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Journal of Microbiological Methods

Zhaojie Li, Shouguang Chen, Chenggang Xu, Lingyan Ju, Fengling Li
Vibrio parahaemolyticus which naturally inhabits marine and estuarine environment represents pathogenic strains (virulence genes tdh or trh positive) and non-pathogenic strains (virulence genes negative). In this study, a rapid method for subtyping pathogenic and non-pathogenic V. parahaemolyticus was established using fourier transform infrared (FTIR) spectroscopy with chemometric analysis. This method targeted three strains of genotypes of V. parahaemolyticus including tdh positive, trh positive and virulence gene-negative (nonpathogenic) V...
November 7, 2018: Journal of Microbiological Methods
Lindsey P Burbank, Brandon C Ortega
Xylella fastidiosa is an insect-transmitted bacterial plant pathogen which causes a variety of economically important diseases worldwide. Molecular identification of X. fastidiosa is used for quarantine screening, surveillance, and research applications; many of which require subspecies level differentiation of pathogen isolates. This study describes quantitative PCR (qPCR) and isothermal amplification assays which can rapidly identify X. fastidiosa isolates belonging to the fastidiosa and multiplex subspecies...
November 5, 2018: Journal of Microbiological Methods
Leonhard Menschner, Uta Falke, Nicole Töpfner, Reinhard Berner
The susceptibility protocol established in this study takes into account that complex media are capable to buffer H2 O2 which otherwise may adulterate test results. Furthermore, we demonstrate that clinical isolates of Staphylococcus aureus showed a higher resistance against H2 O2 than Streptococcus pyogenes after 30 mins of incubation but not after 24 h.
November 2, 2018: Journal of Microbiological Methods
Katarzyna Pieczul, Agnieszka Perek, Krzysztof Kubiak
The study describes a novel diagnostic protocol based on a loop-mediated isothermal DNA amplification (LAMP) for identification of wheat grains infection by Tilletia laevis, Tilletia caries (common bunt) and Tilletia controversa (draft bunt). The presented data showed that the LAMP analysis is a simple, specific and rapid method that could be used for detection of Tilletia spp. in contaminated grain samples. The lowest DNA concentration required for the successful detection of Tilletia spp. strains were estimated to be 0...
October 28, 2018: Journal of Microbiological Methods
Jinqiang Hu, Runna Huang, Yi Wang, Xiangke Wei, Zhangcun Wang, Yao Geng, Jianzhou Jing, Hui Gao, Xincheng Sun, Caiwen Dong, Chunpeng Jiang
In the current study, a duplex PCR-ELISA method was developed targeting the specific genes, invA of Salmonella spp. and rfbE of Escherichia coli O157: H7, to detect one or both bacteria in food. In brief, PCR product amplified by PCR primer labeled with digoxin at the 5'-end and a probe labeled with biotin at the 3'-end can form dimer by nucleic acid hybridization which can be captured by binding of biotin to streptomycin coated in ELISA plate before using enzyme-labeled anti-digoxin antibody and substrate to develop color...
October 26, 2018: Journal of Microbiological Methods
Maria Rosaria Iovene, Francesca Martora, Enza Mallardo, Marco De Sio, Davide Arcaniolo, Chiara Del Vecchio, Chiara Pagliuca, Giuseppe Signoriello, Mariateresa Vitiello
The objective was to investigate the diagnostic accuracy of our microbiological protocol to simplify the evaluation of bacterial prostatitis in the clinical practice. Our findings show the possibility to apply our alternative enrichment semen culture method to detect prostatic bacterial infection with higher sensitivity than the gold standard M&S technique.
October 26, 2018: Journal of Microbiological Methods
Leonardo Alves Rusak, Rodrigo de Castro Lisboa Pereira, Isabelle Geoffroy Freitag, Cristina Barroso Hofer, Ernesto Hofer, Marise Dutra Asensi, Deyse Christina Vallim
Yersinia enterocolitica, a member of the Enterobacteriaceae family, is a zoonotic agent that causes gastrointestinal diseases and some extraintestinal disorders in humans. Y. enterocolitica ssp. palearctica bioserotype 4/O:3 is the primary pathogenic bioserotype in Europe, where it has a high public health relevance. The isolation and identification of Y. enterocolitica from various sources on selective media have been seldom successful due to several reasons. In an attempt to overcome the problems associated with traditional culture-based methods, we developed a single duplex PCR assay for the detection of Y...
October 23, 2018: Journal of Microbiological Methods
Kundi Yang, Mengyang Xu, Fanyi Zhong, Jiangjiang Zhu
Lactobacillus, the major genus of lactic acid bacteria group, plays functional roles in the human body, for example, convert sugars to lactic acid. They are the significant microbiota which can be found at a number of human body sites, such as the digestive system, urinary system, and genital system. A number of Lactobacillus species are often used as probiotics and can benefit host health when administered in adequate amounts. Due to their diverse functional characteristics, it is essential to have identification and high-resolution typing techniques to support the need in health and nutritional research of Lactobacillus species...
October 23, 2018: Journal of Microbiological Methods
Jaeyoun Jang, Niels Bohse Hendriksen, Hans H Jakobsen, Ulrich Gosewinkel
Characterization of airborne bacterial cells requires efficient collection, concentration, and analysis techniques, particularly to overcome the challenge of their dilute nature in outdoor environments. This study aims to establish a rapid and reliable approach for quantification of bacteria in air samples. To do this, a high volume impingement sampler was applied to collect airborne bacteria from a wastewater treatment plant (WWTP). The bacterial cell density was estimated by a Cytosense flow cytometer (Cytobouy) and compared to quantitative PCR (qPCR) data based on 16S rRNA genes...
October 17, 2018: Journal of Microbiological Methods
Katrine Lekang, Kenan Hadziavdic, Katrine Sandnes Skaar, Inge Jonassen, Eric M Thompson, Christofer Troedsson
There is increasing interest in finding new, more efficient methods for routine monitoring of anthropogenic effects on benthic biodiversity and ecosystems. A range of molecular methods have been developed for assessing biodiversity the last decades. Particularly interesting are microarrays targeting phylogenetic marker genes, such as the small subunit of ribosomal RNA in eukaryotes (18S rRNA). This method can detect a large number of taxonomic groups in several samples simultaneously within a relatively short time and has the potential for incorporation in automated remote sensing pipelines...
October 15, 2018: Journal of Microbiological Methods
Ahmet Murat Saytekin, Seyyal Ak
This study aimed to develop direct PCR methods, which enable the diagnosis of brucellosis agents from ruminant aborted fetus samples at species and genus levels, and determine the applicability of the newly developed methods. For this purpose, 137 lung, 137 liver, and 52 fetal stomach fluid samples belonging to 166 ruminant aborted fetuses (326 samples in total) were examined. Firstly, agent isolation and identification were performed and species-specific multiplex PCR (m-PCR) from the culture was applied to the samples...
October 15, 2018: Journal of Microbiological Methods
Runze Li, Dinesh Dhankhar, Jie Chen, Thomas C Cesario, Peter M Rentzepis
Antibiotics are drugs that react against, kill, or inhibit the growth of bacteria. The method most often employed to evaluate the effectiveness of an antibiotic to kill bacteria requires at least 16 to 24 h for bacterial incubation. The requirement of long periods of time for the determination of the number of bacteria still alive after antibiotic treatment, may, in many cases, be detrimental to the patient's health. In addition, with increasing of bacterial antibiotic resistance, the need to utilize methods for distinguishing between live and dead bacteria within a short period of time after treatment with antibiotic agents, is becoming more crucial...
October 15, 2018: Journal of Microbiological Methods
Ondrej Šedo, Lenka Radolfová-Křížová, Alexandr Nemec, Zbyněk Zdráhal
A set of 204 taxonomically well-defined strains belonging to 17 Acinetobacter spp., including 11 recently described species (A. albensis, A. bohemicus, A. colistiniresistens, A. courvalinii. A. dispersus, A. gandensis, A. modestus, A. proteolyticus, A. seifertii, A. variabilis, and A. vivianii) and six species of the so-called haemolytic clade (A. beijerinckii, A. gyllenbergii, A. haemolyticus, A. junii, A. parvus, and A. venetianus), were subjected to MALDI-TOF mass spectrometric profiling. The identification outputs were evaluated using the current version (8...
October 15, 2018: Journal of Microbiological Methods
T L Masters, C A Hilker, P R Jeraldo, A V Bhagwate, K E Greenwood-Quaintance, B W Eckloff, N Chia, A D Hanssen, M P Abdel, J Z Yao, J Jen, R Patel
With the emergence of RNA sequencing technologies, metatranscriptomic studies are rapidly gaining attention as they simultaneously provide insight into gene expression profiles and therefore disease association pathways of microbial pathogens and their hosts. This approach, therefore, holds promise for applicability in infectious disease diagnostics. A challenge of this approach in the clinical setting is the low amount and quality of RNA, especially microbial RNA in most clinically-infected specimens. Here, we compared two commercially available stranded cDNA library preparation kits, the NuGEN Ovation SoLo RNA-Seq System and the Illumina TruSeq Stranded Total RNA, using RNA extracted from synovial and sonicate fluids from a subject with periprosthetic joint infection...
October 14, 2018: Journal of Microbiological Methods
Ji Eun Lee, Sung Jin Jo, Kang Gyun Park, Hyun Soo Suk, Sung Il Ha, Jeong Seob Shin, Yeon-Joon Park
BACKGROUND: We evaluated the two in-house sample preparation methods (saponin method (SAP) and [saponin + Sputazyme] method (SSPZ)) for direct identification of microorganisms using MALDI-TOF MS from positive blood culture bottles. Also, we evaluated the [saponin + Sputazyme] method for direct antimicrobial susceptibility testing (AST) using Vitek 2 system. METHODS: For direct identification, 163 prospective, monomicrobial positive blood culture bottles and 25 contrived blood culture bottles spiked with 25 infrequently isolated bacterial strains were included...
October 12, 2018: Journal of Microbiological Methods
Akio Aono, Kinuyo Chikamatsu, Hiroyuki Yamada, Yuriko Igarashi, Yoshiro Murase, Akiko Takaki, Satoshi Mitarai
We modified Wayne's pyrazinamidase test against Mycobacterium tuberculosis to indirectly measure pyrazinamidase activity via pyrazinoic acid in liquid medium. The modified pyrazinamidase test was easy to perform and its results were in complete agreement with those of the conventional Wayne's method, highlighting its potential application in phenotypic pyrazinamide susceptibility testing.
October 11, 2018: Journal of Microbiological Methods
Shannon Hunter, Rebecca McDougal, Mike J Clearwater, Nari Williams, Peter Scott
A high-throughput assay was developed to screen Phytophthora species for fungicide sensitivity using optical density measurements for unbiased, automated measurement of mycelial growth. The efficacy of the optical density assay (OD) to measure phosphite sensitivity in Phytophthora species was compared to two widely used methods, radial growth (RG) and dry weight (DW) assays. Three isolates of each of Phytophthora cinnamomi, P. multivora and P. pluvialis, with known phosphite exposure and three isolates of each species with no prior phosphite exposure, were screened for phosphite sensitivity using the three assays...
October 9, 2018: Journal of Microbiological Methods
Polina Ilina, Xiaochu Ma, Herman O Sintim, Päivi Tammela
The continuing emergence and spread of antibiotic-resistant bacteria is worrisome and new strategies to curb bacterial infections are being sought. The interference of bacterial quorum sensing (QS) signaling has been suggested as a prospective antivirulence strategy. The AI-2 QS system is present in multiple bacterial species and has been shown to be correlated with pathogenicity. To facilitate the discovery of novel compounds interfering with AI-2 QS, we established a high-throughput setup of whole-cell bioreporter assay, which can be performed in either 96- or 384-well format...
October 6, 2018: Journal of Microbiological Methods
Giuseppe Magistro, Christiane Magistro, Christian G Stief, Sören Schubert
Here we present a simple and rapidly achievable protocol for gene silencing in Escherichia coli (E. coli). In this procedure, antisense RNA (asRNA) of 400-nucleotides (nt) length and absolute complementarity to the target is produced by an expression plasmid. The designed asRNA should ideally cover at least the -10 site of the promoter and the Shine-Dalgarno sequence, and additional 300-bp of the following open reading frame of the target gene. We show that the transcription process of the target is not affected at all, whereas the translation process is impaired...
October 5, 2018: Journal of Microbiological Methods
Christian Brito-Silva, Jaime Pizarro-Cerda, Fernando Gil, Daniel Paredes-Sabja
Clostridium difficile infections are one of the leading causes of hospital-acquired infections. C. difficile spores are considered the morphotype of transmission and recurrent infection due to its natural spore resistance properties. The outermost spore layer, the exosporium, provides the first contact with the environment and the host. However, molecular biology studies on exosporium proteins are lacking primarily due to difficulties in over-expressing these proteins under soluble conditions. In this work, we have developed a protocol to express soluble exosporium proteins of C...
October 4, 2018: Journal of Microbiological Methods
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