type strain showed that the strains from cockles were genetically different to those from oysters and also different to the V. aestuarianus type strain. Moreover, the cockle and oyster strains were classified into two different, but close, groups both separated from the type strain by: (1) analyses of the ldh gene sequences; (2) DDH assays between 12/122 3T3T (LMG 31436T=DSM 109723T), a representative cockle strain, 02/041T (CIP 109791T=LMG 24517T) representative oyster strain and V. aestuarianus type strain LMG 7909T; (3) average nucleotide identity values calculated on the genomes; and (4) phenotypic traits. Finally, results of MALDI-TOF analyses also revealed specific peaks discriminating the three representative strains. The toxicity of representative strains of these cockle isolates was demonstrated by experimental infection of hatchery-produced cockles. The data therefore allow us to propose two novel subspecies of Vibrio aestuarianus: Vibrio aestuarianus subsp. cardii subsp. nov. for the cockle strains and Vibrio aestuarianus subsp. francensis subsp. nov. for the Pacific oyster strains, in addition to an emended description of the species Vibrio aestuarianus.


Assuntos
Cardiidae/microbiologia , Filogenia , Vibrio/classificação , Animais , Técnicas de Tipagem Bacteriana/métodos , Composição de Bases , DNA Bacteriano/genética , França , Hibridização de Ácido Nucleico , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Vibrio/isolamento & purificação
3.
Methods Mol Biol ; 2220: 17-29, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32975763

RESUMO

Listeria monocytogenes is a major food-borne pathogen and causative agent of a fatal disease, listeriosis. Stringent regulatory guidelines and zero tolerance policy toward this bacterium necessitate rapid, accurate, and reliable methods of identification and subtyping. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) has recently become a method of choice for routine identification of pathogens in clinical settings and has largely replaced biochemical assays. Identification relies on well-curated databases such as SARAMIS. Extensive use of SARAMIS to generate consensus mass spectra, in conjunction with statistical analysis, such as partial least square-discriminant analysis and hierarchical cluster analysis, is useful in subtyping bacteria. While MALDI-ToF MS has been extensively used for pathogen detection, its application in bacterial subtyping has been limited. The protocol describes a MALDI-ToF MS workflow as a single tool for simultaneous identification and subtyping of L. monocytogenes directly from solid culture medium.


Assuntos
Listeria monocytogenes/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Técnicas de Tipagem Bacteriana/economia , Técnicas de Tipagem Bacteriana/métodos , Humanos , Listeria monocytogenes/química , Listeria monocytogenes/classificação , Listeriose/microbiologia , Software , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/economia , Fatores de Tempo , Fluxo de Trabalho
4.
Methods Mol Biol ; 2182: 45-50, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32894486

RESUMO

CRISPR typing is a newly developed method used to reveal the genetic relationship of bacterial isolates from different resources. For Salmonella, CRISPR typing can not only reveal the phylogenic difference among isolates belonging to the identical serotype, but also show good correspondence with Salmonella serotypes. Here we describe the protocol of CRISPR typing method used in Salmonella, and the approaches to analyze the genetic relationship among different strains.


Assuntos
Técnicas de Tipagem Bacteriana/métodos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Salmonella/genética , Filogenia , Sorogrupo
5.
Methods Mol Biol ; 2182: 187-196, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32894497

RESUMO

Salmonella is recognized as a major human foodborne pathogen and threat to public health world widely. It is important to carry out epidemiological investigations to determine the primary sources of bacterial contamination. Pulsed-field gel electrophoresis (PFGE) is an important method of the molecular typing, and play an important role in tracking the sources of infection and epidemic control. The PFGE is currently considered as "gold standard" of molecular typing methods for bacterial foodborne pathogen. Here, we describe the PFGE protocol to type the Salmonella from pork.


Assuntos
Técnicas de Tipagem Bacteriana/métodos , Eletroforese em Gel de Campo Pulsado/métodos , Tipagem Molecular/métodos , Salmonella/genética , DNA Bacteriano/genética , Carne de Porco/microbiologia
6.
PLoS One ; 15(9): e0238991, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32946486

RESUMO

BACKGROUND: Invasive Staphylococcus aureus infections are a common cause of morbidity and mortality in children. In the early 2000's the proportion of infections due the methicillin-resistant S. aureus (MRSA) increased rapidly. We described the clinical and molecular epidemiology of invasive S. aureus disease in a pediatric population. METHODS: We prospectively identified children in Utah with invasive S. aureus infections. Medical records were reviewed to determine diagnosis and clinical characteristics. Isolates were genotyped using multi-locus sequence typing. The presence of genes encoding the Panton-Valentine leukocidin (PVL) was determined using polymerase chain reaction. RESULTS: Over a 4-year period between January 2009 and December 2012, we identified 357 children, hospitalized at Primary Children's Hospital, with invasive S. aureus infections and isolates available for the study. Methicillin-susceptible S. aureus (MSSA) caused 79% of disease, while MRSA caused only 21% of disease. Mortality associated with invasive S. aureus infection was 3.6%. The most common diagnoses were osteoarticular infections (38%) followed by central line associated blood stream infections (19%) and pneumonia (12%). We identified 41 multi-locus sequence types. The majority of isolates belonged to 6 predominant clonal complexes (CC5, CC8, CC15, CC30, CC45, CC59). PVL was present in a minority (16%) of isolates, of which most were ST8 MRSA. CONCLUSIONS: MSSA was the primary cause of invasive S. aureus infections at our institution throughout the study period. A limited number of predominant strains accounted for the majority of invasive disease. The classic virulence factor PVL was uncommon in MSSA isolates. Further study is needed to improve our understanding of S. aureus virulence and disease pathogenesis.


Assuntos
Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Infecções Estafilocócicas/epidemiologia , Staphylococcus aureus/genética , Antibacterianos/uso terapêutico , Técnicas de Tipagem Bacteriana/métodos , Criança , Pré-Escolar , Feminino , Genótipo , Humanos , Masculino , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Testes de Sensibilidade Microbiana , Epidemiologia Molecular/métodos , Tipagem de Sequências Multilocus/métodos , Infecções Estafilocócicas/genética , Staphylococcus aureus/patogenicidade , Utah/epidemiologia , Fatores de Virulência/genética
7.
PLoS One ; 15(7): e0234475, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32663215

RESUMO

BACKGROUND: Neisseria meningitidis is a significant cause of morbidity and mortality worldwide. Meningococcal isolates have a highly dynamic population structure and can be phenotypically and genetically differentiated into serogroups and clonal complexes. The aim of this study was to describe the phenotypic and genotypic characteristics of invasive isolates recovered in Colombia from 2013 to 2016. METHODOLOGY: A total of 193 invasive isolates were analyzed. Phenotypic and genotypic characteristics were determined by serotyping, antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing. RESULTS: Based on the results, meningococcal serogroups C, B and Y were responsible for 47.9%, 41.7%, and 9.4% of cases, respectively, and the distribution of serogroups B and C changed over time. Fifteen clonal groups and 14 clonal complexes (cc) were identified by PFGE and genome sequencing. The main clonal group included serogroup B isolates with sequence type (ST)-9493 and its four single-locus variants, which has only been identified in Colombian isolates. The clonal population structure demonstrates that the isolates in this study mainly belong to four clonal complexes: ST-11 cc, ST-32 cc, ST-35 cc and ST-41/44 cc. Thirty-eight penA alleles were identified, but no correlation between MICs and specific sequences was observed. CONCLUSION: This study shows that most meningococcal isolates recovered from patients with invasive meningococcal disease in Colombia are strains associated with distinct globally disseminated hyperinvasive clones.


Assuntos
Infecções Meningocócicas/epidemiologia , Infecções Meningocócicas/genética , Neisseria meningitidis/genética , Adolescente , Adulto , Técnicas de Tipagem Bacteriana/métodos , Criança , Pré-Escolar , Colômbia/epidemiologia , Eletroforese em Gel de Campo Pulsado/métodos , Feminino , Genótipo , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Neisseria meningitidis/isolamento & purificação , Neisseria meningitidis/patogenicidade , Sorogrupo , Sorotipagem
8.
J Med Microbiol ; 69(8): 1105-1113, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32597748

RESUMO

Introduction. Burkholderia cepacia complex (Bcc) bacteria, currently consisting of 23 closely related species, and Burkholderia gladioli, can cause serious and difficult-to-treat infections in people with cystic fibrosis. Identifying Burkholderia bacteria to the species level is considered important for understanding epidemiology and infection control, and predicting clinical outcomes. Matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-TOF) is a rapid method recently introduced in clinical laboratories for bacterial species-level identification. However, reports on the ability of MALDI-TOF to accurately identify Bcc to the species level are mixed.Aim. The aim of this project was to evaluate the accuracy of MALDI-TOF using the Biotyper and VITEK MS systems in identifying isolates from 22 different Bcc species and B. gladioli compared to recA gene sequencing, which is considered the current gold standard for Bcc.Methodology. To capture maximum intra-species variation, phylogenetic trees were constructed from concatenated multi-locus sequence typing alleles and clustered with a novel k-medoids approach. One hundred isolates representing 22 Bcc species, plus B. gladioli, were assessed for bacterial identifications using the two MALDI-TOF systems.Results. At the genus level, 100 and 97.0 % of isolates were confidently identified as Burkholderia by the Biotyper and VITEK MS systems, respectively; moreover, 26.0 and 67.0 % of the isolates were correctly identified to the species level, respectively. In many, but not all, cases of species misidentification or failed identification, a representative library for that species was lacking.Conclusion. Currently available MALDI-TOF systems frequently do not accurately identify Bcc bacteria to the species level.


Assuntos
Burkholderia cepacia/isolamento & purificação , Burkholderia gladioli/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Animais , Técnicas de Tipagem Bacteriana/métodos , Burkholderia cepacia/classificação , Burkholderia gladioli/classificação , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , Análise de Fourier , Humanos , Tipagem de Sequências Multilocus , Filogenia , Recombinases Rec A/genética , Alinhamento de Sequência
9.
PLoS One ; 15(6): e0233990, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32497069

RESUMO

OBJECTIVES: Typing of Chlamydia trachomatis (CT) is traditionally performed by characterising the ompA gene, resulting in more than a dozen different genovars, A to L. Type L is associated with Lymphogranuloma venereum (LGV) and commonly screened for using PCR, targeting the chromosomal pmpH gene. We aimed to develop and validate a new CT/LGV plasmid-based typing assay targeting the pgp3 gene, to increase sensitivity and thus reduce the number of non-typeable results. METHODS: The new pgp3 PCR assay using LNA probes to detect point mutations was analytically and prospectively validated in a routine diagnostic laboratory setting. For the analytical tests, quantified nucleotide constructs (gBlocks) were used to perform limit of detection analyses. Quality control panel samples from 2018 and 2019 for CT were also tested. For the clinical study patient samples which were collected in two months in 2018 were tested simultaneously using the pmpH PCR and the pgp3 PCR. RESULTS: Analytically, the assay proved to be 100% specific relative to the previously used LGV typing assay targeting the single copy pmpH gene but it was much more sensitive to detect non-LGV CT. In the quality control panel 2 nonLGV samples and 7 LGV samples were solely positive with the pgp3 PCR and not with the pmpH PCR. None of the samples from analytical specificity panels were positive, indicating 100% specificity. In a prospective panel of 152 clinical samples, 142 (93%) were successfully typed with the pgp3 PCR compared to 78% with the pmpH PCR. The pgp3 PCR was fully concordant with the pmpH PCR to identify all LGV subtypes and detected an increased number of clinical samples of non-LGV subtype. CONCLUSION: We developed and validated a sensitive and specific plasmid-based typing assay to discriminate LGV from non-LGV CT subtypes. This is useful in a clinical setting to quickly determine the optimal treatment for Chlamydia trachomatis infections.


Assuntos
Chlamydia trachomatis/genética , Linfogranuloma Venéreo/microbiologia , Plasmídeos/genética , Reação em Cadeia da Polimerase/métodos , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana/métodos , Genes Bacterianos , Humanos
10.
J Appl Microbiol ; 129(5): 1193-1206, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32426861

RESUMO

AIMS: Development of a novel hierarchical Mycobacterium avium subsp. paratuberculosis (MAP) typing approach and characterization of MAP field cultures in Central Germany. METHODS AND RESULTS: By combining single nucleotide polymorphisms (SNPs) and mycobacterial interspersed repetitive unit-variable number tandem repeat, we developed a highly discriminating and phylogenetically accurate hierarchical MAP typing approach. Moreover, a novel stepwise workflow was employed to reduce the number of SNP reactions required making the typing approach more affordable. MAP field cultures (n = 142) from dairy herds in Central Germany were classified as cattle type and showed a high level of heterogeneity. Intra-herd multiple genotypes were evident in (13-25%) of the investigated herds. CONCLUSIONS: The hierarchical MAP typing approach proved to be useful in fine discrimination between MAP cultures within limited geographical regions. This could potentially be used in unravelling MAP transmission chains in the respective regions. The observed heterogeneity in some herds is assumed to be due to either multiple introductions through inter-herd trade or intra-herd evolution over time. SIGNIFICANCE AND IMPACT OF THE STUDY: Future MAP epidemiological studies will benefit from the advantages of the novel hierarchical typing approach. The SNP number reduction approach employed here could be extrapolated for other analogous pathogens.


Assuntos
Técnicas de Tipagem Bacteriana/veterinária , Doenças dos Bovinos/microbiologia , Mycobacterium avium subsp. paratuberculosis/classificação , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Paratuberculose/microbiologia , Animais , Técnicas de Tipagem Bacteriana/métodos , Bovinos , Doenças dos Bovinos/epidemiologia , DNA Bacteriano/genética , Genótipo , Alemanha/epidemiologia , Repetições Minissatélites/genética , Mycobacterium avium subsp. paratuberculosis/genética , Paratuberculose/epidemiologia , Filogenia , Polimorfismo de Nucleotídeo Único/genética
11.
PLoS One ; 15(4): e0230976, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32240218

RESUMO

Acinetobacter baumannii is an opportunistic pathogen of intensive care unit (ICU) patients. A. baumannii colonizes many parts of the body including the gastrointestinal tract. Endemic and epidemic strains are polyclonal. There is no clarity on the origin of polyclonality of A. baumannii. The objective of the study was to define the genetic relatedness of serial isolates and the origin of polyclonality. Serial rectal isolates from ICU patients whose rectum was colonized on ≥5 sampling occasions were selected. From a total of 32 eligible colonized patients, isolates from a subgroup of 13 patients (a total of 108 isolates) showing different patterns of colonization as revealed by pulsed-field gel electrophoresis (PFGE) were studied. The isolates were analyzed by PFGE pulsotypes, sequence types (STs) by multi-locus sequence typing (MLST) and clonal complex (CC) by eBURST analysis. Serial isolates constituted a mixture of identical, related and unrelated pulsotypes. Analysis by STs and CCs were less discriminatory. The data suggest a combination of an initial colonizing isolate undergoing mutation as well as colonization by independent isolates. Further clarity on the origin of diversity should be better obtained by whole-genome sequencing.


Assuntos
Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/genética , Acinetobacter baumannii/isolamento & purificação , Mutação/genética , Reto/microbiologia , Técnicas de Tipagem Bacteriana/métodos , Infecção Hospitalar/microbiologia , Eletroforese em Gel de Campo Pulsado , Humanos , Unidades de Terapia Intensiva , Kuweit , Centros de Atenção Terciária
12.
Sci Rep ; 10(1): 4301, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32152417

RESUMO

This study aims to validate the current diagnostic method for the clinical detection of gastroenteritis. We analyzed 400 stool samples to detect three of the most common enteropathogens: Salmonella spp., Campylobacter spp., and Yersinia enterocolitica. All specimens were tested with a routine clinical diagnosis algorithm and with five real-time PCR assays. A total of 98 specimens (24.5%) were positive for enteropathogens. We found 24 samples positive for Salmonella enterica, 71 positive for Campylobacter spp., and 4 positive for Yersinia enterocolitica. All evaluated methods exhibited a good performance in identifying Salmonella and Yersinia enterocolitica, being the highest positive percent agreement (PPA) value of 95.8% and 100%, respectively. The clinical algorithm showed the highest PPA value identifying Salmonella, due to the enrichment in selenite broth. However, the evaluated methods showed notable differences in the identification of Campylobacter species, obtaining a wide range of PPA values: 59.2%-100%. The clinical algorithm showed the lowest PPA value since it was only able to detect Campylobacter jejuni and Campylobacter coli species. This study revealed the importance of implementing the real-time PCR technique in a clinical algorithm: it improved the accuracy of the diagnosis and provided results in a shorter time compared to routine clinical methods.


Assuntos
Algoritmos , Técnicas de Tipagem Bacteriana/métodos , Infecções por Campylobacter/diagnóstico , Fezes/microbiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Infecções por Salmonella/diagnóstico , Yersiniose/diagnóstico , Adolescente , Campylobacter/isolamento & purificação , Infecções por Campylobacter/epidemiologia , Infecções por Campylobacter/microbiologia , Criança , Pré-Escolar , Meios de Cultura , Feminino , Humanos , Masculino , Técnicas de Diagnóstico Molecular/métodos , Curva ROC , Salmonella/isolamento & purificação , Infecções por Salmonella/epidemiologia , Infecções por Salmonella/microbiologia , Espanha/epidemiologia , Yersiniose/epidemiologia , Yersiniose/microbiologia , Yersinia enterocolitica/isolamento & purificação
13.
Talanta ; 212: 120778, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32113541

RESUMO

Tailor-made Escherichia coli (E. coli) receptors were created with microcontact imprinted technique and binding events of E. coli were carried out by a surface plasmon resonance (SPR) sensor in aqueous solution and in urine mimic in real time and label-free. N-methacryloyl-(l)-histidine methyl ester (MAH) was selected as a functional monomer to design tailor-made E. coli receptors on the polymeric film and during the formation of the polymeric film on a chip surface, Ag nanoparticles (AgNPs) were entrapped into the polymer mixture in order to lower the detection limit of biomimetic SPR based sensor. The polymeric film was characterized with atomic force microscopy (AFM), scanning electron microscopy (SEM), ellipsometer and contact angle measurements. Limit of detection (LOD) was found 0.57 CFU/mL and feasibility of the biomimetic sensor was investigated in urine mimic.


Assuntos
Técnicas de Tipagem Bacteriana/métodos , Escherichia coli/isolamento & purificação , Ácidos Polimetacrílicos/química , Ressonância de Plasmônio de Superfície/métodos , Infecções Urinárias/diagnóstico , Biomimética/métodos , Histidina/análogos & derivados , Histidina/química , Limite de Detecção , Nanopartículas Metálicas/química , Metacrilatos/química , Impressão Molecular , Prata/química , Urina/microbiologia
14.
Talanta ; 212: 120781, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32113544

RESUMO

Existing techniques for the detection of Group A Streptococcus pyogenes (GAS) have drawbacks in rapidness, accuracy or in high-cost. Considering the clinical importance of GAS, we have developed a culture-free detection method based on pyrrolidonyl arylamidase (PYR) activity with the aid of magnetic gold nanoparticles (AuNPs). GAS is the reason for pharyngitis and sampling starts from the throat with cotton swabs. After swab sampling, the target was collected with antibody modified magnetic AuNPs and transferred into 500 µL of PYR-broth without any antigen extraction or pure colony isolation. Then, the assay was finished by adding 25 µL of 4-(dimethylamino)-cinnamaldehyde (DMACA) reagent after 4-h incubation. A red color formation was evaluated as the presence of GAS comparing to blank, however, image analysis was employed for the interpretation of color changes clearly. For this purpose, a formula related to image data was proposed and analytical validation parameters were defined. Thus, the correlation was found to be linear with the R2 of 0.9685 between the log of bacteria concentration and the image data with the limit of detection of 3.3 × 102 CFU/mL of GAS. In addition, the assay worked efficiently in the abundance interference of Enterococcus faecalis. The results represent a new feature to nanoparticles eliminating the selective growth media for a bacteria and this study provided a detection with intact cells of bacteria without any antigen or DNA/RNA extraction. The proposed work has been the most similar to the gold standard but a faster method in this field.


Assuntos
Aminopeptidases/análise , Proteínas de Bactérias/análise , Ensaios Enzimáticos/métodos , Nanopartículas de Magnetita/química , Streptococcus pyogenes/isolamento & purificação , Anticorpos Imobilizados/imunologia , Técnicas de Tipagem Bacteriana/métodos , Ouro/química , Imunoensaio/métodos , Streptococcus pyogenes/enzimologia , Streptococcus pyogenes/imunologia
15.
PLoS One ; 15(3): e0230550, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32196527

RESUMO

Many Lactobacillus species are frequently isolated from dairy products, animal guts, and the vaginas of healthy women. However, sequencing-based identification of isolated Lactobacillus strain is time/cost-consuming and lobor-intensive. In this study, we developed a multiplex PCR method to distinguish six closely related species in the Lactobacillus acidophilus group (L. gasseri, L. acidophilus, L. helveticus, L. jensenii, L. crispatus, and L. gallinarum), which is based on species-specific primer sets. Altogether, 86 genomes of 9 Lactobacillus species from the National Center of Biotechnology Information (NCBI) database were compared to detect species-specific genes and design six species-specific primer sets. The PCR conditions of the individual primer sets were optimized via gradient PCR methods. A final multiplex PCR condition was also optimized for a mixture of all six primer sets mixed. When identifying a single strain, the optimized multiplex PCR method can specifically detect one of the six species, but no band was amplified at least from the other Lactobacillus and Enterococcus species. These results indicated that species-specific primer sets designed from the genome comparison could identify one strain within the six Lactobacillus species by a single PCR reaction. Using the method described here, we will be able to save time, cost, and labor during species identification and screening of commercially important probiotic lactobacilli.


Assuntos
DNA Bacteriano/genética , Lactobacillus acidophilus/genética , Animais , Técnicas de Tipagem Bacteriana/métodos , Primers do DNA/genética , Genoma Bacteriano , Humanos , Lactobacillus acidophilus/classificação , Reação em Cadeia da Polimerase Multiplex/métodos , Filogenia , Probióticos/análise
16.
Appl Microbiol Biotechnol ; 104(7): 3157-3166, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32047991

RESUMO

Foodborne pathogens have become ongoing threats in the food industry, whereas their rapid detection and classification at an early stage are still challenging. To address early and rapid detection, hyperspectral microscope imaging (HMI) technology combined with convolutional neural networks (CNN) was proposed to classify foodborne bacterial species at the cellular level. HMI technology can simultaneously obtain both spatial and spectral information of different live bacterial cells, while two CNN frameworks, U-Net and one-dimensional CNN (1D-CNN), were employed to accelerate the data analysis process. U-Net was used for automating cellular regions of interest (ROI) segmentation, which generated accurate cell-ROI masks in a shorter timeframe than the conventional Otsu or Watershed methods. The 1D-CNN was employed for classifying the spectral profiles extracted from cell-ROI and resulted in a higher accuracy (90%) than k-nearest neighbor (81%) and support vector machine (81%). Overall, the CNN-assisted HMI technology showed potential for foodborne bacteria detection.


Assuntos
Técnicas de Tipagem Bacteriana/métodos , Microbiologia de Alimentos/métodos , Microscopia , Redes Neurais de Computação , Algoritmos , Doenças Transmitidas por Alimentos/microbiologia , Processamento de Imagem Assistida por Computador , Aprendizado de Máquina , Microscopia/métodos , Análise Espectral
17.
Nat Microbiol ; 5(3): 455-464, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32042129

RESUMO

Surveillance of drug-resistant bacteria is essential for healthcare providers to deliver effective empirical antibiotic therapy. However, traditional molecular epidemiology does not typically occur on a timescale that could affect patient treatment and outcomes. Here, we present a method called 'genomic neighbour typing' for inferring the phenotype of a bacterial sample by identifying its closest relatives in a database of genomes with metadata. We show that this technique can infer antibiotic susceptibility and resistance for both Streptococcus pneumoniae and Neisseria gonorrhoeae. We implemented this with rapid k-mer matching, which, when used on Oxford Nanopore MinION data, can run in real time. This resulted in the determination of resistance within 10 min (91% sensitivity and 100% specificity for S. pneumoniae and 81% sensitivity and 100% specificity for N. gonorrhoeae from isolates with a representative database) of starting sequencing, and within 4 h of sample collection (75% sensitivity and 100% specificity for S. pneumoniae) for clinical metagenomic sputum samples. This flexible approach has wide application for pathogen surveillance and may be used to greatly accelerate appropriate empirical antibiotic treatment.


Assuntos
Antibacterianos/farmacologia , Técnicas de Tipagem Bacteriana/métodos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Genômica , Bases de Dados Factuais , Humanos , Testes de Sensibilidade Microbiana/métodos , Epidemiologia Molecular , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/isolamento & purificação , Fenótipo , Sensibilidade e Especificidade , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/isolamento & purificação
18.
PLoS One ; 15(1): e0226238, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31978059

RESUMO

Campylobacter jejuni infection is one of the most frequently reported foodborne bacterial diseases worldwide. The main transmission route of these microorganisms to humans is consumption of contaminated food, especially of chicken origin. The aim of this study was to analyze the genetic relatedness of C. jejuni from chicken sources (feces, carcasses, and meat) and from humans with diarrhea as well as to subtype the isolates to gain better insight into their population structure present in Poland. C. jejuni were genotyped using multilocus sequence typing (MLST) and sequence types (STs) were assigned in the MLST database. Among 602 isolates tested, a total of 121 different STs, including 70 (57.9%) unique to the isolates' origin, and 32 STs that were not present in the MLST database were identified. The most prevalent STs were ST464 and ST257, with 58 (9.6%) and 52 (8.6%) C. jejuni isolates, respectively. Isolates with some STs (464, 6411, 257, 50) were shown to be common in chickens, whereas others (e.g. ST21 and ST572) were more often identified among human C. jejuni. It was shown that of 47 human sequence types, 26 STs (106 isolates), 23 STs (102 isolates), and 29 STs (100 isolates) were also identified in chicken feces, meat, and carcasses, respectively. These results, together with the high and similar proportional similarity indexes (PSI) calculated for C. jejuni isolated from patients and chickens, may suggest that human campylobacteriosis was associated with contaminated chicken meat or meat products or other kinds of food cross-contaminated with campylobacters of chicken origin. The frequency of various sequence types identified in the present study generally reflects of the prevalence of STs in other countries which may suggest that C. jejuni with some STs have a global distribution, while other genotypes may be more restricted to certain countries.


Assuntos
Técnicas de Tipagem Bacteriana/métodos , Infecções por Campylobacter/microbiologia , Campylobacter jejuni/genética , Diarreia/diagnóstico , Doenças Transmitidas por Alimentos/microbiologia , Carne/análise , Tipagem de Sequências Multilocus/métodos , Animais , Infecções por Campylobacter/epidemiologia , Campylobacter jejuni/classificação , Campylobacter jejuni/isolamento & purificação , Galinhas , Diarreia/genética , Diarreia/microbiologia , Doenças Transmitidas por Alimentos/epidemiologia , Humanos , Carne/microbiologia , Polônia/epidemiologia , Prevalência
19.
Food Microbiol ; 87: 103394, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31948635

RESUMO

Salmonella is a major pathogen having a public health and economic impact in both humans and animals. Six serotypes of the Salmonella genus are mentioned in the Belgian and European regulation as to be rapidly excluded from the food chain (EU regulation N°2160/2003, Belgian royal decree 27/04/2017). The reference method for Salmonella serotyping, including slide-agglutination and biochemical tests, is time-consuming, expensive, not always objective, and therefore does not match the fast identification criteria required by the legislation. In this study, a molecular method, using genetic markers detected by Multiplex Oligonucleotide Ligation - PCR and Luminex technology, was developed for the identification of the 6 Salmonella serotypes and their variants subjected to an official control. The resulting method was validated with the analysis of 971 Salmonella isolated from different matrixes (human, animal, food or environment) and 33 non-Salmonella strains. The results were compared with the reference identifications, achieving an accuracy of 99.7%. The cost-effective high-throughput genoserotyping assay is performed in 1 day and generates objective results, thanks to the automatic interpretation of raw data using a barcode system. In conclusion, it is fully adapted to the implementation in first line laboratories and meets the requirements of the regulation.


Assuntos
Técnicas de Tipagem Bacteriana/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Oligonucleotídeos/genética , Salmonella/isolamento & purificação , Animais , Microbiologia Ambiental , Humanos , Salmonella/classificação , Salmonella/genética , Infecções por Salmonella/microbiologia , Sensibilidade e Especificidade
20.
Chem Commun (Camb) ; 56(11): 1717-1720, 2020 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-31942593

RESUMO

A novel sensor array based on a (+)AuNP/AuNC nanocomposite was constructed for the selective discrimination of 10 types of Gram-negative bacteria (including 3 types of antibiotic-resistant strains) at a low concentration level of OD600 = 0.015. By recognizing the triple optical patterns of Gram-negative bacteria with the assistance of LDA, the sensor array is able to group the bacteria with respect to their species to each other.


Assuntos
Técnicas de Tipagem Bacteriana/métodos , Bactérias Gram-Negativas/isolamento & purificação , Nanopartículas Metálicas/química , Análise por Conglomerados , Análise Discriminante , Ouro/química , Bactérias Gram-Negativas/classificação , Bactérias Gram-Positivas/classificação , Bactérias Gram-Positivas/isolamento & purificação , Humanos , Nanocompostos/química , Espalhamento de Radiação , Espectrometria de Fluorescência , Urina/microbiologia , Vancomicina/química
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