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Journal of Microbiological Methods

Bo Zhang, Yibeibaihan Maimaiti, Chunyan Liu, Jing Li, Haiye Wang, Haojie Lin, Zhaohui Deng, Xinhong Lu, Xin Zhang
OBJECTIVE: Clinical diagnosis of bloodstream infection diseases depends on the blood culture results. Bacterial identification by traditional methods is time-consuming. This study aimed to utilize molecular beacon-based fluorescence in situ hybridization (MB-FISH) for rapid and direct detection of Staphylococcus aureus in positive blood cultures. METHODS: Three molecular beacon probes (MB1, MB2 and MB3) were designed and synthesized to target the 16S rRNA gene fragment of S...
February 15, 2019: Journal of Microbiological Methods
Shuai Zhi, Jonas Szelewicki, Kim Ziebell, Brendon Parsons, Linda Chui
Shiga toxin-producing E. coli (STEC) is a gastrointestinal pathogen and has been recognized as one of the serious problems in public health. Shiga toxin genes (stx) can be grouped into different types according to their differences in sequence and biological activities. The two main groups of stx are stx1 and stx2 with each group containing several subtypes. It is essential to develop rapid stx1 /stx2 subtyping assays and accurate stx2 general detection assays to provide a quicker turn-around time to predict disease outcome and also to provide data for surveillance purposes...
February 14, 2019: Journal of Microbiological Methods
Y W Yang, M Zhao, L Q Zhang, P Qiao, X Bai, X X Zhang, R R Walcott, W Guan, T C Zhao
Bacterial fruit blotch (BFB) of cucurbits, caused by Acidovorax citrulli, is a major threat to commercial watermelon and melon production worldwide. At present, there are at least two genetically distinct sub-populations (group I and II) of A. citrulli that differ in host preference among cucurbit species and copper sensitivity. In this study, we analyzed the pilA gene sequences of 103 A. citrulli strains from China and other countries. Based on these data, we classified all tested A. citrulli strains into three types...
February 8, 2019: Journal of Microbiological Methods
Basil Britto Xavier, Gesuele Renzi, Christine Lammens, Abdessalam Cherkaoui, Herman Goossens, Jacques Schrenzel, Stephan Harbarth, Surbhi Malhotra-Kumar
Horizontal transfer of plasmid-mediated antibiotic resistance has rarely been documented in vivo. Utilizing long-read (Oxford Nanopore) and short-read (Illumina) sequencing, we confirmed that a gut-colonizing Escherichia coli and a hypervirulent Klebsiella pneumoniae ST23, isolated from a surgical site culture of a patient receiving cefuroxime therapy, harboured a 100% identical blaCTX-M-14 -harbouring plasmid, indicative of a potential transfer in vivo.
February 7, 2019: Journal of Microbiological Methods
Erick Mateus-Barros, Aylan K Meneghine, Inessa Lacativa Bagatini, Camila C Fernandes, Luciano T Kishi, Armando A H Vieira, Hugo Sarmento
In recent years, the rapid advances of culture-independent methods and new molecular tools have revolutionized our understanding of microbial biodiversity and ecological functions. DNA extraction from microbial communities is a critical step in this process and several methods have been proposed and used, but the influence of the extraction method on the outcome and ultimately on ecological inferences from the results is not yet precisely determined. Here, we compared two of the most commonly used extraction methods in aquatic microbial ecology, and investigated whether the two methods yielded comparable results for community ecology analyses...
February 6, 2019: Journal of Microbiological Methods
Adel A Alhowyan, Mohammad A Altamimi, Mohd Abul Kalam, Abdul Arif Khan, Mohamed Badran, Ziyad Binkhathlan, Musaed Alkholief, Aws Alshamsan
Itraconazole (ITZ) loaded Poly-(D, L-lactic-co-glycolic acid, PLGA) nanoparticles (PLGA-NPs) stabilized by D-α-Tocopherol polyethylene-glycol succinate-1000 (TPGS) were developed by nanoprecipitation and single emulsion solvent evaporation methods to improve antifungal activity of ITZ by enhancing its solubility, and hence bioavailability. Encapsulation efficiency, drug loading, in-vitro release, ex-vivo permeation and antifungal activity were performed for the optimized PLGA-NPs. Characterization of PLGA-NPs were performed by scanning electron microscopy, dynamic light scattering, differential scanning calorimetry, Fourier transform infrared spectroscopy, and powder X-ray diffractometry...
February 6, 2019: Journal of Microbiological Methods
Xinjie Song, Shruti Shukla, Myunghee Kim
Cronobacter species are foodborne pathogens that pose a high risk in infant formula and can cause fatality rates of 40-80% in infected infants. To develop a rapid and easy detection method for Cronobacter species, especially in powdered infant formula (PIF), an immunoliposome-based immunochromatographic strip assay was developed using an anti-Cronobacter immunoglobulin G (IgG)-conjugated liposome and an anti-Cronobacter IgG-coated nitrocellulose membrane. The developed assay could detect Cronobacter species in both pure culture and artificially contaminated PIF...
February 6, 2019: Journal of Microbiological Methods
Dmitrii M Bubnov, Tigran V Yuzbashev, Tatiana V Vybornaya, Alexander I Netrusov, Sergey P Sineoky
The introduction of chromosomal mutations into the E. coli genome using λRed-mediated recombineering includes two consecutive steps-the insertion of an antibiotic resistance gene and the subsequent excision of the marker. The second step usually requires a counterselection method, because the efficiency of recombination is not high enough to find recombinants among non-recombinant cells. Most counterselection methods require the introduction of additional mutations into the genome or the use of expensive chemicals...
February 6, 2019: Journal of Microbiological Methods
M Chitra, N Pappa
Interest in low cost cellulase production has become a major challenge in recent years for biorefineries. Fed-batch fermentation of Trichoderma strains for the production of low cost cellulase is carried out on complex media that has various soluble and insoluble substrates. The lack of direct estimation of biomass in the presence of insoluble substrates is one of the major concerns for controlling bioprocesses in industries. In this paper, a Multiphase Artificial Neural Network (MANN) based dynamic soft sensor is developed to predict the biomass concentration of Trichoderma during fed batch fermentation in the presence of insoluble substrates...
February 5, 2019: Journal of Microbiological Methods
Abhik Mojumdar, Anjani Kumar Upadhyay, Vishakha Raina, Lopamudra Ray
Chitin is one of the most abundant biopolymers present in the environment. Chitosan being its major derivative can be obtained by hydrolysis of chitin, especially by microbial degradation. Estimation of resulting chitosan produced by chitin degradation is crucial to the process. Usefulness of the method of Badawy (Badawy, 2012) for estimation of chitosan is limited by interference resulting from susceptibility to variation in the pH of the sample and thiobarbituric acid. This work presents an improvement of the method proposed by Badawy for colorimetric determination of chitosan by using 3, 5-Dinitrosalicylic acid (DNSA) reagent instead of thiobarbituric acid, after one step depolymerization and deamination of chitosan with sodium nitrite (NaNO2 )...
February 3, 2019: Journal of Microbiological Methods
Balaji Venkatesagowda, Robert F H Dekker
A rapid and sensitive method has been devised in order to detect and estimate the synthesis of the alcohol oxidase (AOX) enzyme by the fungi, by way of the use of two chemical complexes namely, acetylacetone (3,5-Diacetyl-1,4-dihydroludine) and acetylacetanilide (3,5-Di-N-phenylacetyl-1,4-dihydroludine). This method involves the use of the AOX enzyme that could specifically oxidize methanol, giving rise to a μmol equivalent each of formaldehyde (HCHO) and hydrogen peroxide (H2 O2 ) as the end products. Further, the formaldehyde, thus produced was allowed to interact with the neutral solutions of acetylacetone and the ammonium salt, gradually developing a yellow color, owing to the synthesis and release of 3,5-Diacetyl-1,4-dihydroludine (Yellow product; λ = 420 nm; λex/em  = 390/470 nm) and the product, so generated was quantified spectrophotometrically by measureing its absorbance at 412 nm...
February 1, 2019: Journal of Microbiological Methods
Qingwei Yao, Haiqin Chen, Shunxian Wang, Xin Tang, Zhennan Gu, Hao Zhang, Wei Chen, Yong Q Chen
Screening oleaginous microorganisms capable of accumulating considerable lipids is essential for industrial lipid production. Here we demonstrated forty-seven filamentous fungal isolates were obtained from eight soil samples using a new screening strategy with both triphenyltetrazolium chloride (TTC), a redox indicator used for testing oil presence, and cerulenin, an inhibitor of fatty acid synthase (FAS), supplemented in screening medium. Among these fungal isolates, nineteen have high lipid content (>20% dry biomass weight) and were affiliated with the genus Mortierella by morphology identification and phylogenetic analysis based on ITS gene sequences...
January 29, 2019: Journal of Microbiological Methods
N Jackson, T Wu, S Adams-Sapper, T Satoorian, M Geisberg, N Murthy, L Lee, L W Riley
Gram-negative bacteria (GNB) are important causes of community (CA) and hospital (HA)- associated infections. Here we describe the development of an indirect ELISA (I-ELISA), which can be used to detect and differentiate the Enterobacteriaceae Escherichia coli, and glucose non-fermenter Pseudomonas aeruginosa from other GNB species. The I-ELISA utilizes six antibodies for bacterial speciation, which were grouped according to their bacterial targets; Enterobacteriaceae (SL-EntA and CH1810 mAb), Escherichia coli (SL-EcA and 6103-46 mAb), Pseudomonas aeruginosa (SL-PaA and SL-PaB)...
January 29, 2019: Journal of Microbiological Methods
Pauline Ract, Redouane Dahoumane, Salah Gallah, Philippe Morand, Isabelle Podglajen, Fabrice Compain
The chromogenic βLACTA™ test was evaluated to detect ceftazidime resistance in P. aeruginosa isolates from patients with cystic fibrosis. Best results were obtained after one hour of incubation with low sensitivity (64.1%), high specificity (98.3%), and negative and positive predictive values of 80.3% and 96.2%, respectively.
January 29, 2019: Journal of Microbiological Methods
Roopali Rajput, Paras Singh, Rohit Sarin, Prabhpreet Sethi, Sangeeta Sharma
BACKGROUND: Confirmatory diagnosis of extra-pulmonary tuberculosis remains a true challenge owing to difficulty in procuring appropriate specimen, inefficient laboratory methods and paucibacillary nature of infection. These obstructions become all the very difficult in pediatric EPTB cases, due to non-specific clinical signs and symptoms, low sensitivity of smear microscopy and culture, lack of awareness among clinicians, etc. AIM OF THE STUDY: The present study aimed to evaluate the diagnostic accuracy of rapid and cost-effective loop-mediated isothermal amplification (LAMP) assay for EPTB diagnosis in children...
January 28, 2019: Journal of Microbiological Methods
Hai-Feng Hu, Hai-Yan Zhou, Mei-Xin Wang, Yuan-Shan Wang, Ya-Ping Xue, Yu-Guo Zheng
(R)-2-(4-hydroxyphenoxy)propionic acid ((R)-HPOPA) is an important intermediate for the synthesis of optically pure aryloxyphenoxypropionic acid herbicides. Regioselective hydroxylation of (R)-2-phenoxypropionic acid ((R)-POPA) by microbes is one of the most useful methods for the industrial production of (R)-HPOPA. In this study, we designed and optimized a rapid throughput assay for screening (R)-HPOPA producing bacterial/fungal strains which can regioselectively hydroxylate (R)-POPA. (R)-HPOPA could react with 4-aminoantipyrine (4-AAP) in the presence of potassium hexacyanoferrate (K3 [Fe(CN)6 ]) to form indoxyl antipyrine, an orange-red chromophore, that can easily spectrophotometrically be determined at 550 nm...
January 28, 2019: Journal of Microbiological Methods
Jinqiang Hu, Runna Huang, Yanting Sun, Xiangke Wei, Yi Wang, Chunpeng Jiang, Yao Geng, Xincheng Sun, Jianzhou Jing, Hui Gao, Zhangcun Wang, Caiwen Dong
Salmonella Typhimurium (S. Typhimurium) can cause serious foodborne diseases. In this study, an assay combining recombinase polymerase amplification (RPA) with lateral flow dipsticks (LFD) was developed to detect S. Typhimurium in milk. The RPA forward primers STF1, STF2, STF3, the reverse primer STR labeled with digoxin, and the probe STProb labeled with FAM were designed and screened to produce RPA products for LFD detection. The RPA reaction volume, temperature, and time were then optimized, and the sensitivity and specificity of the developed method were analyzed...
January 28, 2019: Journal of Microbiological Methods
Jana Schmidt, Sandra Krohn, Laura Buenger, Katharina Zeller, Hartmut Schneider, Maud Treuheit, Thorsten Kaiser, Dirk Ziebolz, Thomas Berg, Rainer Haak
Microbial analyzes of carious dentine samples, especially in terms of interventions, represent a challenge due to difficulties in carious dentine sampling particularly with small bacterial DNA contents. Therefore, information about the quantitative reduction of bacterial DNA as well as microbial shifts and differences in diversity correlating with treatment interventions are scarce. In this study, carious dentine samples were collected in a first step in the course of a selective caries excavation at two different deep dentine caries lesions in three patients...
January 23, 2019: Journal of Microbiological Methods
Satoshi Higashide, Otomi Cho, Yuko Matsuda, Daiki Ogishima, Sanae Kurakado, Takashi Sugita
A rapid detection method for Lactobacillus crispatus and Lactobacillus iners, which are important for maintaining a healthy vaginal environment, was developed using loop-mediated isothermal amplification (LAMP). The LAMP assay had a lower limit of detection of 10 fg DNA and could detect both species within 45 min.
January 22, 2019: Journal of Microbiological Methods
Athenia L Oldham, Vince Sandifer, Kathleen E Duncan
Three replicate seawater samples were collected on three different days, filtered immediately and preserved with one of two guanidinium thiocyanate-based preservatives (DNAzol™ or RNA Lysis Buffer™ plus β-mercaptoethanol (RLA+)) and were kept frozen while being shipped to a lab. In parallel, a carboy of seawater was collected on each of the three days and maintained at ambient temperature while being shipped to a lab. Upon receipt the samples were filtered and treated in the same manner as for immediate preservation...
January 21, 2019: Journal of Microbiological Methods
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