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Mycobacterium tuberculosis complex (MTBC) whole genome sequencing (WGS) turnaround time and WGS success rates are highly influenced by DNA extraction protocols even from cultures. Efficient mycobacterial lysis is crucial for obtaining sufficient DNA from cultures to facilitate reliable genomic drug susceptibility prediction and accurate genotyping with WGS. We compared four DNA extraction protocols from BD BACTEC™ Mycobacterial Growth Indicator Tubes (MGIT) for WGS with a focus on the lysis step: protocol A) column-based protocol without mechanical lysis; protocol B) an adapted protocol including a bead beating step; protocol C) DNA extraction from primary received cultures using bead beating: and protocol D) DNA extraction from pre MGIT-positive (enriched) cultures. Protocol B increased DNA yield approximately 60-fold, and significantly improved the sequencing success rate. The increased yield also allowed DNA extraction from primary cultures with high success rates (protocol C). Additionally, by using pre-positive enriched MGIT cultures, we demonstrated that bead beating opens the possibility of reliable WGS up to five days before a MGIT tube would be flagged positive (protocol D). The most optimal bead beating-based DNA extraction was also evaluated for Nanopore sequencing. Shortening bead beating duration to 15 s resulted in longer read lengths (N50 from 1.4 kb to 2.6 kb) while still providing efficient lysis. Furthermore, AmpureXP bead beating-based DNA capture / purification proved to be as efficient as Qiagen column-based DNA extraction, further simplifying and shortening the DNA extraction protocol. Adding a mechanical lysis step to our routine MTBC DNA extraction protocol has allowed us to reduce the turnaround time while maintaining DNA quality sequencing success rates., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Kristin Kremer, Noud Hermans, Richard Anthony reports financial support was provided by Netherlands National Postcode Lottery. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Four enzymatic DNA library preparation kits were compared for sequencing Shiga toxin-producing E. coli. All kits produced high quality sequence data which performed equally well in the downstream analyses for surveillance and outbreak detection. Important differences were noted in the workflow user-friendliness and per sample cost., (Published by Elsevier B.V.)

Accurate detection of vector-borne pathogens (VBPs) is extremely important as the number of reported cases in humans and animals continues to rise in the US and abroad. Validated PCR assays are currently the cornerstone of molecular diagnostics and can achieve excellent analytical sensitivity and specificity. However, the detection of pathogens at low parasitemia still presents a challenge for VBP diagnosis, especially given the very low volume of specimens tested by molecular methods. The objective of this study is to determine if a commercially available microbial enrichment kit, used prior DNA extraction, is capable of expanding the overall microbial community and increasing detectable levels of VBPs in canine blood samples through host DNA depletion. This study used EDTA-whole blood samples from dogs naturally infected with varying parasitemia levels of either Anaplasma phagocytophilum, Babesia gibsoni, or Ehrlichia ewingii. For two VBPs, EDTA-blood samples were diluted to determine the effect of microbial concentration at low parasitemia. Paired EDTA-blood samples from each dog were subjected to traditional, automated DNA extraction with or without the microbial concentrating kit (MolYsis®) prior DNA extraction. Relative amounts of pathogen DNA in paired samples were determined by real-time PCR and Next-Generation Sequencing targeting conserved regions of 16S rRNA (for bacteria) and 18S rRNA (for protozoa). Results from the three molecular methods suggest that the microbial concentrating kit did not improve the detection of VBPs, although significantly reduced the presence of host DNA. Alternative methods for VBP enrichment in clinical samples prior to molecular testing should continue to be investigated, as it may significantly improve clinical sensitivity and reduce the number of false-negative results., (Copyright © 2021 Elsevier B.V. All rights reserved.)

A rapid method that provides information on the viability of organisms is needed to protect public health and ensure that remediation efforts following a release of a biological agent are effective. The rapid viability-polymerase chain reaction (RV-PCR) method combines broth culture and molecular methods to provide results on whether viable organisms are present in less than 15 h. In this study, a modified RV-PCR (mRV-PCR) method was compared to a membrane-filtration culture method for the detection of viable Bacillus spores in water matrices. Samples included small and large volumes of chlorine and non‑chlorine treated tap water. Large volume water samples (up to 100 L), were processed by ultrafiltration using a semi-automated waterborne pathogen concentrator, followed by centrifugation as a secondary concentration technique. The concentrated samples were analyzed by mRV-PCR and culture methods. The overall agreement between the mRV-PCR and culture methods when seed concentrations were greater than 10 spores per sample volume analyzed was 96%. The total time from the start of sample processing to the final sample result for the mRV-PCR method was decreased by approximately 2 h, in comparison to the previously published RV-PCR method because of the incorporation of shorter, more efficient primary and secondary concentration steps and a shorter DNA extraction technique. Overall, this study confirmed that RV-PCR is a promising approach for identifying viable Bacillus spores in small- and large-volume water samples and for producing results in less time than traditional culture methods., (Published by Elsevier B.V.)

Introduction: Rapid, sensitive, and specific diagnostic methods are indispensable for sexually transmitted infections (STIs). In this study, a multiplex PCR-dipstick DNA chromatography assay for diagnosis of four STI pathogens, namely Neisseria gonorrhoeae (N. gonorrhoeae), Mycoplasma hominis (M. hominis), Ureaplasma (U. urealyticum and U. parvum), and Chlamydia trachomatis (C. trachomatis), was established and evaluated., Methods: Based on the hybridization of probes and interaction between streptavidin and biotin, PCR products were visualized through hybridization of specific probes and enzymatic color generation. The sensitivity and specificity of all four pathogens were evaluated. Clinical performance of the test was evaluated using 295 specimens, and comparisons among results were determined via culture or colloidal gold assay., Results: No cross-reactions were observed, confirming the high specificity of this method. The limit of detection (LOD) of the four STI pathogens was 100 copies/μL. The sensitivity between PCR-dipstick DNA chromatography and culture or colloidal gold assay ranged from 84.6% to 100%. The specificity was between 93.5% and 96.6%, positive predictive value ranged from 53.6% to 86.7%, negative predictive value was over 98.3%, kappa value ranged from 0.676 to 0.864 (Cohen's kappa coefficient test), and the agreement rate was over 93.5%., Conclusion: In conclusion, PCR-dipstick DNA chromatography serves as a rapid, sensitive, and specific method for simultaneous diagnosis of four STI pathogens., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Numerous factors associated with sample preparation, DNA extraction, primer choice, sequencing platform and data analysis can affect the accuracy of 16S rRNA sequencing results. The DNA extraction method is considered critical for the success of sequencing as it can be the source of considerable variations in the analysis of the microbiome. In this study, the impact of various DNA extraction methods on the results of analysis of bacterial communities in cheese was evaluated. DNA was isolated from Mozzarella as a model cheese using optimized bead-based homogenization followed by different extraction procedures. Five commercial kits and two open-formula DNA extraction protocols were evaluated for amplicon sequencing of a 16S rRNA fragment of ~1460 bp. In addition, model cheese samples artificially contaminated by defined concentrations of Listeria monocytogenes and Escherichia coli, as representatives of Gram positive and Gram negative bacteria, were analysed. Six out of seven DNA extraction procedures were found to be able to provide amplifiable bacterial DNA suitable for 16S rRNA sequence analysis, but individual extraction procedures led to variable results. In particular, lysis supported with bead-beating led to a higher proportion of G+ bacteria in relative abundance profiles, probably because of the more efficient cell wall disruption. Artificially added bacterial species were reliably detected with a quantitative response. The results demonstrated a risk in comparing the data on bacterial communities in cheese when different DNA extraction protocols are used and highlighted the need to choose a standardized approach when comparison across multiple sequencing runs is required., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Detection of low abundance human health pathogens in environmental samples is a challenge for water monitoring. This limitation can be overcome by the introduction of multiple displacement amplification (MDA) where a minute amount of genetic material can be amplified using a phi-29 DNA polymerase. However, the genetic makeup and the concentration of the polynucleotides might influence the amplification process due to inherent assay bias. Herein, a series of experiments were designed to demonstrate the effect of genome length, guanidine and cytosine content, and template concentration on the efficiency of MDA. Quantitative polymerase chain reaction (qPCR) was performed to quantify pre- and post-MDA concentrations of selected genes. Linear regression between pre- and post-MDA log gene copies L -1 of both environmental and lab-grown samples showed a positive correlation (F = 77.59, P < 0.001, R 2  = 0.7, slope = 1.01). Correlation between relative polynucleotide increase after MDA and target organism length and gene target guanidine and cytosine (G + C) content (F = 4.3, P = 0.02) shows that lower G + C and higher genome length is favored in the MDA process. The MDA process was shown to favor a longer genome over a shorter genome (1.19 and 1.04 change in log gene copy L -1 , respectively) and a lower G + C content over a higher G + C content (1.11 and 0.61 change in log gene copy L -1 , respectively). There was no MDA bias observed when polynucleotides had the same G + C and genome length but different initial concentrations. This study highlights the need for increased caution when interpreting relative abundance of organisms amplified by MDA such as in next generation sequencing., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Amplification of high GC content genes by PCR is a major challenge during the creation of recombinant GC-rich DNA constructs. This may be due to the difficulty in DNA denaturation or the possibility of forming secondary structures from DNA templates. Tools have been described to address the technical problems associated with the amplification of shorter sequences (<1000 bp). However, obstacles of synthesizing larger-sized GC-rich sequences by PCR continue to exist. This study aims to investigate the amplification of long and high GC content genes by PCR from the Mycobacterium bovis, a genome with GC content >60%, in comparison to amplifying a gene from the Listeria monocytogenes genome, a genome with a 37.8% GC content. Three PCR protocols were designed and experimented at various conditions with two M. bovis genes, Mb0129, a large gene of 1794 bp with 77.5% GC content, mpb83, a smaller gene of 663 bp in length with moderate GC content of 63%, together with LMHCC&#95;RS00060, a large L. monocytogenes gene of 1617 bp with a lower GC content of 41.5%. The result demonstrated the superiority of the 2-step PCR protocol over other protocols in PCR amplification of Mb0129 when specific high fidelity DNA polymerases were used in the presence of an enhancer. The study highlighted the importance of manipulating the cycling conditions to perform the annealing and extension steps at higher temperatures while adjusting the ramp speed at a lower speed for a successful PCR amplification of a large GC-rich DNA template. A final PCR protocol was developed and enabled the amplification of 51 GC-rich targets. This can be a valuable tool for the amplification of long GC-rich DNA sequences for various downstream applications., (Copyright © 2020. Published by Elsevier B.V.)

We prepared a newer growth medium, banana peel extract agar (BPEA) containing the extracts of chopped banana peels for the selective cultivation of Cryptococcus neoformans. Over the medium, the growth resulted in the development of light to the dark brown coloured colonies indicating the chromogenic potential of the BPEA. The organism grown over BPEA was subsequently confirmed as C. neoformans by phenotypic as well as by molecular method. This medium, being cost-effective, may be used in resource-poor settings of the developing or underdeveloped countries for selective isolation of C. neoformans., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Many species of intestinal bacteria are present in moderate numbers in the faecal microbiota, which is dominated by obligate anaerobes. Little is known regarding the detection sensitivity of next-generation sequencing for these microbes in samples of complex microbiota., Methods: Twenty stool samples from six healthy infants, who were followed from 1 week to 1 year of age, were previously cultured quantitatively for total population counts, as well as for counts of relevant facultative bacteria and a limited selection of obligate anaerobes that are prevalent in the neonatal microbiota. The same samples were analysed by Next-generation sequencing (NGS, pyrosequencing) of the 16S rRNA gene (V1-V3 regions; average read length, 500 nucleotides; average number of reads per sample, 30,000). We used the bacterial culture data to determine the lowest bacterial populations that could be detected by NGS. Different DNA extraction kits (QIAamp DNA Stool Mini, ZR Faecal DNA MiniPrep, and PowerSoil DNA Isolation) were compared for efficacy in extracting DNA from Gram-negative and Gram-positive Type strains., Results: NGS yielded one read per 10 6  CFU/g faeces of the Gram-negative commensal gut bacteria Bacteroides and Enterobacteriaceae, but only one read per 10 8  CFU/g faeces of Gram-positive bifidobacteria. The Gram-positive facultative bacteria Enterococcus was often undetectable by DNA-based methods despite being present at >10 6  CFU/g faeces. The DNA extraction kits tested varied considerably in their ability to extract DNA from bacterial samples, and showed considerably lower efficacies in extracting DNA from Gram-positive than from Gram-negative bacteria., Conclusions: NGS has lower sensitivity for detecting Gram-positive bacteria than Gram-negative bacteria, due at least in part to inefficient extraction of DNA from Gram-positive bacteria. Therefore, enzymatic lysis may enhance the yield of DNA and increase the sensitivity of NGS methods for Gram-positive bacteria, and the inclusion of positive and negative controls during DNA extraction is indicated for validation purposes. The differential extraction of DNA from bacterial samples by different DNA extraction kits may limit comparability between studies on the gut microbiota. Finally, quantitative culture methods detect certain bacteria with greater sensitivity than NGS techniques, and thus culture- and DNA-based methods can be used in tandem to define the complex composition of the gut microbiota with greater accuracy., Competing Interests: Declaration of Competing Interest AW and IA holds shares in Flora Innovation, a small researcher-driven company investigating potential preventive treatments against immune-regulated diseases. The other authors declare no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

We report the development of ddPCR assays for single and simultaneous detection of the bacterial pathogens Flavobacterium psychrophilum and Yersinia ruckeri in water from land-based recirculation aquaculture systems (RAS), producing Atlantic salmon (Salmo salar) smolt. The method was tested and verified for use in water analyses from RAS production sites, and proved to be specific and with sensitivity 0.0011 ng DNA for F. psychrophilum and 1.24 ng for Y. ruckeri. These bacteria are important fish pathogens that have caused reoccurring salmonid infection disease in RAS. Monitoring pathogen levels in water samples could be a useful alternative surveillance strategy to evaluate operational risk assessment connected to stress factors. Water quality is essential for fish health and growth in RAS production in general, and high or increasing levels of these pathogens in the RAS water may generate an early indication of unfavourable conditions in the RAS environment, and give directions to operational actions. This approach may reduce fish mortality, reduce production loss, and offer more effective and targeted preventive measures within RAS production., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

This report describes the development, optimization, and validation of a ddPCR assay for the detection of Bartonella spp. DNA within several sample matrices, including clinical blood samples from patients with or without documented Bartonella spp. bacteremia. The Bartonella spp. ddPCR assay was developed based upon previously published TaqMan-based qPCR assays that can amplify DNA of over 25 Bartonella spp. Host DNA (housekeeping gene) amplification serves as a reference target to facilitate quantification. The efficiency, sensitivity, and specificity of the Bartonella spp. ddPCR assay was assessed by direct comparison with the current qPCR methods used by the Intracellular Pathogens Research Laboratory (North Carolina State University, North Carolina, USA), and Galaxy Diagnostics (Research Triangle Park, North Carolina, USA). Bartonella spp. ddPCR assay parameters were successfully optimized to detect Bartonella concentrations equivalent to 0.5 bacterial genome copies per microliter of blood (0.001 pg/ul of bacterial DNA). The number of droplets detected (resolution) for each concentration was consistent across each of four assessed time points. The Bartonella spp. ddPCR assay detected 16 species/strains including B. henselae; B. quintana; B. vinsonii subsp. berkhoffii (genotypes I, II, III and IV); B. vinsonii subsp. vinsonii; B. melophagi; B. volans; B. monaki; B. alsatica; B. bovis; B. elizabethae; B. clarridgeiae; and B. koehlerae. Bartonella DNA was detected in only one previously negative patient sample (119/120 negative; 99% specificity). The ddPCR sensitivity (53/112) was significantly better than qPCR (6/112) when testing patient blood and enrichment blood culture samples. The development of commercial ddPCR systems with integrated technologies has significantly streamlined the DNA detection process, making it more efficient and standardized for clinical diagnostic testing. The assay described in this work is the first step toward the development of a multiplex ddPCR assay (i.e., using the QX One from Bio-Rad) for the simultaneous detection and absolute quantification of multiple vector-borne pathogens (such as Babesia, Bartonella and Borrelia) within clinical samples., Competing Interests: Declaration of Competing Interest Dr. Ricardo Maggi is a co-founder and the Chief Technical Officer for Galaxy Diagnostics Inc. Dr. Jennifer Miller is the Director of Research and Development and Assistant Laboratory Director for Galaxy Diagnostics Inc. In conjunction with Dr. S. Sontakke and North Carolina State University, E. B. Breitschwerdt holds US Patent No. 7,115,385; Media and Methods for Cultivation of Microorganisms, which was issued on October 3rd, 2006. He is a co-founder, shareholder and Chief Scientific Officer for Galaxy Diagnostics, a company that provides advanced diagnostic testing for the detection of Bartonella spp. infections. Toni Richardson has no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Real-time PCR tests have been widely used to detect mycobacterial infection. The quality of extracted DNA is crucial for obtaining accurate results of the real-time PCR tests, and automated extraction methods are faster and more effective than manual extraction. The novel Real-Prep automated extraction system has not yet been verified by direct comparisons to existing methods. In this study, we compared it with manual extraction, and the Nextractor system, an automated extraction method commonly used in Korea. From August to December 2018, 238 specimens, including sputum, bronchial washing, pericardial fluid, bronchial aspiration, pleural fluid, and closed pus samples, were collected and examined at Yeungnam University Hospital. After decontamination, smear microscopy, and culturing, DNA was extracted using the three methods. The DNA extraction efficiency (total amount of DNA [ng]/input specimen volume [μL]) and purity (A260/280 ratio), which indicates the presence of contaminants, were compared. Real-time PCR tests were conducted using the DNA extracted by each method. The cycle threshold, which is inversely related to the initial amount of mycobacterial DNA, and the percentage agreement between the PCR results of the three methods were evaluated. Our study revealed that the DNA extraction efficiency of the Real-Prep system was higher than that of manual extraction. There was no significant difference in DNA purity between the methods, and the percentage agreement for Mycobacterium tuberculosis and non-tuberculous mycobacteria among all three methods was almost perfect. The performance of the Real-Prep system was similar to that of the Nextractor system and superior to that of manual extraction. The Real-Prep system, a new automated nucleic acid extraction device, has a clear benefit because of its relative speed and low hands-on time. Therefore, the Real-Prep system is a useful substitute for manual DNA extraction, which has the potential to reduce workloads in laboratories and as a sensitive non-tuberculous mycobacteria detection method throughout the world., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

DNA-SIP (DNA-based stable isotope probing) is increasingly being employed in soil microbial ecology to identify those microbes assimilating the 13 C/ 15 N labelled substrate. Isopycnic gradient centrifugation is the primary experimental process for conducting DNA-SIP. However, diverse centrifugal conditions have been used in various recent studies. In order to get the optimum conditions of centrifugation for DNA-SIP, centrifugation time (36, 42, 48, 60 h), speed (45,000, 55,000 rpm) and the initial buoyant density (1.69, 1.71, 1.725 g ml -1 ), as were used extensively in related studies, were tested in this experiment with the Vti 65.2 rotor. DNA with either 13 C-labelling or unlabelled was extracted from a paddy soil pre-incubated with either 13 C-labelled or natural abundance glucose. After ultracentrifugation, the gene abundance of bacterial 16S rRNA, fungal 18S rRNA, bacterial and archaeal amoA within the fractioned DNA was detected. The results showed that centrifugation for 48 h was enough for the DNA to reach stabilization in the CsCl solution. The initial density of the mixed solution was best adjusted to 1.71 g ml -1 to ensure that most of the genes were concentrated on the middle fractions of the density gradient. Increasing the centrifugation speed would increase the density gradient of fractions; therefore, 45,000 rpm (184,000 g) was recommended so as to obtain the more widespread pattern of DNA in the centrifugal tube. We hope these findings will assist future researchers to conduct optimum ultracentrifugation for DNA-SIP., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

There is increasing interest in methodologies for the simultaneous concentration and detection of multiple targets in individual samples. The aim of this study was to investigate the potential presence of E. coli DNA in beef extract powder used as part of a procedure to concentrate water samples for the simultaneous detection of bacteria, viruses and protozoa. DNA from E. coli was detected in five out of six beef extract lots tested, demonstrating the limitations of its inclusion when being used in assays that will be used for the detection of E. coli in water samples. Further work is required to clarify if this phenomenon also occurs for other microorganisms of interest in water., (Copyright © 2019. Published by Elsevier B.V.)

Successful and efficient extraction of high quality, high molecular weight genomic DNA from the environmental samples is an essential primary step to understand the genetic, metabolic and evolutionary characteristics of the microbial communities. Deep mine biofilm samples that contain high amounts of mucoid exopolysaccharide often pose difficulties to obtaining refined community DNA. To circumvent this hindrance, we report our "MINES" method which we developed for optimal biofilm DNA recovery suitable for all types of high-resolution downstream applications. The method is also suitable for samples collected from landfill compost, kitchen digest (KD), and for Gram-positive Geobacillus sp. strain WSUCF1 and Gram-negative E. coli DH5α strains. In one form of the method, use of a gentle preprocessing technique to loosen the mucoid layer, combined with a multi-lytic polyzyme treatment to maximize yields from all cell types in the biofilm sample, yielded >1 μg of high molecular weight DNA (16-20 kb) per gram of the biofilm sample, with an A260/280 and A260/230 ratio of about 2. Furthermore, amplification of 16S rRNA genes as well as restriction digestion with BamHI and HindIII suggest that the newly developed method can minimize any inhibitory effects of contaminants. Results indicate that it is an appropriate methodology for the extraction of total genomic DNA for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging. IMPORTANCE: Our present knowledge of microorganisms and their enzymes from deep mine subsurfaces is based largely on laboratory studies of pure microbial cultures. These methods tend only to hit nearly 1% of the entire microbial community. In this regard, metagenomics, has emerged as a strategic approach to explore unculturable microbes through the sequencing and analysis of DNA extracted from the environmental samples. This research paper discusses our "MINES" method for genomic DNA extraction from deep biosphere biofilm samples., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This study aimed to assess the feasibility of using RFLP of PCR-amplified 16S rRNA gene (s) by using universal primers 27f/1492r and a combination of three restriction enzymes, AluI, CfoI, and TaqI, for a low-cost, rapid screen for a primarily differentiation of isolates of the complex of aerobic spore-forming bacteria commonly found in honey samples. The described method produced unique and distinguishable patterns to differentiate among 80 isolates belonging to 26 different species of Bacillus, Brevibacillus, Lysinibacillus, Rummeliibacillus, and Paenibacillus reported in honey and other apiarian sources., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Analyses of bacterial DNA in faecal samples are becoming ever more common, yet we still do not know much about bird microbiomes. These challenges partly lie in the unique chemical nature of their faeces, and in the choice of sample storage method, which affects DNA preservation and the resulting microbiome composition. However, there is little information available on how best to preserve avian faeces for microbial analyses. This study evaluates five widely used methods for preserving nucleic acids and inferring microbiota profiles, for their relative efficacy, cost, and practicality. We tested the five methods (in-situ bead-beating with a TerraLyzer instrument, silica-bead desiccation, ethanol, refrigeration and RNAlater buffer) on 50 fresh faecal samples collected from captive House sparrows (Passer domesticus). In line with other studies, we find that different storage methods lead to distinct bacterial profiles. Storage method had a large effect on community composition and the relative abundance of dominant phyla such as Firmicutes and Proteobacteria, with the most significant changes observed for refrigerated samples. Furthermore, differences in the abundance of aerobic or facultatively aerobic taxa, particularly in refrigerated samples and those stored in ethanol, puts limits on comparisons of bacterial communities across different storage methods. Finally, the methods that did not include in-situ bead-beating did not recover comparable levels of microbiota to the samples that were immediately processed and preserved using a TerraLyzer device. However, this method is also less practical and more expensive under field work circumstances. Our study is the most comprehensive analysis to date on how storage conditions affect subsequent molecular assays applied to avian faeces and provides guidance on cost and practicality of methods under field conditions., (Copyright © 2019. Published by Elsevier B.V.)

Pollution of various environmental matrices by antibiotic resistance genes (ARGs) has become a growing threat to human health. For the quantitative analysis of the presence of ARGs, there is a need for sensitive and robust qPCR assays which can detect various genes from different types of DNA extracts. Fourteen ARGs were selected as target genes in this study including: bla TEM , bla OXA-1 and bla CTX-M coded for resistance to β-lactams; ermB for macrolides; tetA, tetG, tetM, tetQ, tetW and tetX for tetracyclines; sul I and sul II for sulfonamides; drfA1 and drfA12 d for trimethoprim; and integron gene intI 1 and intI 2. Chemically synthesized double-stranded gene fragments were modified using molecular biology methods and used as real-time PCR standards as well as to establish in-house qPCR assays. The ermB gene from a naturally occurring plasmid was used to compare the performance of qPCR assay with the chemically synthesized ermB. Additionally, environmental water, soil and faeces samples were used to validate the established qPCR assays. Importantly, the study proves the usefulness of rapidly synthesized oligonucleotides serving as qPCR standards for ARG analysis and provides comparable sensitivity and reliability to a traditional amplicon standard., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Human gut microbiome analysis through faecal sampling typically involves five stages: sample collection, storage, DNA extraction, next generation sequencing and bioinformatics analysis. Of these, the first three are considered irreversible. This feasibility study describes an assessment of methodologies used for faecal DNA extraction and sample handling, using the parameters DNA yield, purity and resultant microbial profile. Six DNA extraction techniques, including commercially available kits and manual protocols were compared on human faecal samples (n = 3). Different extraction techniques produced significant variance in DNA yield (range 2.7-164 ng/mg faeces) and microbial diversity profiles, with considerable variation in phyla dominance (Firmicutes (P < 0.001), Bacteroidetes (P = 0.003), Actinobacteria (P = 0.003), One-way ANOVA). The most effective method, with the highest DNA yield, was a simple and inexpensive extraction technique named MetaHIT. Using this method, DNA was extracted from separate faecal samples (n = 3) and had been aliquoted to seven storage conditions including three stabilizing buffers and three temperature conditions, for a period of 120-h, with storage at -80 °C as a control treatment. DNA yield and purity was not statistically different between the control and remaining treatments. 16S rDNA-based diversity profile was largely comparable across the treatments with only minor differences in genera between samples stored at room temperature in air and - 80 °C control. Overall these results suggest that the choice of DNA extraction method has a greater influence on the resultant microbial diversity profile than the short-term storage method., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Early detection of infectious bacteria is a necessity for combating infectious diseases. Due to low infectious dose of Shigella, rapid and sensitive detection is needed. Compared to the presented genes, Spa gene can be introduced as a novel sequence for all species of Shigella detection. Herein, the possibility of Spa genes for detection of four species of Shigella was investigated for the first time by AuNPs-based optical genosensing system. In this method, AuNP-DNA probes were hybridized with Spa gene sequence. When the complementary target is present, it prevents the aggregation of the complex under acid environment and the solution remains red whereas in the absence of the specific sequence, it turns to purple. Therefore, visual detection is possible with bare eye. The comparison of this Optical DNA biosensor and PCR-based method showed that the proposed method is simple, cost-effective, rapid operation, with high or comparable detection limit of (LOD and LOQ: 8.14 and 26.6 ng mLl -1 , respectively), without need of any expensive techniques, and equipments compared to the conventional methods. In conclusion, the described method may develop into a platform that could be utilized for detection of various bacterial species with high accuracy and prompt screening of samples., (Copyright © 2019. Published by Elsevier B.V.)

Xylella fastidiosa (Xf) is a quarantine plant pathogen bacterium originating from the Americas and that has emerged in Europe in 2013. Xf can be detected directly on plant macerate using molecular methods such as real-time PCR, which is a sensitive technique. However, some plants may contain components that can act as PCR reaction inhibitors, which can lead to false negative results or an underestimation of the bacterial concentration present in the analyzed plant sample. Droplet digital PCR (ddPCR) is an innovative tool based on the partitioning of the PCR reagents and the DNA sample into thousands of droplets, allowing the quantification of the absolute number of target DNA molecules present in a reaction mixture, or an increase of the detection sensitivity. In this study, a real-time PCR protocol, already used for Xf detection in the framework of official surveys in the European Union, was transferred and optimized for Xf detection using ddPCR. This new assay was evaluated and compared to the initial real-time PCR on five plant matrices artificially inoculated and on naturally infected plants. In our conditions, this new ddPCR enabled the detection of Xf on all artificially inoculated plant macerates with a similar limit of detection, or a slight benefit for Quercus ilex. Moreover, ddPCR improved diagnostic sensitivity as it enabled detection of Xf in samples of Polygala myrtifolia or Q. ilex that were categorized as negative or close to the limit of detection using the real-time PCR. Here, we report for the first time a ddPCR assay for the detection of the bacterium Xf., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The objective of this study was to establish a novel isothermal amplification method for detection of heat-labile enterotoxin (LT-I)-producing Escherichia coli. Loop-mediated isothermal amplification (LAMP), cross-priming amplification (CPA), and isothermal multiple-self-matching-initiated amplification (IMSA) were developed and evaluated. Optimal conditions, specificity, and sensitivity tests were performed and compared to qPCR findings. All three methods could produce ladder-like products with LT-I positive samples, while no products were generated with the negative controls. The amplified products could be directly visualized as negative or positive in the isothermal amplification (IAM) tube, which saved time and prevented the possibility of cross-contamination. The detection limits of each assay were similar, and all three assays could directly detect the DNA of Escherichia coli in clinical samples successfully. This is the first report on the application of CPA and IMSA methods for the detection of LT-I. The findings suggest that the three assays may be important tools for the rapid detection of enterotoxigenic Escherichia coli (ETEC) in the clinic., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Community-acquired pneumonia (CAP) is a common cause of pediatric hospital admission. Empiric antibiotic therapy for hospitalized children with serious CAP now targets the most likely pathogen(s), including those that may demonstrate significant antibiotic resistance. Cell-free plasma next-generation sequencing (CFPNGS) was first made available for Pediatric Infectious Diseases physicians in June 1, 2017, to supplement standard-of-care diagnostic techniques. A retrospective chart review was performed for children hospitalized with CAP between June 1, 2017, and January 22, 2018, to evaluate the impact of CFPNGS. We identified 15 hospitalized children with CAP without other underlying medical conditions for whom CFPNGS was performed. CFPNGS identified a pathogen in 13 of 15 (86%) children compared with 47% for those using standard culture and PCR-based methods alone. Changes in antibiotic management were made in 7 of 15 (47%) of children as a result of CFPNGS., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Identification of bacteria in new or existing commercial microbial-based products (MBPs) is important for compliance with government regulations and for human and environmental risk assessment. Research was performed to develop effective methods to identify bacteria present in a MBP using a combined approach of conventional enrichment culture technique and denaturing gradient gel electrophoresis (DGGE) followed by clonal sequencing or next generation sequencing (NGS). Genomic DNA obtained from un-enriched or enriched MBP in MacConkey broth, Azide Dextrose broth, Peptone Water mixed with Polymixine B and Gram Negative (GN) media under three different temperatures (22 °C, 28 °C and 37 °C) were sequenced in two methods for the V3 and V6 hypersensitive regions of 16S ribosomal DNA (rDNA) and compared. Enrichment followed by DGGE and clonal sequence analysis identified 20 bacterial genera in all enriched and un-enriched media. In contrast, NGS was able to identify 114 bacterial families and 134 genera both in V3 and V6 regions. In clonal sequence analysis, in comparison to the un-enriched MBP, the MacConkey broth enriched for Escherichia or Shigella and Morganella species, GN medium enriched for Proteus and Morganella species and Azide Dextrose broth enriched for Vagococcus and Enterococcus species at both 28 °C and 37 °C. Moreover, the enrichment facilitated NGS to record higher numbers of families and genera in all enrichment cultures, comparatively higher variations in V3 region than in V6. More prominently, NGS identified 14 genera and 9 species in the family Enterobacteriaceae compared to only 5 genera identified in the un-enriched control using V6 region variance in MacConkey broth at 28 °C. Increasing the temperature without enrichment identified specific families by V3 and V6 regions. This study indicates that the polyphasic approach with appropriate enrichment and incubation at different temperatures followed by NGS analysis is a promising method for the identification of viable, non-pathogenic or potential pathogenic bacteria in complex MBPs., (Crown Copyright © 2019. Published by Elsevier B.V. All rights reserved.)

Microbialites are organo-sedimentary structures formed by complex microbial communities that interact with abiotic factors to form carbonate rich fabrics. Extraction of DNA or total RNA from microbialites can be difficult because of the high carbonate mineral concentration and exopolymeric substances. The methods employed until now include substances such as cetyltrimethylammonium bromide, sodium dodecyl sulfate, xanthogenate, lysozyme and proteinase K, as well as mechanical disruption. Additionally, several commercial kits have been used to improve DNA and total RNA extraction. This minireview presents different methods applied for DNA and RNA extraction from microbialites and discusses their advantages and disadvantages. Moreover, extraction of all 'omes (DNA, RNA, Protein, Lipids, polar metabolites) using multiomic extraction methods (MPlex), as well as the state of art for extraction of viruses from microbialites, are also discussed., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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. We extracted DNA from 15 different shallow lakes with phenol:chloroform, a classical and widely used extraction method, and with the PowerSoil DNA isolation Kit, often suggested as the standard DNA extraction method, with some adaptations for aquatic environments. We found that although only 5% of all OTUs showed significant differences in pairwise comparisons (using the 15 lakes as replicates), these OTUs accounted for >35% (on average) of the relative abundance. Diversity and richness did not differ significantly between the two extraction methods, but the beta-dispersion of the communities indicated that the organic extraction yielded more homogeneous communities, while the kit extraction generated variability. Consequently, we conclude that despite the small number of OTUs with significant differences, their impact on the community composition obtained was not negligible, and therefore the results from these two extraction methods were not comparable., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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. Second, after selective caries excavation and sampling of carious dentine, an intervention was performed by applying dental materials onto the remaining carious dentine followed by a restoration of the study teeth with composite fillings. After 8 weeks, remaining carious dentine was sampled and analyzed as described above. The microbial community before and after therapy was analyzed by conventional culture compared to bacterial DNA analyses using 16S rRNA gene based real-time PCR and terminal restriction fragment length polymorphism (T-RFLP) for fingerprinting community changes within carious dentine samples. An ultra-pure workflow allowed the valid comparison of even small carious dentine samples with low DNA contents and the differentiation between intact cell-derived and cell-free bacterial DNA. Intra- and inter-subject related differences in the bacterial DNA content and its composition in deep dentine caries were determined considering the first visits. The ratio of cell-free bacterial DNA and DNA from intact cells decreased in two of three subjects included in the current study from visit 1 to visit 2 with the test substance (1:200 to 1:17) and the control substance (1:82 to 1:7). T-RFLP revealed changes in the bacterial diversity and composition shifts after treatment as well as between cell-free bacterial DNA and DNA derived from intact cells. The approach of differentiation and quantification of cell-free and intact cell-derived bacterial DNA is reasonable within the investigation of carious dentine samples, especially when considering the effect of an intervention. T-RFLP is principally suitable for the analysis of microbial shifts within carious dentine samples., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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. Significantly more DNA was obtained from samples immediately preserved with DNAzol than the corresponding shipped samples for 2 of the 3 days. More DNA was extracted from DNAzol preserved samples but more RNA was obtained from RLA+ preserved samples. A protocol was designed to extract both DNA and RNA from split samples preserved with RLA+ and cDNA was synthesized from the RNA. Three high-throughput 16S rRNA gene libraries were constructed, one from DNA preserved with DNAzol, one from DNA preserved with RLA+ and one from cDNA (RLA+ preserved). Greater alpha diversity was found for libraries constructed from immediately preserved vs. shipped samples for both preservation types, with immediate preservation with DNAzol obtaining the highest level of diversity. Libraries constructed from immediately preserved (RLA+) DNA had greater alpha diversity than libraries constructed from shipped preserved (RLA+) DNA or cDNA. Unifrac measures of beta diversity showed clearer separation of sample types and a greater % variance explained for weighted than for unweighted principal coordinate analysis (PCoA) plots, indicating sample types varied more in their relative abundance of taxa than the presence/absence of particular taxa. We recommend immediate preservation of seawater samples, with DNAzol as the preferred preservative if quantification via qPCR will be performed or the highest alpha diversity is desired but preservation with RLA+ if RNA will be extracted., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Although isolation and identification of bacteria in a clinical specimen constitute essential steps for the diagnosis of bacterial infection, positive results of the bacterial culture are not always attained, despite observing the bacteria by Gram staining. As bacteria phagocytosed by the leukocytes are considered as the causative agents of infectious diseases, this study aims to introduce a new approach for the collection of only bacteria phagocytosed by the neutrophils in an animal model using laser capture microdissection (LCM) followed by the DNA identification using polymerase chain reaction (PCR). We inoculated representative bacteria (Escherichia coli and Staphylococcus aureus) into the abdominal cavities of specific pathogen-free C57BL/6 J mice. After 6 h inoculation, we collected the fluid samples from the peritoneal cavities of mice and demonstrated peritonitis by the increase of neutrophils. Then, we smeared the neutrophils on the membrane slides and collected single-cell phagocytosing bacteria by LCM. The supernatant of the cell lysate was supplied for the PCR reaction to amplify the 16S rRNA gene, and we validated the DNA sequences specific for the inoculated bacteria. In addition, PCR using specific primers for E. coli and S. aureus identified each species of bacteria. Hence, this study suggests that the combination of LCM and PCR could be a novel approach to determine bacteria in infectious diseases. Nevertheless, further investigation is warranted to test various additional bacterial taxa to demonstrate the general applicability of this method to clinical samples., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We developed the com1 gene based loop-mediated isothermal amplification (LAMP) assay for the detection of Coxiella burnetii and validated it by screening DNA isolated from serum samples collected from animals and humans. The detection of Coxiella by LAMP assay was comparable with the com1 based-PCR., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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. In addition, the Mayer-Scholl m-PCR method was modified and termed 'modified Mayer-Scholl', and genus specific Bcsp31 PCR was also modified with minor changes. Four different methods were applied to direct examination samples and the obtained results were compared. The conventional culture method was set as the standard method to which sensitivities and specificities of the molecular methods were calculated. According to the assessments on the basis of fetus (n = 166), sensitivity and specificity values for modified Mayer-Scholl m-PCR method were 94.11% and 98.76%, and the same indicators for the modified Bcsp31 PCR were 95.29% and 98.76%, respectively. When all organ samples were taken into account (n = 326), sensitivity and specificity values for the modified Mayer-Scholl m-PCR method were 85.38% and 98.06%, and for the modified Bcsp31 PCR, they were 83.62% and 98.06%, respectively. As a result, it was found that the diagnostic power of the tests were 'high' when results were evaluated at fetus level. On the other hand, it was found to be 'clinically useful' when evaluated at organ level. We concluded that species level identifications can be made through the modified Mayer-Scholl method, which is a direct m-PCR method, with a high diagnostic power by specifying DNAs belonging to Brucella species directly from clinical samples., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Salmonella enterica serovar Indiana (S. Indiana) was the most frequently reported foodborne pathogen, which has a broad host range including poultry, swine, and humans. Traditional methods used for the detection of S. Indiana from contaminated food products are time-consuming and labor-intensive. Therefore, rapid detection methods with high sensitivity and specificity are vitally important to prevent the spread of S. Indiana. In this study, we developed a nearly instrument-free, simple molecular method which incorporates cross-priming amplification (CPA) combined with a nucleic acid detection strip (NADS) for sensitive detection of S. Indiana. A set of CPA primers was designed based on S. Indiana specific nucleotide sequences and the specificity of CPA-NADS was tested against 42 bacterial strains. The results showed that this method was highly specific for detection of S. Indiana. The sensitivity of CPA-NADS was evaluated and compared with that of the serovar-specific PCR method and the real-time PCR method. The limit of detection of the CPA method was 8.997 fg/μL for genomic DNA and 6.2 × 10 1  CFU/mL for bacteria in pure culture. An application of the CPA assay was conducted with 90 inoculated specimens by S. Indiana. The accuracy of CPA-NADS was consistent with the results of the traditional culture-based methods in inoculated specimens. This method showed a higher sensitivity than the serovar-specific PCR method did and was more convenient to perform. In conclusion, we demonstrated that the CPA-NADS system offers high specificity, sensitivity, rapidity, and a simple detection tool for screening S. Indiana., (Copyright © 2018. Published by Elsevier B.V.)

Determination of cell numbers in filamentous bacteria, such as Streptomyces, is challenging due to the tangled and twisted structure of the filaments and formation of cell clumps in liquid cultures. Here, we developed a conidia-based approach, in which fluorescence of conidia, after staining with the DNA-binding stain SYBR Green 1, was related to SYBR Green 1 fluorescence of DNA in Streptomyces. When cell number in Streptomyces filaments, determined by the conidia assay, was compared to number obtained by a qPCR assay, 34 to 62% of cells in the Streptomyces filaments were recovered. The difference in numbers probably reflects an insufficient extraction of DNA from the Gram-positive bacteria, rather than underestimation of the actual cell number by the conidia-based determination. The conidia-based approach appears to be a fast and reliable procedure for counting cell numbers in Streptomyces filaments but it can also be used for other filamentous bacteria, if proper standard curves can be made., (Copyright © 2018 Elsevier B.V. All rights reserved.)

DNA-based monitoring of pathogens in aerosol samples requires extraction methods that provide high recovery of DNA. To identify a suitable method, we evaluated six DNA extraction methods for recovery of target-specific DNA from samples with four bacterial agents at low abundance (<10,000 genome copies per detection assay). These methods differed in rigor of cell disruption, approach for DNA capture, and extent of DNA purification. The six methods varied 1000-fold in the recovery of DNA from spores or cells of surrogates of Bacillus anthracis, Yersinia pestis, Burkholderia pseudomallei, and Francisella tularensis, each at about 10 5  CFU per sample. A custom method using paramagnetic Dynabeads for DNA capture greatly outperformed the other five methods. The cDynabead method provided about 80% recovery of target-specific DNA. The cDynabead method and a filtration method were further evaluated for DNA recovery from bacterial agents spiked on filters (c.a. 10 5  CFU of each agent per filter quadrant) that were subsequently used to collect background outdoor air particulates for 24-h. The filtration method generally failed to recover detectable quantities of target DNA from the spiked filters, suggesting at least a 100-fold loss of target DNA during extraction, whereas the custom cDynabead method consistently yielded DNA sufficient for target detection., (Published by Elsevier B.V.)

In the process of developing a point-of-care testing device, we fabricated a plane heating element using carbon paste capable of thermal lysis. The plane heating element can generate heat of 150 °C within 60 s at 0.4 A power supply within 8 W. The developed plane heating element was very effective in the extraction of DNA from both Gram-negative and Gram-positive bacteria which was further confirmed by agarose gel electrophoresis. In this approach, we aimed to eliminate expensive, time-consuming and labor-intensive procedures for the lysis of bacterial cells to extract nucleic acids to detect pathogenic bacteria accurately and swiftly., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Tuberculosis (TB), caused by infections of the Mycobacterium tuberculosis (MTB) complex, is the ninth leading cause of death worldwide, and several molecular approaches for MTB species identification and the detection of mutations associated with drug resistance have been developed to date. We previously developed a diagnostic assay for drug susceptibility testing that can detect mutations conferring resistance to anti-TB drugs using allele-specific primer extension on a microsphere-based platform for multiplex polymerase chain reaction. The aim of the present study was to optimize this diagnostic assay based on the evaluation of three methods for extracting mycobacterial DNA from clinical samples. Mycobacterial DNA of 81 samples was digested and decontaminated by N-acetyl-l-cysteine-2% NaOH and then extracted using three methods: "in-house" 5% Chelex-100 chelating resin, InstaGene Matrix, and MagPurix TB DNA Extraction Kit. The former two methods are manual extraction methods, whereas the MagPurix TB DNA Extraction Kit is an automated extraction method used with the MagPurix 12 s automated nucleic acid purification system. The extracted DNA was then subjected to our diagnostic assay, and the results were compared among methods. The magnetic bead method exhibited a higher extraction efficiency and resulted in greater diagnostic efficacy than the two resin-based methods with respect to both target gene detection and acid-fast bacilli smear grades. Therefore, the MagPurix TB DNA Extraction Kit is the optimal MTB DNA extraction method for our diagnostic assay of TB drug susceptibility testing., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Uropathogenic Escherichia coli (UPEC) cause the majority of community-acquired urinary tract infections (UTIs). Quantitation of bacteriuria (the number of bacteria in urine) is important for diagnostic approaches and in diverse research applications. Most UPEC strains express hemolysin, the expression of which has been correlated with the severity of UTI in murine models of infection. In this study, we sought to develop and optimise a quantitative Polymerase Chain Reaction (qPCR) assay for enumeration of hemolysin-positive UPEC in urine. Using recombinant plasmid pJET1.2::hlyD, which we termed pGU2470, the sensitivity range and linearity of amplification of qPCR was determined using primers and a probe targeting hlyD. Whole-cell preparations containing UPEC were quantified for hlyD copy number using C T values and compared to standards prepared with a known amount of pGU2470. We compared the efficiency of the assay for analysis of human and mouse urine because mouse models of human UTI are frequently used for investigating UPEC UTI. Urine samples were collected from healthy adults and mouse infection assays and used to assess any potential inhibitory effects of urine on the qPCR. The linear quantitative range of the qPCR (i.e. sensitivity) in detecting UPEC genomes was 10 3 copies/ml; qPCR-derived estimates of UPEC bacteriuria (based on number of genomes detected) were, on average, ten-fold higher that culture-based estimates. Finally, the frequencies of positive and negative predictions of UPEC in urine using the qPCR were equivalent to colony count methods. This assay provides an alternative to culture-based approaches for quantitation of UPEC bacteriuria in research studies., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Symbioses between bacteria and insects are often associated with changes in important biological traits that can significantly affect host fitness. To a large extent, studies of these interactions have been based on physiological changes or induced phenotypes in the host, and the genetic mechanisms by which symbionts interact with their hosts have only recently become better understood. Learning about symbionts has been challenging in part due to difficulties such as obtaining enough high quality genomic material for high throughput sequencing technology, especially for symbionts present in low titers, and in small or difficult to rear non-model hosts. Here we introduce a new method that substantially increases the yield of bacterial DNA in minute arthropod hosts, and requires less starting material relative to previous published methods., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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. The universal amplicon sequencing provided 1) accurate estimates of nitrifier composition, 2) clustering of the samples based on these compositions consistent with sample origin, 3) estimates of the relative abundance of the guilds correlated with those obtained from the targeted approaches and within ~1.2 orders of magnitude of them, but with measurable bias that should be considered when comparing estimates from both approaches. In contrast, the diversity and richness estimations using the universal 16S rRNA based amplicon sequencing were likely limited by the sequencing depth; therefore, we suggest preferring targeted approaches for assessing nitrifiers diversity and richness or using sequencing depth larger than those currently typically practiced. Overvall, we conclude that universal amplicon sequencing provides, in a single analysis, useful information on the abundance and composition of diverse guilds in complex environmental communities., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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. The assay was specific for L. intracellularis, as no cross-reaction was observed with other pathogens. The detection limit of the real-time LAMP assay was 1.4 × 10 -1 pg of L. intracellularis DNA, which was the same as that of real-time PCR and approximately 100 times more sensitive than that of conventional PCR. Of the 136 clinical samples, L. intracellularis DNA was identified in 60 samples by real-time LAMP, which was the same as real-time PCR and higher than conventional PCR (36.8%, 50/136). The specific, sensitive and rapid real-time LAMP assay developed in this study could be a useful alternative tool in point-of-care (POC) diagnosis of L. intracellularis infection., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Use of the 16S rRNA gene in microbiota studies is limited by the lack of taxonomic and functional resolution. High resolution analyses are particularly important for understanding transmission and persistence of bacteria. The aim of our work was therefore to compare a novel reduced metagenome sequencing (RMS) approach with 16S rRNA gene sequencing to determine both the metagenome genetic diversity and the mother-to-child sharing of the microbiota in a cohort of 17 mother-child pairs. We found that although both approaches gave comparable results with respect to sample separation and taxonomy, RMS gave higher resolution and the potential for genomic-/functional assignment. Using RMS we estimated that the metagenome size increased from about 60 Mbp for 4-day-old children to about 225 Mbp for mothers. The 4-day-old children shared 7% of the metagenome sequences with the mothers, while the metagenome sequence sharing was >30% among the mothers. We found 15 genomes shared across >50% of the mothers, of which 10 belonged to Clostridia. Only Bacteroides showed a direct mother-child association, with B. vulgatus being abundant in both 4-day-old children and mothers. For the functional assignments, we identified a significant association between antibiotic usage during labor, and quantity of Fosfomycin resistance genes. In conclusion, our results show a higher functional and taxonomic resolution for RMS compared to 16S rRNA gene sequencing, where RMS enabled a detailed description of mother to child gut microbiota transmission - supporting a late recruitment of most gut bacteria and an effect of antibiotic treatment during labor on infant antibiotic resistance gene patterns., (Copyright © 2018. Published by Elsevier B.V.)

For some environments such as planktonic/aqueous environments, the separation of bacteria cells from eukaryotic cells prior to DNA extraction using filtration is relatively straightforward. However, for woodchips, the bacteria are attached/embedded within the wood matrix, which prevents easy removal of bacterial cells. In this study, a method for the selective extraction of DNA from bacteria inhabiting Eucalyptus spp. woodchips has been developed. The objective was to compare milled and unmilled woodchips processed via three detachment methods, viz., sonication, vortexing and shaking followed by filtration using Teflon filters according to three relevant criteria: DNA yield, DNA purity and quality of DNA. Highest DNA yield was obtained by milling and vortexing for 10 min (77.50 ± 5.17 ng/μl), followed by milling and vortexing for 2 min (61.00 ± 6.56 ng/μl), unmilled and vortexing for 10 min (38.67 ± 5.17 ng/μl) and milled and shaking for 2 h (31.62 ± 5.17 ng/μl). The lowest DNA yield was obtained by using unmilled woodchips and 5 min of sonication treatment (7.00 ± 1.22 ng/μl). There was no significant difference in DNA purity for milled or unmilled woodchips processed via the three detachment methods. Duration of cell detachment treatment did not significantly influence DNA yield and purity. Following optimisation experiments, it was possible to extract bacterial DNA using milled woodchips and 10 minute vortexing devoid of DNA from the host background and other associated eukaryotes and of sufficient quality and quantity for metagenomic analysis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Purpose: The aim of this study was to evaluate two non-commercial Real-Time PCR assays for the detection of microorganisms in amniotic fluid followed by identification by pyrosequencing., Methods: We collected 126 amniotic fluids from 2010 to 2015 for the evaluation of two Real-Time PCR assays for detection of bacterial DNA in amniotic fluid (16S Universal PCR and Ureaplasma spp. specific PCR). The method was developed in the Department of Microbiology of the University Hospital La Paz., Results: Thirty-seven samples (29.3%) were positive by PCR/pyrosequencing and/or culture, 4 of them were mixed cultures with Ureaplasma urealyticum. The Universal 16S Real-Time PCR was compared with the standard culture (81.8% sensitivity, 97.4% specificity, 75% positive predictive value, 98% negative predictive value). The Ureaplasma spp. specific Real-Time PCR was compared with the Ureaplasma/Mycoplasma specific culture (92.3% sensitivity, 89.4% specificity, 50% positive predictive value, 99% negative predictive value) with statistically significant difference (p=0.005)., Conclusions: Ureaplasma spp. PCR shows a rapid response time (5h from DNA extraction until pyrosequencing) when comparing with culture (48h). So, the response time of bacteriological diagnosis in suspected chorioamnionitis is reduced., (Copyright © 2017 Elsevier B.V. All rights reserved.)

DNA extraction for molecular biological applications usually requires target optimized extraction procedures depending on the origin of the samples. For environmental samples, a range of different procedures has been developed. We compared the applicability and efficiency of ten selected DNA extraction methods published in recent literature using four different environmental samples namely: activated sludge from a domestic wastewater treatment plant, river sediment, anaerobic digestion sludge and nitrifying enrichment culture. We assessed the suitability of the extraction procedures based on both DNA yield and quality. DNA quantification was performed by both ultra violet (UV) spectrophotometry and fluorescence spectrophotometry after staining with PicoGreen. In our study, DNA yields based on UV measurement were overestimated in most cases while DNA yields from fluorescence measurements correlated well with the sample load on agarose gels of crude DNA. The quality of the DNA extracts was determined by gel electrophoresis of crude DNA and PCR products from 16S rDNA with the universal primer set 27f/1525r. It was observed that gel electrophoresis of crude DNA was not always suitable to evaluate DNA integrity and purity since interfering background substances (e.g. humic substances) were not visible. Therefore, we strongly recommend examining the DNA quality of both crude DNA and 16S rDNA PCR products by gel electrophoresis when a new extraction method is established. Summarizing, we found four out of ten extraction procedures being applicable to all tested samples without noticeable restrictions. The procedure G (according to the standard method 432&#95;10401 of the Lower Saxony State Office for Consumer Protection and Food Safety) had the broadest application range over procedure J (published by Wilson, 2001). These were followed by procedures F (Singka et al., 2012) and A (Bourrain et al., 1999). All four extraction procedures delivered reliable and reproducible crude DNA and PCR products. From an economical point of view, all procedures tested during this study were cheaper compared to commercial DNA extraction kits., (Copyright © 2017 Elsevier B.V. All rights reserved.)

We evaluated the performance of two multiplex, real-time PCR tests (Anyplex II MTB/MDR and MTB/XDR; Seegene, Seoul, Korea), designed to detect the Mycobacterium tuberculosis complex (MTC) and drug-resistance mutations associated with isoniazid, rifampicin, fluoroquinolones, and second-line injectable drugs. We analyzed 122 clinical isolates with the Anyplex II MTB/MDR test, 68 of which were also tested with the Anyplex II MTB/XDR test. The Anyplex II MTB/MDR and MTB/XDR tests showed the following respective sensitivities and specificities: 68.8% and 100% for detecting isoniazid resistance, 93.8% and 100% for rifampicin, 82.8% and 100% for levofloxacin, 75.0% and 100% for kanamycin, and 92.6% and 100% for MTC identification. These kits correctly identified 61.8% of multi-drug resistant M. tuberculosis isolates and 64.7% of extensively drug-resistant M. tuberculosis isolates, and enabled semi-automatic detection of drug-resistant MTC in 3 hours. The Anyplex II kits could be useful as rule-in tests for detecting MTC and drug resistance., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Brucella canis, a gram-negative, facultative intracellular and zoonotic bacterium causes canine brucellosis. Direct methods are the most appropriate for the detection of canine brucellosis and bacterial isolation from blood samples has been employed as gold-standard method. However, due to the delay in obtaining results and the biological risk of the bacterial culturing, the polymerase chain reaction (PCR) has been successfully used as an alternative method for the diagnosis of the infection. Sample preparation is a key step for successful PCR and protocols that provide high DNA yield and purity are recommended to ensure high diagnostic sensitivity. The objective of this study was to evaluate the performance of PCR for the diagnosis of B. canis infection in 36 dogs by testing DNA of whole blood obtained through different extraction and purification protocols. Methods 1 and 2 were based on a commercial kit, using protocols recommended for DNA purification of whole blood and tissue samples, respectively. Method 3 was an in-house method based on enzymatic lysis and purification using organic solvents. The results of the PCR on samples obtained through three different DNA extraction protocols were compared to the blood culture. Of the 36 dogs, 13 (36.1%) were positive by blood culturing, while nine (25.0%), 14 (38.8%), and 15 (41.6%) were positive by PCR after DNA extraction using methods 1, 2 and 3, respectively. PCR performed on DNA purified by Method 2 was as efficient as blood culturing and PCR performed on DNA purified with in-house method, but had the advantage of being less laborious and, therefore, a suitable alternative for the direct B. canis detection in dogs., (Copyright © 2017. Published by Elsevier B.V.)

A highly sensitive and specific Chlamydia trachomatis (CT) diagnostic test was developed by combining filtration isolation of nucleic acid (FINA) extraction with quantitative polymerase chain reaction including an internal control to identify test inhibition. A pilot study of 40 clinical specimens yielded 100% sensitivity and specificity., (Copyright © 2017 Elsevier Inc. All rights reserved.)