Simple lysis of bacterial cells for DNA-based diagnostics using hydrophilic ionic liquids

Martzy, Roland; Bica-Schröder, Katharina; Pálvölgyi, Ádám Márk; Kolm, Claudia; Jakwerth, Stefan; Kirschner, Alexander K. T.; Sommer, Regina; Krska, Rudolf; Mach, Robert L.; Farnleitner, Andreas H.; Reischer, Georg H.

doi:10.1038/s41598-019-50246-5

Cited by 36 publications

(25 citation statements)

References 32 publications

(31 reference statements)

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“…Then, the tubes were centrifuged at 1000 rpm for 5 minutes and collected the supernatant. The DNA quality was determined by optical density at 260/280 nm, and 5 microliters of aliquot were used for PCR [15].…”

Section: Bacteria Dna Extractionmentioning

confidence: 99%

Diversity of Colony Morphotypes, Biochemical Characteristics, and Drug Susceptibility Patterns of Burkholderia pseudomallei Isolated from Humans, Animals, and Environmental Sources in Thailand

et al. 2022

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Melioidosis is an infection that occurs in humans and a wide range of animals by Burkholderia pseudomallei as a causative pathogen, which inhabits environments of tropical and subtropical climate zones, particularly in Thailand. The standard laboratory diagnosis is by culture and biochemical characteristic differentiation. This study aimed to determine the diversity of colony morphology, biochemical characteristics, and drug susceptibility patterns of a B. pseudomallei collection isolated from humans, animals, soil, and water in Thailand. According to the previous characterization of colony morphotypes from Type I to VII, Type I is predominant with B. pseudomallei from humans and animals with a similar water source. Meanwhile, Type III is predominant from soil sources. The results confirmed the hypothesis that infection might be from water instead of soil exposure. The typical biochemical phenotypes were consistent among different isolate sources, including cytochrome oxidase (positive), triple iron sugar (K/N or A/N), sulfide (negative), indole (negative), non-motile, glucose oxidizing, and DNase (positive). The biochemical characteristic results supported the data for the selection of the prepopulated biochemical tests in clinical and research laboratories. The antimicrobial susceptibility patterns by standard disk diffusion showed mode values of inhibition zones for selected drugs that had lower trends in B. pseudomallei isolated from the soil than other sources. The results were consistent with MIC50 and MIC90 values of ceftazidime, which was highest in B. pseudomallei from the soil than other sources. HIGHLIGHTS The previous study reported the characterization of Burkholderia pseudomallei colony morphotypes from Type I to VII. However, there were no reports of the diversity of colony morphology and antibiotic susceptibility pattern from diverse sources Type I is predominant with B. pseudomallei from humans and animals with a similar water source, which confirmed the hypothesis that infection might be from water instead of soil exposure The mode of antimicrobial inhibition zones for selected drugs had lower trends in the isolates from the soil than other sources. The results indicated the soil isolates showed a trend in increasing ceftazidime resistance compared with other sources GRAPHICAL ABSTRACT

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Section: Bacteria Dna Extractionmentioning

confidence: 99%

Diversity of Colony Morphotypes, Biochemical Characteristics, and Drug Susceptibility Patterns of Burkholderia pseudomallei Isolated from Humans, Animals, and Environmental Sources in Thailand

et al. 2022

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“…Physical lysis can include freeze-thaw ( Shao et al, 2012 ), heat, cavitation ( Liu et al, 2020 ), and ultrasonic ( Han et al, 2019 ) methods. Chemical lysis uses ionic alkali solutions or detergents to disrupt the bacterial cell membrane ( Chapela et al, 2007 ; Martzy et al, 2019 ). Enzymatic lysis is accomplished through the application of enzymes such as proteinase k and lysozyme ( Gill et al, 2016 ).…”

Section: Wet Lab Considerationsmentioning

confidence: 99%

Considerations and best practices in animal science 16S ribosomal RNA gene sequencing microbiome studies

Weinroth

Belk

Dean

et al. 2022

Journal of Animal Science

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Microbiome studies in animal science using 16S rRNA gene sequencing have become increasingly common in recent years as sequencing costs continue to fall and bioinformatic tools become more powerful and user-friendly. The combination of molecular biology, microbiology, microbial ecology, computer science, and bioinformatics—in addition to the traditional considerations when conducting an animal science study—makes microbiome studies sometimes intimidating due to the intersection of different fields. The objective of this review is to serve as a jumping-off point for those animal scientists less familiar with 16S rRNA gene sequencing and analyses and to bring up common issues and concerns that arise when planning an animal microbiome study from design through analysis. This review includes an overview of 16S rRNA gene sequencing, its advantages, and its limitations; experimental design considerations such as study design, sample size, sample pooling, and sample locations; wet lab considerations such as field handing, microbial cell lysis, low biomass samples, library preparation, and sequencing controls; and computational considerations such as identification of contamination, accounting for uneven sequencing depth, constructing diversity metrics, assigning taxonomy, differential abundance testing, and, finally, data availability. In addition to general considerations, we highlight some special considerations by species and sample type.

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“…There are few studies which reported isolation of DNA from gram positive bacteria even without using lysozyme [35,36] Yuan et al [37] reported that lysozyme alone is not efficient in extracting DNA from human microbiome (especially gram positive bacteria). Shahriar et al [38] 5).…”

Section: Gram +Ve Gram -Ve Gram Variable Othersmentioning

confidence: 99%

Optimization of Soil DNA Extraction Protocol Using Na2EDTA, SDS, Heating, Vortexing and CaCl2 and Its Validation for Metagenomic Studies

Pasha

Krishnaraj

2020

IJECC

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Aim: Several methods described previously for isolation and purification of soil DNA. Most of these protocols use combination of techniques or methods but the role and contribution of each individual method or component used is not clearly discussed. This study aims at analysing the effect of individual components used in extraction of DNA from soil and finally to optimize soil DNA isolation protocol and its validation by using 16SrDNA sequence analysis. Methods and Results: The soil was washed with anionic buffers before lysis step to reduce humic substances and release microbial cells from soil matrix, then the cells were lysed using combination of SDS, heating and vortexing and finally humic substances were removed using chemical flocculation. Pre-lysis washing of soil with 100 mmol l-1 Na2EDTA proved good for releasing microbial cells from soil matrix. Heating the soil sample at 75°C yielded good quantity (15.73 µg g-1 soil) DNA followed by 2% SDS (10.28 µg g-1 soil) and vortexing at 1400 rpm (8.94 µg g-1 soil). Combination of heating, SDS and vortexing yielded 25 µg DNA per gram of soil. Different concentrations of chemical flocculants like AlNH4(SO4)2, FeCl3, CaCl2 and MgCl2 were used to reduce humic substances. Flocculation with 100 mmol l-1 CaCl2 removed 5.2 mg humic substances without significant loss of DNA. 16S rDNA sequence analysis of DNA extracted from soil reveals presence of all the common soil bacterial species indicating the protocol is unbiased. Conclusion: Combination of chemical (SDS) and physical (heating and vortexing) methods yield good DNA whereas addition of enzyme (lysozyme) did not show significant effect on cell lysis. The digestion of isolated DNA with restriction enzyme and amplification of 16S rDNA using Taq DNA polymerase indicates the isolated DNA is pure enough for metagnomic analysis. 16Sr DNA sequencing of soil DNA indicates that this protocol can extract good quality and quantity DNA from range of bacteria present in soil varying in their cell wall composition. The optimised protocol is unbiased, very simple, does not need special equipments and many samples can be processed simultaneously.

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Simple lysis of bacterial cells for DNA-based diagnostics using hydrophilic ionic liquids

Cited by 36 publications

References 32 publications

Diversity of Colony Morphotypes, Biochemical Characteristics, and Drug Susceptibility Patterns of Burkholderia pseudomallei Isolated from Humans, Animals, and Environmental Sources in Thailand

Diversity of Colony Morphotypes, Biochemical Characteristics, and Drug Susceptibility Patterns of Burkholderia pseudomallei Isolated from Humans, Animals, and Environmental Sources in Thailand

Considerations and best practices in animal science 16S ribosomal RNA gene sequencing microbiome studies

Optimization of Soil DNA Extraction Protocol Using Na2EDTA, SDS, Heating, Vortexing and CaCl2 and Its Validation for Metagenomic Studies

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