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Journal of Microbiological Methods

Anne-Marie Van den Abeele, Dirk Vogelaers, Peter Vandamme, Elke Vanlaere, Kurt Houf
Arcobacters are considered emerging gastrointestinal pathogens. Rapid, reliable and species-specific identification of these bacteria is important. Biochemical tests commonly yield negative or variable results. Molecular methods prove more reliable but are time consuming and lack specificity. Matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is a fast, cheap and robust technique that has revolutionized genus and species identification in clinical microbiology. The performance of an in vitro diagnostic (RUO) spectral database of MALDI-TOF MS for the identification of human clinically relevant Arcobacter isolates was validated and compared to an in house created Reference Spectral database (RS) containing a representative set of deposited Arcobacter strains of zoonotic interest...
July 12, 2018: Journal of Microbiological Methods
Junjun Shang, Yan Li, Ruiheng Yang, Ying Wang, Wenjun Mao, Lihua Tang, Yingying Wu, Takehito Nakazawa, Yoichi Honda, Yan Li, Dapeng Bao
We introduced a site-directed mutation in the sdi1 gene and used it as a selective marker for the polyethylene glycol-mediated transformation of Pleurotus eryngii monokaryon protoplasts. The transformants displayed obvious and stable resistance to the fungicide carboxin indicating that the mutant Pesdi1 gene is an efficient selective marker.
July 12, 2018: Journal of Microbiological Methods
Kássia de Carvalho Dias, Denise Lins de Sousa, Paula Aboud Barbugli, Paulo Sérgio Cerri, Vehid Max Salih, Carlos Eduardo Vergani
The aim of this study was to (i) design, develop and validate a practical and physiologically relevant reconstituted in vitro oral mucosa tissue model and (ii) to assess its applicability in in vitro host-pathogen interactions with C. albicans and S. aureus. Co-culture organotypic constructions were created by incorporating specific numbers of keratinocytes (NOK-si) onto cellularised, collagen gel scaffolds containing human gingival fibroblasts incubated in KGM media and cultured for 14 days. The detection of the appropriate oral mucosa/epithelial structure was evaluated by histology (hematoxylin and eosin (HE), periodic acid-Schiff (P...
July 11, 2018: Journal of Microbiological Methods
Mano Sivaganesan, Manju Varma, Shawn Siefring, Richard Haugland
An obstacle to establishing widely useful data acceptance criteria for U.S. Environmental Protection Agency (EPA) qPCR methods has been the unavailability of standardized reference materials. Earlier versions of EPA Methods 1609 and 1611 for enterococci used cellular reference materials for quantifying enterococci in unknown test samples, however, EPA updates to these fundamentally DNA-based analysis methods have shifted toward the use of DNA standards. This report describes the application of droplet digital PCR (ddPCR) analysis for the quantification of a set of synthetic plasmid DNA standards that have been made available for updated EPA Methods 1609...
July 11, 2018: Journal of Microbiological Methods
A M Nogacka, P Ruas-Madiedo, E Gómez, G Solís, N Fernández, M Suárez, A Suárez, N Salazar, C G de Los Reyes-Gavilán, M Gueimonde
Several in vitro screening tests have been used for selecting probiotic strains; however they often show low predictive value and only a limited number of strains have demonstrated functionality in vivo. The most used in vitro tests represent a very simplified version of the gut environment, especially since they do not consider the accompanying microbiota. Therefore, there is a need to develop sensitive and discriminating in vitro models including the microbiota. Here we developed an in vitro model to discriminate among microbiotas/fecal waters from different population groups...
July 10, 2018: Journal of Microbiological Methods
Raelene Engelberg, Ashley Danielson, Shirley Wang, Manu Singh, Alvan Wai, John Sorensen, Kangmin Duan, Georg Hausner, Ayush Kumar
Antibiotic resistance of bacteria is a considerable challenge to human health in the 21st century. With our discovery pipeline for new and effective antibiotics rapidly drying out, innovative approaches are needed to find new antimicrobials. Soil fungi are known to produce a variety of antimicrobials but rapid screening of fungi that produce such compounds remains a challenge. In this work, we used a hyper-susceptible strain of Pseudomonas aeruginosa to create a luminescent-reporter strain to be used as a screening tool to select fungi producing antimicrobials...
July 7, 2018: Journal of Microbiological Methods
Brijesh B Karakkat, Kurt Hockemeyer, Margot Franchett, Megan Olson, Cortney Mullenberg, Paul L Koch
Root-infecting fungal pathogens such as Gaeumannomyces avenae, Ophiosphaerella korrae, and Magnaporthiopsis poae cause extensive damage to amenity turfgrasses in temperate climates. The diseases they cause are difficult to diagnose by visual symptoms or microscopic inspection, and traditional polymerase chain reaction-based assays require large financial investments in equipment such as thermal cyclers and highly trained staff. The primary objective of this research was to develop fast and accurate detection assays for the three pathogens listed above that did not require the use of thermal cycling equipment...
June 28, 2018: Journal of Microbiological Methods
Susanna Pecchia, Daniele Da Lio
The 'Nucleic Acid Lateral Flow Immunoassay' (NALFIA) using a generic 'Lateral Flow Device' (LFD), combined with PCR employing labelled primers (PCR-NALFIA), enables to circumvent the use of electrophoresis, making the diagnostic procedure more rapid and easier. If the specific amplicon is present in the sample, a coloured band, with an intensity proportional to the amplicon concentration, will develop on the LFD strip in addition to the control band. Species-specific primers for M. phaseolina based on the rDNA intergenic spacer (IGS) were developed and their specificity was checked and confirmed using 20 isolates of M...
June 27, 2018: Journal of Microbiological Methods
E Bussalleu, G C Althouse
Serratia marcescens is a bacterial contaminant that can be spermicidal when present in extended boar semen that is typically stored prior to breeding use at 15 to 18 °C for several days. This particular contaminant appears to originate from carrier boars, where it resides in the preputial cavity, but has also been shown to then easily contaminate the semen-processing laboratory. Screening for carrier boars to date has been performed through detection of S.marcescens in ejaculates using traditional agar plate culture techniques...
June 27, 2018: Journal of Microbiological Methods
Rohit Khandelwal, Sonal Agarwal, Divya Singhi, Preeti Srivastava, Virendra Swarup Bisaria
Zymomonas mobilis ZM4 is a gram negative ethanologenic bacterium used in several biotechnological applications. Metabolic engineering in this bacterium is limited because of the available genome engineering tools. In the present study, we report genome engineering in this bacterium using bacteriophage lambda Red genes. Stability of plasmid replicons RK2 (pSIM9) and pBBR1 (pSIM7) containing the lambda Red genes was found to be 78% and 74%, respectively. We demonstrate successful deletion of pyruvate decarboxylase gene by recombineering in Z...
June 26, 2018: Journal of Microbiological Methods
Karim Helmi, Fabienne David, Patrick Di Martino, Marie-Pierre Jaffrezic, Valérie Ingrand
The control of Legionella proliferation in cooling tower water circuits requires regular monitoring of water contamination and effective disinfection procedures. In this study, flow cytometry was assessed to monitor water contamination and disinfection treatment efficiency on bacterial cells regarding nucleic acid injury (SYBR® Green II), cell integrity (SYBR® Green II and propidium iodide) and metabolism activity (ChemChrome V6). A total of 27 cooling tower water samples were analyzed in order to assess water contamination levels regarding viable populations: standard culture, ATP measurement and flow cytometry methods were compared...
June 26, 2018: Journal of Microbiological Methods
Esmaeil Forouzan, Parvin Shariati, Masoumeh Sadat Mousavi Maleki, Ali Asghar Karkhane, Bagher Yakhchali
Next Generation Sequencing (NGS) technologies are revolutionizing the field of biology and metagenomic-based research. Since the volume of metagenomic data is typically very large, De novo metagenomic assembly can be effectively used to reduce the total amount of data and enhance quality of downstream analysis, such as annotation and binning. Although, there are many freely available assemblers, but selecting one suitable for a specific goal can be highly challenging. In this study, the performance of 11 well-known assemblers was evaluated in the assembly of three different metagenomes...
June 25, 2018: Journal of Microbiological Methods
Warawan Wongboot, Kazuhisa Okada, Siriporn Chantaroj, Watcharaporn Kamjumphol, Shigeyuki Hamada
Acute diarrheal diseases are causes of global public health concern, especially in developing countries. A variety of diarrhea-associated microbial species, including bacteria, viruses, and protozoa, have been recognized. Simplified methods for detecting a wide range of diarrheagenic enteric microbes can clarify the etiology and aid in the diagnosis of diarrheal diseases. Here, we report a quantitative real-time (q)PCR-based method for simultaneous detection of 24 targets from 19 microbes suspected of causing diarrhea in stool specimens...
June 18, 2018: Journal of Microbiological Methods
Jess Vergis, Richa Pathak, Manesh Kumar, R Sunitha, S V S Malik, S B Barbuddhe, Deepak B Rawool
We explored and evaluated for the first time colorimetric nitrocefin assay in conjunction with the double disc test and PCR assay. We suggested the use of nitrocefin assay for rapid screening of ESBL-production by Enteroaggregative Escherichia coli.
June 18, 2018: Journal of Microbiological Methods
Johan Gimonet, Anne-Catherine Portmann, Coralie Fournier, Leen Baert
This work shows that an incubation time reduced to 4-5 h to prepare a culture for DNA extraction followed by an automated DNA extraction can shorten the hands-on time, the turnaround time by 30% and increase the throughput while maintaining the WGS quality assessed by high quality Single Nucleotide Polymorphism analysis.
June 16, 2018: Journal of Microbiological Methods
Bernice J Klotoe, Barbara Molina-Moya, Harrison Magdinier Gomes, Michel K Gomgnimbou, Lorenna Oliveira Suzarte, Maria H Féres Saad, Sajid Ali, José Dominguez, Edita Pimkina, Elena Zholdybayeva, Christophe Sola, Guislaine Refrégier
Several diagnostic tests are being developed to detect drug resistance in tuberculosis. In line with previous developments detecting rifampicin and isoniazid resistance using microbead-based systems (spoligoriftyping and TB-SPRINT), we present here an assay called TB-EFI detecting mutations involved in resistance to ethambutol, fluoroquinolones and the three classical injectable drugs (kanamycin, amikacin and capreomycin) in Mycobacterium tuberculosis. The proposed test includes both wild-type and mutant probes for each targeted locus...
June 15, 2018: Journal of Microbiological Methods
Cristina García-Timermans, Peter Rubbens, Frederiek-Maarten Kerckhof, Benjamin Buysschaert, Dmitry Khalenkow, Willem Waegeman, Andre G Skirtach, Nico Boon
Raman spectroscopy has gained relevance in single-cell microbiology for its ability to detect bacterial (sub)populations in a non-destructive and label-free way. However, the Raman spectrum of a bacterium can be heavily affected by abiotic factors, which may influence the interpretation of experimental results. Additionally, there is no publicly available standard for the annotation of metadata describing sample preparation and acquisition of Raman spectra. This article explores the importance of sample manipulations when measuring bacterial subpopulations using Raman spectroscopy...
June 14, 2018: Journal of Microbiological Methods
Yanan Li, Jianchang Wang, Jinfeng Wang, Libing Liu, Ruoxi Zhang, Ruihan Shi, Qingan Han, Jiguo Sun, Wanzhe Yuan
Porcine proliferative enteritis is a common diarrheal disease characterized by thickening of the intestinal mucosa in swine due to enterocyte proliferation, which is caused by Lawsonia intracellularis. In this study, a real-time loop-mediated isothermal amplification (LAMP) assay was developed to detect L. intracellularis based on the conserved region of the 16S ribosomal RNA gene. The optimal reaction conditions of the real-time LAMP was 65 °C for 60 min. The LAMP products could be detected by both real-time turbidity and direct visual inspection...
June 13, 2018: Journal of Microbiological Methods
Dmitrii M Bubnov, Tigran V Yuzbashev, Tatiana V Vybornaya, Alexander I Netrusov, Sergey P Sineoky
Plasmid-based systems are the most appropriate for multistep lambda Red (λRed)-mediated recombineering, such as the assembly of strains for biotechnological applications. Currently, the widely used λRed-expressing plasmids use a temperature-sensitive origin of replication or temperature shift control of λRed expression. In this work, we have constructed a new, conditionally replicating vector that can be efficiently eliminated from the host strain through passaging in medium containing isopropyl-β-d-thiogalactopyranoside...
June 7, 2018: Journal of Microbiological Methods
Vaibhav Diwan, Hans-Jørgen Albrechtsen, Barth F Smets, Arnaud Dechesne
Universal (i.e., targeting most bacteria/prokaryotes) 16S rRNA gene based amplicon sequencing is widely used for assessing microbial communities due to its low cost, time efficiency, and ability to provide a full overview of the community. However, it is currently unclear if it can yield reliable information on specific microbial guilds, as obtained by using primer sets targeting functional genes or specific16S rRNA gene sequences. Here, we compared the relative abundance, diversity, richness, and composition of selected guilds (nitrifiers), obtained from universal 16S rRNA gene based amplicon sequencing and from guild targeted approaches...
May 30, 2018: Journal of Microbiological Methods
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