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Journal of Microbiological Methods

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https://www.readbyqxmd.com/read/28545919/molecular-diagnosis-of-salmonella-typhi-and-its-virulence-in-suspected-typhoid-blood-samples-through-nested-multiplex-pcr
#1
Solai Ramatchandirane Prabagaran, Vellingiri Kalaiselvi, Naganathan Chandramouleeswaran, Krishnan Nair Geetha Deepthi, Kootallur Narayanan Brahmadathan, Mariappa Mani
A nested multiplex polymerase chain reaction (PCR) based diagnosis was developed for the detection of virulent Salmonella typhi in the blood specimens from patients suspected for typhoid fever. After the Widal test, two pairs of primers were used for the detection of flagellin gene (fliC) of S. typhi. Among them, those positive for fliC alone were subjected to identification of genes in Via B operon of Salmonella Pathogenesity Island (SPI-7) where four primer pairs were used to detect tviA and tviB genes. Among 250 blood samples tested, 115 were positive by fliC PCR; 22 of these were negative for tviA and tviB...
May 22, 2017: Journal of Microbiological Methods
https://www.readbyqxmd.com/read/28533107/ehs-matrix-incubated-in-media-containing-penicillin-retains-sufficient-concentrations-of-antibiotic-to-inhibit-growth-of-susceptible-microorganisms
#2
Mark P Murphy, Dagmara A Niedziela, Orla M Keane
In studying the interaction between bacteria and host cells in vitro, the latter are frequently cultured on commercially available biotic matrices such as Matrigel® or Geltrex®. To avoid contamination, host cells are commonly grown in the presence of antibiotics. However, we present here the finding that cell culture on such a matrix in the presence of antibiotics interferes with the outcome of subsequent infection experiments by virtue of diminished bacterial survival. By comparing outcomes for penicillin-susceptible and resistant strains of Staphylococcus aureus, we show that residual penicillin remains in the matrix despite the antibiotics' withdrawal from culture...
May 19, 2017: Journal of Microbiological Methods
https://www.readbyqxmd.com/read/28527886/immuno-pcr-a-new-technique-for-the-serodiagnosis-of-tuberculosis
#3
REVIEW
Promod K Mehta, Bhawna Dahiya, Suman Sharma, Netrapal Singh, Renu Dharra, Zoozeal Thakur, Neeru Mehta, Krishna B Gupta, Mahesh C Gupta, Dhruva Chaudhary
Rapid and accurate diagnosis of tuberculosis (TB) is essential to control the disease. The conventional microbiological tests have limitations and there is an urgent need to devise a simple, rapid and reliable point-of-care (POC) test. The failure of TB diagnostic tests based on antibody detection due to inconsistent and imprecise results has stimulated renewed interest in the development of rapid antigen detection methods. However, the World Health Organization (WHO) has emphasized to continue research for designing new antibody-based detection tests with improved accuracy...
May 17, 2017: Journal of Microbiological Methods
https://www.readbyqxmd.com/read/28522389/crispr-dcas9-mediated-inhibition-of-gene-expression-in-staphylococcus-aureus
#4
Xiaoyun Dong, Yingli Jin, Di Ming, Bangbang Li, Haisi Dong, Lin Wang, Tiedong Wang, Dacheng Wang
The understanding of the genetic mechanism of Staphylococcus aureus requires efficient tools, however, genetic manipulation in S. aureus is always laborious and time-consuming. Here we proposed a novel CRISPR/dCas9 interference method for the rapid knockdown of target genes. Furthermore, multiple genes can be repressed simultaneously by using this method.
May 15, 2017: Journal of Microbiological Methods
https://www.readbyqxmd.com/read/28514671/evaluation-of-improved-is6110-lamp-assay-for-diagnosis-of-pulmonary-and-extra-pulmonary-tuberculosis
#5
Deepali Joon, Manoj Nimesh, Mandira Varma-Basil, Daman Saluja
In the present study, IS6110 loop mediated isothermal amplification (LAMP) assay was modified using dUTP-UNG (uracil-DNA N-glycosylase) strategy to prevent carryover contamination, and was evaluated using clinical specimens. The clinical specimens were collected from 236 suspected patients of pulmonary tuberculosis and 315 specimens of suspected patients of extra pulmonary tuberculosis. DNA was extracted from specimens and used as template for nucleic acid amplification. The results were evaluated with culture method as gold standard...
May 14, 2017: Journal of Microbiological Methods
https://www.readbyqxmd.com/read/28511895/diagnostic-performance-of-anyplex-ii-mtb-mdr-xdr-for-detection-of-resistance-to-first-and-second-line-drugs-in-mycobacterium-tuberculosis
#6
F Pérez-García, M J Ruiz-Serrano, P López Roa, Fermín Acosta, Laura Pérez-Lago, D García-De-Viedma, E Bouza
PURPOSE: Genotypic methods have considerably improved the diagnosis of multidrug-resistant (MDR) tuberculosis. One of these tests is Anyplex II MTB/MDR/XDR (Anyplex). Our aim was to evaluate the diagnostic performance of this multiplex PCR. METHODS: We conducted our study on 47 MDR tuberculosis and 14 pan-susceptible strains. We evaluated the ability of Anyplex to detect resistance mutations in rpoB (rifampin [RIF]), katG and inhA (isoniazid [INH]), gyrA (fluoroquinolones [FLQ]), and rrs and eis (aminoglycosides [AMG])...
May 13, 2017: Journal of Microbiological Methods
https://www.readbyqxmd.com/read/28506638/ionic-liquid-based-reagents-improve-the-stability-of-midterm-fecal-sample-storage
#7
Lilan Hao, Zhongkui Xia, Huanming Yang, Jian Wang, Mo Han
Fecal samples are widely used in metagenomic research, which aims to elucidate the relationship between human health and the intestinal microbiota. However, the best conditions for stable and reliable storage and transport of these samples at room temperature are still unknown, and whether samples stored at room temperature for several days will maintain their microbiota composition is still unknown. Here, we established and tested a preservation method using reagents containing imidazolium- or pyridinium-based ionic liquids...
May 13, 2017: Journal of Microbiological Methods
https://www.readbyqxmd.com/read/28495585/rapid-separation-of-very-low-concentrations-of-bacteria-from-blood
#8
Clara M Buchanan, Ryan L Wood, Taalin R Hoj, Mahsa Alizadeh, Colin G Bledsoe, Madison E Wood, Daniel S McClellan, Rae Blanco, Caroline L Hickey, Tanner V Ravsten, Ghaleb A Husseini, Richard A Robison, William G Pitt
A rapid and accurate diagnosis of the species and antibiotic resistance of bacteria in septic blood is vital to increase survival rates of patients with bloodstream infections, particularly those with carbapenem-resistant enterobacteriaceae (CRE) infections. The extremely low levels in blood (1 to 100CFU/ml) make rapid diagnosis difficult. In this study, very low concentrations of bacteria (6 to 200CFU/ml) were separated from 7ml of whole blood using rapid sedimentation in a spinning hollow disk that separated plasma from red and white cells, leaving most of the bacteria suspended in the plasma...
May 8, 2017: Journal of Microbiological Methods
https://www.readbyqxmd.com/read/28487054/reduced-cost-chlamydia-trachomatis-specific-multiplex-real-time-pcr-diagnostic-assay-evaluated-for-ocular-swabs-and-use-by-trachoma-research-programmes
#9
Robert Butcher, Jo Houghton, Tamsyn Derrick, Athumani Ramadhani, Beatriz Herrera, Anna R Last, Patrick A Massae, Matthew J Burton, Martin J Holland, Chrissy H Roberts
INTRODUCTION: Trachoma, caused by the intracellular bacterium Chlamydia trachomatis (Ct), is the leading infectious cause of preventable blindness. Many commercial platforms are available that provide highly sensitive and specific detection of Ct DNA. However, the majority of these commercial platforms are inaccessible for population-level surveys in resource-limited settings typical to trachoma control programmes. We developed two low-cost quantitative PCR (qPCR) tests for Ct using readily available reagents on standard real-time thermocyclers...
May 6, 2017: Journal of Microbiological Methods
https://www.readbyqxmd.com/read/28483549/detection-of-oxa-370-directly-from-rectal-swabs-and-blood-culture-vials-using-an-immunochromatographic-assay
#10
Carolina Silva Nodari, Ana Cristina Gales, Afonso Luís Barth, Cibele Massotti Magagnin, Alexandre Prehn Zavascki, Cecília Godoy Carvalhaes
We evaluated the performance of OXA-48 K-SeT assay for detecting OXA-370 directly from spiked rectal swabs and blood culture vials. The limit of detection of this test was 10(4)UFC/mL for rectal swabs. Detection of the OXA-370-producing isolates was successfully achieved directly from positive blood culture vials independent of growing conditions.
May 5, 2017: Journal of Microbiological Methods
https://www.readbyqxmd.com/read/28479312/a-modified-esbl-nordmann-dortet-poirel-based-protocol-to-optimize-early-sepsis-management
#11
Jorge Arca-Suárez, Fátima Galán-Sánchez, César Del Prado Montoro, Manuel A Rodríguez-Iglesias
We evaluated a modification of a colorimetric test recently described by Dortet et al. (2015) for the rapid detection of ESBL-producing Enterobacteriaceae directly from positive blood cultures that requires less manipulation, materials and hands-on time. The simplified protocol showed a sensitivity and specificity of 100% and 95.7% respectively.
May 4, 2017: Journal of Microbiological Methods
https://www.readbyqxmd.com/read/28477900/detection-and-quantification-of-intracellular-bacterial-colonies-by-automated-high-throughput-microscopy
#12
Christina L Ernstsen, Frédéric H Login, Helene H Jensen, Rikke Nørregaard, Jakob Møller-Jensen, Lene N Nejsum
To target bacterial pathogens that invade and proliferate inside host cells, it is necessary to design intervention strategies directed against bacterial attachment, cellular invasion and intracellular proliferation. We present an automated microscopy-based, fast, high-throughput method for analyzing size and number of intracellular bacterial colonies in infected tissue culture cells. Cells are seeded in 48-well plates and infected with a GFP-expressing bacterial pathogen. Following gentamicin treatment to remove extracellular pathogens, cells are fixed and cell nuclei stained...
May 3, 2017: Journal of Microbiological Methods
https://www.readbyqxmd.com/read/28461023/applications-of-copolymer-for-rapid-identification-of-bacteria-in-blood-culture-broths-using-matrix-assisted-laser-desorption-ionization-time-of-flight-mass-spectrometry
#13
Kazuho Ashizawa, Syota Murata, Takashi Terada, Daisuke Ito, Masaru Bunya, Koji Watanabe, Yoko Teruuchi, Sachio Tsuchida, Mamoru Satoh, Motoi Nishimura, Kazuyuki Matsushita, Yuji Sugama, Fumio Nomura
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) can be used to identify pathogens in blood culture samples. However, sample pretreatment is needed for direct identification of microbes in blood culture bottles. Conventional protocols are complex and time-consuming. Therefore, in this study, we developed a method for collecting bacteria using polyallylamine-polystyrene copolymer for application in wastewater treatment technology. Using representative bacterial species Escherichia coli and Staphylococcus capitis, we found that polyallylamine-polystyrene can form visible aggregates with bacteria, which can be identified using MALDI-TOF MS...
April 29, 2017: Journal of Microbiological Methods
https://www.readbyqxmd.com/read/28457942/identification-and-characterization-of-simple-sequence-repeats-ssrs-for-population-studies-of-puccinia-novopanici
#14
Gabriela K Orquera-Tornakian, Patricia Garrido, Brent Kronmiller, Robert Hunger, Brett M Tyler, Carla D Garzon, Stephen M Marek
Switchgrass (Panicum virgatum L.) can be severely affected by rust disease. Recently switchgrass rust caused by P. emaculata (now confirmed to be Puccinia novopanici) has received most of the attention by the research community because this pathogen is responsible for reducing the biomass production and biofuel feedstock quality of switchgrass. Microsatellite markers found in the literature were either not informative (no allele frequency) or showed few polymorphisms in the target populations, therefore additional markers are needed for future studies of the genetic variation and population structure of P...
April 27, 2017: Journal of Microbiological Methods
https://www.readbyqxmd.com/read/28456552/subtyping-of-canadian-isolates-of-salmonella-enteritidis-using-multiple-locus-variable-number-tandem-repeat-analysis-mlva-alone-and-in-combination-with-pulsed-field-gel-electrophoresis-pfge-and-phage-typing
#15
Kim Ziebell, Linda Chui, Robin King, Suzanne Johnson, Patrick Boerlin, Roger P Johnson
Salmonella enterica subspecies enterica serovar Enteritidis (SE) is one of the most common causes of human salmonellosis and in Canada currently accounts for over 40% of human cases. Reliable subtyping of isolates is required for outbreak detection and source attribution. However, Pulsed-Field Gel Electrophoresis (PFGE), the current standard subtyping method for Salmonella spp., is compromised by the high genetic homogeneity of SE. Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) was introduced to supplement PFGE, although there is a lack of data on the ability of MLVA to subtype Canadian isolates of SE...
April 26, 2017: Journal of Microbiological Methods
https://www.readbyqxmd.com/read/28438642/diagnostic-microarray-for-14-water-and-foodborne-pathogens-using-a-flatbed-scanner
#16
Vidya Srinivasan, Robert D Stedtfeld, Dieter M Tourlousse, Samuel W Baushke, Yu Xin, Sarah M Miller, Trinh Pham, Jean-Marie Rouillard, Erdogan Gulari, James M Tiedje, Syed A Hashsham
Parallel detection approaches are of interest to many researchers interested in identifying multiple water and foodborne pathogens simultaneously. Availability and cost-effectiveness are two key factors determining the usefulness of such approaches for laboratories with limited resources. In this study, we developed and validated a high-density microarray for simultaneous screening of 14 bacterial pathogens using an approach that employs gold labeling with silver enhancement (GLS) protocol. In total, 8887 probes (50-mer) were designed using an in-house database of virulence and marker genes (VMGs), and synthesized in quadruplicate on glass slides using an in-situ synthesis technology...
April 23, 2017: Journal of Microbiological Methods
https://www.readbyqxmd.com/read/28438643/convenient-pcr-method-for-variable-number-tandem-repeat-typing-of-mycobacterium-tuberculosis-clinical-isolates
#17
Junji Seto, Takayuki Wada, Yu Suzuki, Tatsuya Ikeda, Katsumi Mizuta, Satoshi Mitarai, Tadayuki Ahiko
Variable-number tandem-repeat typing for Mycobacterium tuberculosis clinical isolates contributes to evidence-based tuberculosis control. However, cumbersome PCR procedures for the typing have disturbed routine analyses. We proposed a convenient PCR method for the typing using a PCR master mix that provides rapidity and long-term stability of the frozen PCR cocktail.
April 22, 2017: Journal of Microbiological Methods
https://www.readbyqxmd.com/read/28434823/influence-of-serum-and-polystyrene-plate-type-on-stability-of-candida-albicans-biofilms
#18
Purity N Kipanga, Walter Luyten
Adhesion is a crucial initial step in microbial biofilm formation. Firm attachment to a target surface subsequently ensures successful colonization and survival despite turbulent conditions. In the laboratory, polystyrene plates are commonly used in biofilm experiments and the 'washing/rinse steps' before staining are critical for assaying biofilm viability. However, these rinse steps risk the removal (partially or entirely) of the formed biofilm, resulting in inconsistent results. The aim of the present study was to optimize conditions for firmer biofilms, less prone to disruption and thus significantly reducing well-to-well variability...
April 21, 2017: Journal of Microbiological Methods
https://www.readbyqxmd.com/read/28434824/multiple-pathogen-biomarker-detection-using-an-encoded-bead-array-in-droplet-pcr
#19
Prem Kumar Periyannan Rajeswari, Lovisa M Soderberg, Alia Yacoub, Mikael Leijon, Helene Andersson Svahn, Haakan N Joensson
We present a droplet PCR workflow for detection of multiple pathogen DNA biomarkers using fluorescent color-coded Luminex® beads. This strategy enables encoding of multiple singleplex droplet PCRs using a commercially available bead set of several hundred distinguishable fluorescence codes. This workflow provides scalability beyond the limited number offered by fluorescent detection probes such as TaqMan probes, commonly used in current multiplex droplet PCRs. The workflow was validated for three different Luminex bead sets coupled to target specific capture oligos to detect hybridization of three microorganisms infecting poultry: avian influenza, infectious laryngotracheitis virus and Campylobacter jejuni...
April 20, 2017: Journal of Microbiological Methods
https://www.readbyqxmd.com/read/28411150/comparative-diagnostic-efficacy-of-recombinant-llo-and-pi-plc-based-elisas-for-detection-of-listeriosis-in-animals
#20
Rahul D Suryawanshi, Satya Veer Singh Malik, Bhushan Jayarao, Sandeep P Chaudhari, Emily Savage, Jess Vergis, Nitin V Kurkure, Sukhadeo B Barbuddhe, Deepak B Rawool
The present study for the first time evaluates the serodiagnostic efficacy of two recombinant antigens namely, listeriolysin O (rLLO) and phosphatidyl-inositol phospholipase C (rPI-PLC). Indirect ELISA with the above recombinant antigens was used on samples collected from bovines (n=106), goats (n=138) and pigs (n=92) having either a history of abortion, emaciation and/or apparently healthy animals. Isolation of Listeria was attempted from the blood samples using USDA-FSIS method. On screening of test sera by rLLO-based ELISA, antibodies against anti-listeriolysin O (ALLO) were observed in goats (22...
April 12, 2017: Journal of Microbiological Methods
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