Optimization of Surface-Enhanced Raman Spectroscopy Detection Conditions for Interaction between Gonyautoxin and Its Aptamer

This study aimed to optimize the detection conditions for surface-enhanced Raman spectroscopy (SERS) of single-stranded DNA (ssDNA) in four different buffers and explore the interaction between gonyautoxin (GTX1/4) and its aptamer, GO18. The influence of the silver colloid solution and MgSO4 concentration (0.01 M) added under four different buffered conditions on DNA SERS detection was studied to determine the optimum detection conditions. We explored the interaction between GTX1/4 and GO18 under the same conditions as those in the systematic evolution of ligands by exponential enrichment technique, using Tris-HCl as the buffer. The characteristic peaks of GO18 and its G-quadruplex were detected in four different buffer solutions. The change in peak intensity at 1656 cm−1 confirmed that the binding site between GTX1/4 and GO18 was in the G-quadruplex plane. The relative intensity of the peak at 1656 cm−1 was selected for the GTX1/4–GO18 complex (I1656/I1099) to plot the ratio of GTX1/4 in the Tris-HCl buffer condition (including 30 μL of silver colloid solution and 2 μL of MgSO4), and a linear relationship was obtained as follows: Y = 0.1867X + 1.2205 (R2 = 0.9239). This study provides a basis for subsequent application of SERS in the detection of ssDNA, as well as the binding of small toxins and aptamers.

PHIDA: A High Throughput Turbidimetric Data Analytic Tool to Compare Host Range Profiles of Bacteriophages Isolated Using Different Enrichment Methods

Bacteriophages are viruses that infect bacteria and are present in niches where bacteria thrive. In recent years, the suggested application areas of lytic bacteriophage have been expanded to include therapy, biocontrol, detection, sanitation, and remediation. However, phage application is constrained by the phage’s host range—the range of bacterial hosts sensitive to the phage and the degree of infection. Even though phage isolation and enrichment techniques are straightforward protocols, the correlation between the enrichment technique and host range profile has not been evaluated. Agar-based methods such as spotting assay and efficiency of plaquing (EOP) are the most used methods to determine the phage host range. These methods, aside from being labor intensive, can lead to subjective and incomplete results as they rely on qualitative observations of the lysis/plaques, do not reflect the lytic activity in liquid culture, and can overestimate the host range. In this study, phages against three bacterial genera were isolated using three different enrichment methods. Host range profiles of the isolated phages were quantitatively determined using a high throughput turbidimetric protocol and the data were analyzed with an accessible analytic tool “PHIDA”. Using this tool, the host ranges of 9 Listeria, 14 Salmonella, and 20 Pseudomonas phages isolated with different enrichment methods were quantitatively compared. A high variability in the host range index (HRi) ranging from 0.86–0.63, 0.07–0.24, and 0.00–0.67 for Listeria, Salmonella, and Pseudomonas phages, respectively, was observed. Overall, no direct correlation was found between the phage host range breadth and the enrichment method in any of the three target bacterial genera. The high throughput method and analytics tool developed in this study can be easily adapted to any phage study and can provide a consensus for phage host range determination.

Honey Mead Fermentation from Thai Stingless Bee (Tetragonula leaviceps) Honey using Ethanol Tolerant Yeast

Honey mead is a well-known conventional alcoholic beverage made by microbial fermentation of diluted honey. The selection of prospective yeasts for inoculation of honey-must with regard to honey mead quality determines the quality of mead production. The yeast consortium tolerant to ethanol stress was selected for this study using an enrichment technique. The activity of the invertase enzyme and the level of ethanol tolerance have been investigated. Thai stingless bee honey was used as a substrate, and the selected ethanol tolerant yeast consortium was used for mead fermentation. The results revealed that the PP03 had the highest invertase activity of 75.13±9.16 U/mL and the highest ethanol tolerance level of 12%. This is the first study using an ethanol tolerant yeast consortium to ferment honey mead from Thai stingless bee honey.

Isolation and Identification of Phenol-Degrading Bacteria from Oil-Contaminated Sites

This work is aimed at isolating and identifying phenol-degrading bacteria from oil-contaminated sites. Five soil samples from three auto-mechanic workshops within Katsina metropolis were collected. The samples were analyzed by selective enrichment technique, which resulted in the isolation of four bacterial species. The species were further subjected to the Vitek 2 compact microbiological system analysis. Cupriavidus pauculus, Pontoea spp, Proteus mirabilis 1 and Proteus mirabilis 2 were identified. Result from the present study showed that the bacteria could utilize phenol as their carbon source. Proteus mirabilis 1 and Proteus mirabilis 2 showed lower phenol degradation potential, under similar conditions. Cupriavidus pauculus and Pontoea sp. showed significant increases (p<0.05) in their optical densities. The optical density increment is strongly correlated with increase in colony forming units of the bacteria. This study further showed that the isolates could tolerate high phenol concentrations and may serve as strong putative isolates in bioremediation of phenol-contaminated sites.

High-Throughput and Automated Acoustic Trapping of Extracellular Vesicles to Identify microRNAs With Diagnostic Potential for Prostate Cancer

Molecular profiling of extracellular vesicles (EVs) offers novel opportunities for diagnostic applications, but the current major obstacle for clinical translation is the lack of efficient, robust, and reproducible isolation methods. To bridge that gap, we developed a microfluidic, non-contact, and low-input volume compatible acoustic trapping technology for EV isolation that enabled downstream small RNA sequencing. In the current study, we have further automated the acoustic microfluidics-based EV enrichment technique that enables us to serially process 32 clinical samples per run. We utilized the system to enrich EVs from urine collected as the first morning void from 207 men referred to 10-core prostate biopsy performed the same day. Using automated acoustic trapping, we successfully enriched EVs from 199/207 samples (96%). After RNA extraction, size selection, and library preparation, a total of 173/199 samples (87%) provided sufficient materials for next-generation sequencing that generated an average of 2 × 106 reads per sample mapping to the human reference genome. The predominant RNA species identified were fragments of long RNAs such as protein coding and retained introns, whereas small RNAs such as microRNAs (miRNA) accounted for less than 1% of the reads suggesting that partially degraded long RNAs out-competed miRNAs during sequencing. We found that the expression of six miRNAs was significantly different (Padj &lt; 0.05) in EVs isolated from patients found to have high grade prostate cancer [ISUP 2005 Grade Group (GG) 4 or higher] compared to those with GG3 or lower, including those with no evidence of prostate cancer at biopsy. These included miR-23b-3p, miR-27a-3p, and miR-27b-3p showing higher expression in patients with GG4 or high grade prostate cancer, whereas miR-1-3p, miR-10a-5p, and miR-423-3p had lower expression in the GG4 PCa cases. Cross referencing our differentially expressed miRNAs to two large prostate cancer datasets revealed that the putative tumor suppressors miR-1, miR-23b, and miR-27a are consistently deregulated in prostate cancer. Taken together, this is the first time that our automated microfluidic EV enrichment technique has been found to be capable of enriching EVs on a large scale from 900 μl of urine for small RNA sequencing in a robust and disease discriminatory manner.

A comparison of Salmonella survival and detection using an enrichment technique in dry and wet inoculated rendered chicken fat treated with sodium bisulfate

The differences in the recovery of Salmonella from rendered chicken fat treated with sodium bisulfate (SBS) when inoculated with a dry vs wet inoculum was evaluated. Food grade rendered chicken fat was inoculated with a dry and wet inoculum containing a Salmonella cocktail (S. Enteritidis, S. Heidelberg, and S. Typhimurium). In addition, the effect of an antimicrobial treatment (sodium bisulfate, SBS) against Salmonella spp. in both the aqueous and fat phase of the chicken fat was evaluated. The untreated control samples in the aqueous phase had a consistent level of Salmonella (~7 log) when both the dry and wet inoculum was used. In the SBS treated aqueous phase, Salmonella were not detectable after 6h when the wet inoculum was used; whereas, when using the dry inoculum, the Salmonella were not detectable at 24h. Salmonella were detected for up to 6h in the SBS treated fat phase when the dry inoculum was used as compared to 2h with the wet-inoculum. The 24h fat samples which failed to show growth on trypticase soy agar were enriched for Salmonella isolation followed by confirmation by polymerase chain reaction using primers for the invA gene. Both SBS treated and control samples from the dry inoculated, and the inoculated control from the wet inoculated rendered chicken fat tested positive for Salmonella. However, the SBS treated sample from the wet inoculated fat was negative for Salmonella. The use of dry SBS powder against dry Salmonella inoculum in the fat matrix caused only ~2.8 log reduction after 24h as compared to ~2.2 log reduction in the positive control. However, the recovery of Salmonella from untreated control fat was lower and was not different (P ≤ 0.05) than the SBS treated fat. The results suggest the development of potential VBNC states of Salmonella or the presence of injured cells in rendered chicken fat hence suggesting that testing should include an enrichment and appropriate molecular confirmation instead of agar plating alone

Characterization of hydrocarbon utilizing bacteria in waste engine oil-impacted sites

Changes in soil physicochemical properties and bacterial species present in soil contaminated with waste engine oil were evaluated at three auto-mechanical workshops in Uyo, Nigeria. This work was aimed at isolating and identifying hydrocarbon degrading bacteria from waste engine oil polluted soil, and assessing their hydrocarbon-utilizing ability. Waste engine oil pollution affected soils significantly with increases in soil physicochemical properties, and heterotrophic bacterial population counts. Eight bacterial species Corynebacterium kutscheri, Pseudomonas aeruginosa, Flavobacterium aquatile, Serratia odorifera, Micrococcus agilis, Staphylococcus aureus, Micrococcus luteus and Bacillus substilis were isolated by the selective enrichment technique and screened for hydrocarbon utilization capability in mineral salt media with 1% (v/v) waste engine oil as a sole carbon and energy source. The extent of bacterial growth observed was related to the ability of organisms to biodegrade hydrocarbons present in the medium bacterium species, which showed varying hydrocarbon utilization during the 15 days of incubation. Growth in hydrocarbon medium was the most efficient in cultures of Corynebacterium kutscheri. All isolates also showed variable emulsification ability, with Corynebacterium kutscheri, showing the highest ability. These results demonstrate the presence of indigenous bacteria in hydrocarbon-polluted soils and the potential toward the remediation of hydrocarbons.

Quantification of Naphthalene Dioxygenase (NahAC) and Catechol Dioxygenase (C23O) Catabolic Genes Produced by Phenanthrene-Degrading Pseudomonas fluorescens AH-40

Background: Petroleum polycyclic aromatic hydrocarbons (PAHs) are known to be toxic and carcinogenic for humans and their contamination of soils and water is of great environmental concern. Identification of the key microorganisms that play a role in pollutant degradation processes is relevant to the development of optimal in situ bioremediation strategies. Objective: Detection of the ability of Pseudomonas fluorescens AH-40 to consume phenanthrene as a sole carbon source and determining the variation in the concentration of both nahAC and C23O catabolic genes during 15 days of the incubation period. Methods: In the current study, a bacterial strain AH-40 was isolated from crude oil polluted soil by enrichment technique in mineral basal salts (MBS) medium supplemented with phenanthrene (PAH) as a sole carbon and energy source. The isolated strain was genetically identified based on 16S rDNA sequence analysis. The degradation of PAHs by this strain was confirmed by HPLC analysis. The detection and quantification of naphthalene dioxygenase (nahAc) and catechol 2,3-dioxygenase (C23O) genes, which play a critical role during the mineralization of PAHs in the liquid bacterial culture were achieved by quantitative PCR. Results: Strain AH-40 was identified as pseudomonas fluorescens. It degraded 97% of 150 mg phenanthrene L-1 within 15 days, which is faster than previously reported pure cultures. The copy numbers of chromosomal encoding catabolic genes nahAc and C23O increased during the process of phenanthrene degradation. Conclusion: nahAc and C23O genes are the main marker genes for phenanthrene degradation by strain AH-40. P. fluorescence AH-40 could be recommended for bioremediation of phenanthrene contaminated site.