scholarly journals Use of a DNA Microarray for Detection and Identification of Bacterial Pathogens Associated with Fishery Products

2011 ◽  
Vol 77 (23) ◽  
pp. 8219-8225 ◽  
Author(s):  
Boyang Cao ◽  
Rongrong Li ◽  
Songjin Xiong ◽  
Fangfang Yao ◽  
Xiangqian Liu ◽  
...  
Keyword(s):  
Dna Microarray ◽  
Bacterial Species ◽  
Simultaneous Detection ◽  
Detection Sensitivity ◽  
23S Rrna ◽  
Rrna Gene ◽  
Bacterial Strains ◽  
Content Type ◽  
Detection And Identification ◽  
Fishery Products

ABSTRACTWe established a microarray for the simultaneous detection and identification of diverse putative pathogens often associated with fishery products by targeting specific genes ofListeria monocytogenes,Salmonella,Shigella,Staphylococcus aureus,Streptococcus pyogenes,Vibrio cholerae,Vibrio parahaemolyticus,Vibrio vulnificus, andYersinia enterocoliticaand the 16S-23S rRNA gene internal transcribed spacer (ITS) region ofProteus mirabilisandProteus vulgaris. The microarray contained 26 specific probes and was tested against a total of 123 target bacterial strains that included 55 representative strains, 68 clinical isolates, and 45 strains of other bacterial species that belonged to 8 genera and 34 species, and it was shown to be specific and reproducible. A detection sensitivity of 10 ng DNA or 10 CFU/ml for pure cultures of each target organism demonstrated that the assay was highly sensitive and reproducible. Mock and real fishery product samples were tested by the microarray, and the accuracy was 100%. The DNA microarray method described in this communication is specific, sensitive, and reliable and has several advantages over traditional methods of bacterial culture and antiserum agglutination assays.

scholarly journals Mobile Elements in a Single-Filament Orange Guaymas Basin Beggiatoa (“Candidatus Maribeggiatoa”) sp. Draft Genome: Evidence for Genetic Exchange with Cyanobacteria

2013 ◽  
Vol 79 (13) ◽  
pp. 3974-3985 ◽  
Author(s):  
Barbara J. MacGregor ◽  
Jennifer F. Biddle ◽  
Andreas Teske
Keyword(s):  
Gulf Of California ◽  
Bacterial Species ◽  
Draft Genome ◽  
Genetic Exchange ◽  
23S Rrna ◽  
Rrna Gene ◽  
Homing Endonucleases ◽  
Guaymas Basin ◽  
Content Type ◽  
Group I

ABSTRACTThe draft genome sequence of a single orangeBeggiatoa(“CandidatusMaribeggiatoa”) filament collected from a microbial mat at a hydrothermal site in Guaymas Basin (Gulf of California, Mexico) shows evidence of extensive genetic exchange with cyanobacteria, in particular for sensory and signal transduction genes. A putative homing endonuclease gene and group I intron within the 23S rRNA gene; several group II catalytic introns; GyrB and DnaE inteins, also encoding homing endonucleases; multiple copies of sequences similar to thefdxNexcision elements XisH and XisI (required for heterocyst differentiation in some cyanobacteria); and multiple sequences related to an open reading frame (ORF) (00024_0693) of unknown function all have close non-Beggiatoaceaematches with cyanobacterial sequences. Sequences similar to the uncharacterized ORF and Xis elements are found in otherBeggiatoaceaegenomes, a variety of cyanobacteria, and a few phylogenetically dispersed pleiomorphic or filamentous bacteria. We speculate that elements shared among filamentous bacterial species may have been exchanged in microbial mats and that some of them may be involved in cell differentiation.

scholarly journals Development of a DNA Microarray Method for Detection and Identification of All 15 Distinct O-Antigen Forms of Legionella pneumophila

2013 ◽  
Vol 79 (21) ◽  
pp. 6647-6654 ◽  
Author(s):  
Boyang Cao ◽  
Fangfang Yao ◽  
Xiangqian Liu ◽  
Lu Feng ◽  
Lei Wang
Keyword(s):  
Dna Microarray ◽  
Legionella Pneumophila ◽  
Detection Sensitivity ◽  
Immunological Methods ◽  
Clinical Samples ◽  
Content Type ◽  
O Antigen ◽  
Detection And Identification ◽  
Legionnaires Disease ◽  
Reference Strains

ABSTRACTLegionellais ubiquitous in many environments. At least 50 species and 70 serogroups of the Gram-negative bacterium have been identified. Of the 50 species, 20 are pathogenic, andLegionella pneumophilais responsible for the great majority (approximately 90%) of the Legionnaires' disease cases that occur. Furthermore, of the 15L. pneumophilaserogroups identified, O1 alone causes more than 84% of the Legionnaires' disease cases that occur worldwide. Rapid and reliable assays for the detection and identification ofL. pneumophilain water, environmental, and clinical samples are in great demand.L. pneumophilabacteria are traditionally identified by their O antigens by immunological methods. We have recently developed an O serogroup-specific DNA microarray for the detection of all 15 distinct O-antigen forms ofL. pneumophila, including serogroups O1 to O15. A total of 35 strains were used to verify the specificity of the microarray, including 15L. pneumophilaO-antigen standard reference strains and sevenL. pneumophilaclinical isolates as target strains, seven reference strains of other non-pneumophila Legionellaspecies as closely related strains, and six non-Legionellabacterial species as nonrelated strains. The detection sensitivity was 1 ng of genomic DNA or 0.4 CFU/ml in water samples with filter enrichment and plate culturing. This study demonstrated that the microarray allows specific, sensitive, and reproducible detection ofL. pneumophilaserogroups. To the best of our knowledge, this is the first report of a microarray serotyping method for all 15 distinct O-antigen forms ofL. pneumophila.

scholarly journals Neurotoxin Gene Profiling of Clostridium botulinum Types C and D Native to Different Countries within Europe

2012 ◽  
Vol 78 (9) ◽  
pp. 3120-3127 ◽  
Author(s):  
Cedric Woudstra ◽  
Hanna Skarin ◽  
Fabrizio Anniballi ◽  
Lucia Fenicia ◽  
Luca Bano ◽  
...  
Keyword(s):  
Clostridium Botulinum ◽  
Limit Of Detection ◽  
Bacterial Species ◽  
Simultaneous Detection ◽  
Geographical Location ◽  
Gene Profiling ◽  
Animal Origin ◽  
Bacterial Strains ◽  
Content Type ◽  
Pcr Assays

ABSTRACTClostridium botulinumtypes C and D, as well as their mosaic variants C-D and D-C, are associated with avian and mammalian botulism. This study reports on the development of low-density macroarrays based on the GeneDisc cycler platform (Pall-GeneDisc Technologies) applied to the simultaneous detection of theC. botulinumsubtypes C, C-D, D, and D-C. The limit of detection of the PCR assays was 38 fg of total DNA, corresponding to 15 genome copies. Artificially contaminated samples of cecum showed a limit of detection below 50 spores/g. The tests were performed with a large variety of bacterial strains, includingC. botulinumtypes C (n= 12), C-D (n= 29), D (n= 5), and D-C (n= 10), other botulinum neurotoxin (BoNT)-producingClostridiumstrains (n= 20), non-BoNT-producing clostridia (n= 20), and other bacterial species (n= 23), and showed a high specificity. These PCR assays were compared to previously published real-time PCRs for the detection ofC. botulinumin 292 samples collected from cases of botulism events in four European regions. The majority of the samples originated from wild birds (n= 108), poultry (n= 60), and bovines (n= 56). Among the 292 samples, 144 were positive for either thebont/C-D or thebont/D-C gene by using the GeneDisc arrays. The reliability of the results tallied to 97.94%. Interestingly, only BoNT mosaics, types C-D and D-C, were found in naturally contaminated samples whatever their animal origin and their geographical location. Further investigations should now be performed in order to check that mosaic types dominate in Europe and that acquisition of mosaic types helps in survival or adaptation to particular niche.

scholarly journals Prospective Evaluation of ResistancePlus MG, a New Multiplex Quantitative PCR Assay for Detection of Mycoplasma genitalium and Macrolide Resistance

2017 ◽  
Vol 55 (6) ◽  
pp. 1915-1919 ◽  
Author(s):  
S. N. Tabrizi ◽  
J. Su ◽  
C. S. Bradshaw ◽  
C. K. Fairley ◽  
S. Walker ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Clinical Performance ◽  
Simultaneous Detection ◽  
Mycoplasma Genitalium ◽  
Macrolide Resistance ◽  
23S Rrna ◽  
Rrna Gene ◽  
Resistance Mutations ◽  
Content Type ◽  
Conventional Culture

ABSTRACT Mycoplasma genitalium is a significant pathogen for which first-line treatment is becoming less effective due to increased resistance to macrolides. As conventional culture and antimicrobial susceptibility testing is not feasible for routine detection of this pathogen, molecular markers such as detection of mutations in the 23S rRNA gene have been described to predict resistance. Recently, a novel multiplex quantitative PCR (qPCR) assay, ResistancePlus MG, has been described for the simultaneous detection of Mycoplasma genitalium and macrolide resistance. In the current study, the clinical performance of the assay was evaluated on 1,089 consecutive urine and anogenital swab samples in symptomatic and asymptomatic male and female patients. Overall, 6.0% were positive for M. genitalium , with 63.1% having macrolide resistance-associated mutations. Compared to the laboratory-validated qPCR method targeting the 16S rRNA gene and Sanger sequencing to determine 23S rRNA mutations, the sensitivity and specificity of M. genitalium detection were 98.5% and 100% and for detection of macrolide resistance mutations were 100.0% and 96.2%, respectively. This assay offers a considerable advantage in clinical settings for M. genitalium testing by making the results of macrolide resistance and mutation analyses simultaneously available, which is increasingly important with escalating macrolide resistance.

scholarly journals Characterization of Phascolarctobacterium succinatutens sp. nov., an Asaccharolytic, Succinate-Utilizing Bacterium Isolated from Human Feces

2011 ◽  
Vol 78 (2) ◽  
pp. 511-518 ◽  
Author(s):  
Yohei Watanabe ◽  
Fumiko Nagai ◽  
Masami Morotomi
Keyword(s):  
Bacterial Species ◽  
Short Chain Fatty Acids ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Gi Tract ◽  
Bacterial Strains ◽  
High Sequence Identity ◽  
Healthy Human ◽  
Content Type

ABSTRACTIsolation, cultivation, and characterization of the intestinal microorganisms are important for understanding the comprehensive physiology of the human gastrointestinal (GI) tract microbiota. Here, we isolated two novel bacterial strains, YIT 12067Tand YIT 12068, from the feces of healthy human adults. Phylogenetic analysis indicated that they belonged to the same species and were most closely related toPhascolarctobacterium faeciumACM 3679T, with 91.4% to 91.5% 16S rRNA gene sequence similarities, respectively. Substrate availability tests revealed that the isolates used only succinate; they did not ferment any other short-chain fatty acids or carbohydrates tested. When these strains were cocultured with the xylan-utilizing and succinate-producing bacteriumParaprevotella xylaniphilaYIT 11841T, in medium supplemented with xylan but not succinate, their cell numbers became 2 to 3 orders of magnitude higher than those of the monoculture; succinate became undetectable, and propionate was formed. Database analysis revealed that over 200 uncultured bacterial clones from the feces of humans and other mammals showed high sequence identity (>98.7%) to YIT 12067T. Real-time PCR analysis also revealed that YIT 12067T-like bacteria were present in 21% of human fecal samples, at an average level of 3.34 × 108cells/g feces. These results indicate that YIT 12067T-like bacteria are distributed broadly in the GI tract as subdominant members that may adapt to the intestinal environment by specializing to utilize the succinate generated by other bacterial species. The phylogenetic and physiological properties of YIT 12067Tand YIT 12068 suggest that these strains represent a novel species, which we have designatedPhascolarctobacterium succinatutenssp. nov.

scholarly journals Mutational and Transcriptomic Changes Involved in the Development of Macrolide Resistance in Campylobacter jejuni

2012 ◽  
Vol 57 (3) ◽  
pp. 1369-1378 ◽  
Author(s):  
Haihong Hao ◽  
Zonghui Yuan ◽  
Zhangqi Shen ◽  
Jing Han ◽  
Orhan Sahin ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Dna Microarray ◽  
Ribosomal Proteins ◽  
Macrolide Resistance ◽  
23S Rrna ◽  
Rrna Gene ◽  
Content Type ◽  
23S Rrna Gene ◽  
Resistant Mutants ◽  
Antibiotic Efflux

ABSTRACTMacrolide antibiotics are important for clinical treatment of infections caused byCampylobacter jejuni. Development of resistance to this class of antibiotics inCampylobacteris a complex process, and the dynamic molecular changes involved in this process remain poorly defined. Multiple lineages of macrolide-resistant mutants were selected by stepwise exposure ofC. jejunito escalating doses of erythromycin or tylosin. Mutations in target genes were determined by DNA sequencing, and the dynamic changes in the expression of antibiotic efflux transporters and the transcriptome ofC. jejuniwere examined by real-time reverse transcription-PCR, immunoblotting, and DNA microarray analysis. Multiple types of mutations in ribosomal proteins L4 and L22 occurred early during stepwise selection. On the contrary, the mutations in the 23S rRNA gene, mediating high resistance to macrolides, were observed only in the late-stage mutants. Upregulation of antibiotic efflux genes was observed in the intermediately resistant mutants, and the magnitude of upregulation declined with the occurrence of mutations in the 23S rRNA gene. DNA microarray analysis revealed the differential expression of 265 genes, most of which occurred in the intermediate mutant, including the upregulation of genes encoding ribosomal proteins and the downregulation of genes involved in energy metabolism and motility. These results indicate (i) that mutations in L4 and L22 along with temporal overexpression of antibiotic efflux genes precede and may facilitate the development of high-level macrolide resistance and (ii) that the development of macrolide resistance affects the pathways important for physiology and metabolism inC. jejuni, providing an explanation for the reduced fitness of macrolide-resistantCampylobacter.

scholarly journals Bdellovibrio and Like Organisms Enhanced Growth and Survival of Penaeus monodon and Altered Bacterial Community Structures in Its Rearing Water

2014 ◽  
Vol 80 (20) ◽  
pp. 6346-6354 ◽  
Author(s):  
Huanhuan Li ◽  
Cheng Chen ◽  
Qiuping Sun ◽  
Renliang Liu ◽  
Junpeng Cai
Keyword(s):  
Bacterial Community ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Bacterial Species ◽  
Penaeus Monodon ◽  
Pcr Amplification ◽  
Rrna Gene ◽  
Community Structures ◽  
Bacterial Strains ◽  
Content Type ◽  
Growth And Survival

ABSTRACTIn this study, a 96-h laboratory reduction test was conducted with strain BDHSH06 (GenBank accession no.EF011103) as the test strain forBdellovibrioand like organisms (BALOs) and 20 susceptible marine bacterial strains forming microcosms as the targets. The results showed that BDHSH06 reduced the levels of approximately 50% of prey bacterial strains within 96 h in the seawater microcosms. An 85-day black tiger shrimp (Penaeus monodon) rearing experiment was performed. The shrimp survival rate, body length, and weight in the test tanks were 48.1% ± 1.2%, 99.8 ± 10.0 mm, and 6.36 ± 1.50 g, respectively, which were values significantly (P< 0.05) higher than those for the control,viz., 31.0% ± 2.1%, 86.0 ± 11.1 mm, and 4.21 ± 1.56 g, respectively. With the addition of BDHSH06, total bacterial andVibrionumbers were significantly reduced (P< 0.05) by 1.3 to 4.5 log CFU · ml−1and CFU · g−1in both water and shrimp intestines, respectively, compared to those in the control. The effect of BDHSH06 on bacterial community structures in the rearing water was also examined using PCR amplification of the 16S rRNA gene and denaturing gradient gel electrophoresis (DGGE). The DGGE profiles of rearing water samples from the control and test tanks revealed that the amounts of 44% of the bacterial species were reduced when BDHSH06 was added to the rearing water over the 85-day rearing period, and among these, approximately 57.1% were nonculturable. The results of this study demonstrated that BDHSH06 can be used as a biocontrol/probiotic agent inP. monodonculture.

scholarly journals Development of a DNA Microarray for Molecular Identification of All 46 Salmonella O Serogroups

2013 ◽  
Vol 79 (11) ◽  
pp. 3392-3399 ◽  
Author(s):  
Dan Guo ◽  
Bin Liu ◽  
Fenxia Liu ◽  
Boyang Cao ◽  
Min Chen ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Bacterial Species ◽  
Rapid Identification ◽  
Detection Sensitivity ◽  
Epidemiological Surveillance ◽  
Disease Assessment ◽  
Content Type ◽  
Detection And Identification ◽  
Food Borne

ABSTRACTSalmonellais a major cause of food-borne disease in many countries. Serotype determination ofSalmonellais important for disease assessment, infection control, and epidemiological surveillance. In this study, a microarray system that targets the O antigen-specific genes was developed for simultaneously detecting and identifying all 46SalmonellaO serogroups. Of these, 40 serogroups can be confidently identified, and the remaining 6, in three pairs (serogroups O67 and B, E1 and E4, and A and D1), need to be further distinguished from each other using PCR methods or conventional serotyping methods. The microarray was shown to be highly specific when evaluated against 293Salmonellastrains, 186Shigellastrains, representativeEscherichia colistrains, and 10 strains of other bacterial species. The assay correctly identified 288 (98%) of theSalmonellastrains. The detection sensitivity was determined to be 50 ng genomic DNA per sample. By testing simulated samples in a tomato background, 2 to 8 CFU per gram inoculated could be detected after enrichment. This newly developed microarray assay is the first molecular protocol that can be used for the comprehensive detection and identification of all 46SalmonellaO serogroups. Compared to the traditional serogrouping method, the microarray provides a reliable, high-throughput, and sensitive approach that can be used for rapid identification of multipleSalmonellaO serogroups simultaneously.

scholarly journals Clostridium algifaecis sp. nov., an anaerobic bacterial species from decomposing algal scum

2014 ◽  
Vol 64 (Pt_11) ◽  
pp. 3844-3848 ◽  
Author(s):  
Yu-Fan Wu ◽  
Hui Zheng ◽  
Qing-long Wu ◽  
Hong Yang ◽  
Shuang-Jiang Liu
Keyword(s):  
Novel Species ◽  
Bacterial Species ◽  
Rrna Gene ◽  
Clostridium Tyrobutyricum ◽  
Gram Stain ◽  
Bacterial Strains ◽  
16S Rrna Gene Sequences ◽  
Content Type ◽  
Cellular Fatty Acids ◽  
Link Type

Two anaerobic bacterial strains, MB9-7T and MB9-9, were isolated from decomposing algal scum and were characterized using a polyphasic approach. Phylogenetic analysis of 16S rRNA gene sequences showed that strains MB9-7T and MB9-9 are closely related to each other (99.7 % similarity) and they are also closely related to Clostridium tyrobutyricum (96.5 %). The two strains were Gram-stain positive and rod-shaped. Growth occurred at 20–45 °C, at pH 4.0–8.0 and at NaCl concentrations of up to 2 % (w/v). Acid was produced from glucose, xylose and mannose. Products of fermentation in PYG medium were mainly butyrate, acetate, carbon dioxide and hydrogen. The predominant cellular fatty acids were C14 : 0 and C16 : 0. The cellular polar lipids comprised phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, two glycolipids, one phospholipid, one aminophospholipid and two aminolipids. The DNA G+C contents of strain MB9-7T and MB9-9 were 27.9 and 28.7 mol%, respectively. These results support the assignment of the new isolates to the genus Clostridium and also distinguish them from other species of the genus Clostridium . Hence, it is proposed that strains MB9-7T and MB9-9 represent a novel species of the genus Clostridium , with the suggested name Clostridium algifaecis sp. nov. The type strain is MB9-7T ( = CGMCC 1.5188T = DSM 28783T).

scholarly journals Clinical Evaluation of Three Commercial PCR Assays for the Detection of Macrolide Resistance in Mycoplasma genitalium

2019 ◽  
Vol 58 (2) ◽  
Author(s):  
Chloé Le Roy ◽  
Cécile Bébéar ◽  
Sabine Pereyre
Keyword(s):  
Sensitivity And Specificity ◽  
Internal Control ◽  
Simultaneous Detection ◽  
Mycoplasma Genitalium ◽  
Macrolide Resistance ◽  
University Hospital ◽  
23S Rrna ◽  
Rrna Gene ◽  
Content Type ◽  
Commercial Kits

ABSTRACT As macrolide resistance in Mycoplasma genitalium is increasing worldwide, macrolide resistance-associated mutations should be assessed in M. genitalium-positive specimens. New commercial kits are available for detection of macrolide resistance concurrently with M. genitalium. We prospectively evaluated the handling and clinical performances of three commercial kits for detection of macrolide resistance in M. genitalium. Between August and December 2018, remnants of all urogenital specimens determined to be M. genitalium positive using an in-house real-time PCR assay were prospectively collected at the French National Reference Center for Bacterial Sexually Transmitted Infections, Bordeaux University Hospital, Bordeaux, France. The internal control of each kit was added to the primary specimen before DNA extraction, and the absence of amplification inhibition associated with the addition of the three internal controls was assessed. Specimens were evaluated with four assays: the ResistancePlus MG assay (SpeeDx), the S-DiaMGRes assay (Diagenode), the RealAccurate TVMGres assay (PathoFinder), and amplification and sequencing of the 23S rRNA gene (the reference assay). Overall, 195 M. genitalium-positive specimens were assessed. The positive agreement of M. genitalium detection for each kit ranged between 94.8% and 96.4%. Among 154 specimens with M. genitalium positivity as detected by the three commercial kits and 23S rRNA sequencing data, the clinical sensitivity and specificity ranges of the three commercial kits for detecting macrolide resistance-associated mutations were 95 to 100% and 94.6 to 97.3%, respectively. The sensitivity and specificity values were similar among the kits. The launch of three easy-to-use sensitive and specific commercial kits for simultaneous detection of M. genitalium and macrolide resistance will be useful for resistance-guided therapy.

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