DNA Extraction Research Articles

Impact of bariatric surgery on gut microbiota composition in obese patients compared to healthy controls

Bariatric surgery is vital for sustainable weight loss and metabolic improvement in obese individuals, but its effects on gut microbiota and their role in these benefits require further investigation. Investigate the temporal changes in gut microbiota in obese patients undergoing bariatric surgery (gastric sleeve gastrectomy or Roux-en-Y Gastric Bypass (RYGB)) compared to healthy controls, aiming to understand their role in weight loss and metabolic health improvement. A case-control study included 30 obese patients aged 65–95 undergoing bariatric surgery, and 18 matched healthy controls. Selection criteria were based on age, race, BMI, history of antibiotics, probiotics, and prebiotics usage. Stool samples were collected at baseline, three months, and six months post-surgery for DNA extraction and quantitative real-time PCR analysis to assess gut microbiota changes. Physical activity and dietary intake were evaluated using standardized questionnaires. Statistical analyses were performed using R. Post-surgery, patients showed significant reductions in weight and BMI, with changes in dietary habits and physical activity. Quantitative real-time PCR analysis revealed substantial alterations in bacterial groups such as Bacteroides and Fusobacterium. However, some groups showed no significant changes, indicating a complex interaction between gut microbiota and bariatric surgery. Notable correlations were found between body weight, BMI, and specific bacterial groups like the C. cluster IV and Lactobacillus, particularly in RYGB patients. Bariatric surgery significantly alters gut microbiota, aiding weight loss and metabolic regulation in obese patients. Understanding these changes is crucial for developing effective obesity management strategies, requiring further research to optimize outcomes.

Investigation of the Effects Of ACE Gene Polymorphism on Athletic Performance in Active Adult Males

Studying genes associated with exercise-related physiological systems and metabolic pathways has proven to be an effective method for identifying genetic markers associated with enhanced athletic performance. One of the extensively studied genes in genetics is "Angiotensin I-converting enzyme (ACE)" which has a significant role in regulating blood pressure, plasma volume, and the development of cardiac and skeletal muscle. The objective of this study was to determine the different results and genetic variations in the effects of ACE gene polymorphisms on the performance of the 30m sprint test and the arrowhead agility drill test in active adult males after a 6-week training program. Following the manufacturer's instructions, genomic DNA was extracted from participant mouth swab samples using the Buccalyse DNA Extraction Kit from Isohelix. The Statistical Package for the Social Sciences (SPSS) 25 application was used to perform statistical analysis on the research data. The findings indicate that 45.7% of the participants possess the I/D genotype, 31.4% possess the I/I genotype, and 22.9% possess the D/D genotype. There was no notable disparity in the performance of the arrowhead agility test and the 30 m sprint test among the different genotype groups (p<0.05). Our analysis revealed a lack of association between genetic variants and the values obtained from performance measurements in the test outcomes. Due to the involvement of various genes and variables, further research is needed to improve the predictability of approaches to ACE gene variations, considering their performance-enhancing benefits.

Frequency of Bacteria Causing Exudative Diarrhea in Children Less Than Five Years Old in a Tertiary Hospital, Iran

Background: Exudative diarrhea is a significant global public health issue, particularly affecting children under the age of 5. Identifying the cause of diarrhea is crucial for epidemiological surveillance and, in some cases, for ensuring appropriate treatment for patients. Objectives: The aim of this study was to investigate the frequency of bacteria responsible for exudative diarrhea in samples from children under five years old in a tertiary hospital in Iran. Methods: In this multicenter cross-sectional descriptive study, 104 children with exudative diarrhea who were referred to Mofid Children's Hospital in Tehran, as well as hospitals in Hamedan, Ardebil, and Bandar Abbas, from December 2020 to March 2022 were enrolled. DNA extraction was performed using a commercial kit, and the identification of various causative bacteria was conducted through conventional and real-time PCR. Descriptive and inferential statistics were analyzed using SPSS version 22 software. Results: Most of the children with exudative diarrhea were under 12 months old (31%) or between 12 and 24 months old (22%). Boys made up 66% of the participants. Additionally, 70% of the children had a fever, and 58% experienced vomiting. Furthermore, 56% of the patients were dehydrated. The most prevalent bacterial causes of exudative diarrhea were Shigella spp., followed by Salmonella spp., Campylobacter spp., Clostridium difficile, E. coli-Stx1, and E. coli-Stx2. Conclusions: The findings indicated that *Shigella* spp. was the leading cause of diarrhea in children under five years old. The most common signs and symptoms associated with exudative diarrhea were fever and vomiting, which physicians should consider in their diagnostic and treatment processes.

Petrous bones versus tooth cementum for genetic analysis of aged skeletal remains.

A proper sampling strategy is important to obtain sufficient DNA for successful identification of aged skeletal remains. The petrous bone is the highest DNA-yielding bone in the human body. Because DNA extraction from the petrous bone is very destructive, the demand for other DNA sources is significant. When investigating aged skeletal remains, teeth are usually preserved, and recent studies have shown that DNA in teeth can be best preserved in the dental cementum that surrounds the surface of the tooth root. To extract DNA from the surface of the tooth root, a nondestructive method without grinding was used. Petrous bones and teeth from 60 archaeological adult skeletons were analyzed. The DNA yield, degree of DNA degradation, and STR typing success were compared, and the results showed higher DNA yield and higher amplification success in petrous bones, despite higher degradation of petrous bones' DNA. The greater success of petrous bones is associated with poorly preserved DNA in a quarter of the teeth analyzed. When teeth with badly preserved DNA were excluded from the statistical analysis, no differences in the success of STR loci amplification were observed even if DNA yield was higher in petrous bones, which can be explained by greater degradation of petrous bones' DNA. When teeth are well preserved, they can be used for genetically analyzing aged skeletal remains instead of petrous bones, and a rapid nondestructive extraction method can be applied to shorten the identification process and to physically preserve the biological specimen.

Surgical Face Mask as an Air Sampling Device for Assessing Personal Exposure to Airborne Antimicrobial Resistance Gene-Bearing Bacteria.

In this study, we assessed the feasibility of using a surgical face mask as a sampling device to collect airborne antimicrobial resistance genes (ARGs). The method entails collection of ARG-bearing microbes on face masks, followed by their DNA extraction and quantification by qPCR analysis. Analysis of masks worn by volunteers showed an apparent mask wearing time-dependent accumulation of 16S rRNA gene and select ARGs trapped on masks, highlighting the applicability of the method in monitoring personal ARG exposure through inhalation. The sampling method was then validated for reproducibility and compared with a filter-based sampling method before application in different environmental settings to further assess personal exposure to ARGs. In comparison with the filter-based method, our new sampling method does not require a sampling pump and is more user-friendly. More importantly, it records ARG exposure down to the personalized level; thus, it may be used in routine monitoring of occupational exposure and surveillance of ARG concentrations in indoor environments.

Reliable RNA-seq analysis from FFPE specimens as a means to accelerate cancer-related health disparities research.

Whole transcriptome sequencing (WTS/ RNA-Seq) is a ubiquitous tool for investigating cancer biology. RNA isolated from frozen sources limits possible studies for analysis of associations with phenotypes or clinical variables requiring long-term follow-up. Although good correlations are reported in RNA-Seq data from paired frozen and formalin fixed paraffin embedded (FFPE) samples, uncertainties regarding RNA quality, methods of extraction, and data reliability are hurdles to utilization of archival samples. We compared three different platforms for performing RNA-seq using archival FFPE oropharyngeal squamous carcinoma (OPSCC) specimens stored up to 20 years, as part of an investigation of transcriptional profiles related to health disparities. We developed guidelines to purify DNA and RNA from FFPE tissue and perform downstream RNA-seq and DNA SNP arrays. RNA was extracted from 150 specimens, with an average yield of 401.8 ng/cm 2 of tissue. Most samples yielded sufficient RNA reads >13,000 protein coding genes which could be used to differentiate HPV-associated from HPV-independent OPSCCs. Co-isolated DNA was used to identify patient ancestry. Utilizing the methods described in this study provides a robust, reliable, and standardized means of DNA & RNA extraction from FFPE as well as a means by which to assure the quality of the data generated.

Nucleotide sequence variants, gene expression and serum profile of immune and antioxidant markers associated with bacterial diarrhea susceptibility in Barki lambs

BackgroundDespite the fact that diarrhea is more accurately described as a clinical symptom than a disease. Diarrhea is one of the most important issues in ovine medicine, particularly in lambs, and because of high morbidity and mortality rate, sluggish growth performance, and veterinary costs, it is believed to be a major source of economic loss. Salmonella and enterotoxigenic Escherichia coli are the most common and commercially significant agents responsible for diarrhea.ObjectiveThe objective of this study was to monitor the nucleotide sequence variations, gene expression, serum inflammatory and oxidative stress biomarkers in diarrheic lambs. Another aim was to identify different pathotypes and virulence genes of Salmonella and E. coli causing diarrhea.MethodologyBlood samples were taken from 50 Barki who were diarrheal and 50 who appeared to be healthy, and then divided in 3 portions, with EDTA added to the first part for CBC, DNA and RNA extraction. The second sample received 5000 I.U. of heparin calcium, and a clean plain tube was used for the third component. The second and third sections were centrifuged to extract serum and plasma until the biochemical and immunological analysis was completed. Fecal samples were collected for bacteriological examination, and the bacteria were identified by PCR analysis. PCR-DNA sequencing was conducted for immune (SELL, JAK2, SLC11A1, IL10, FEZF1, NCF4, LITAF, SBD2, NFKB, TNF-α, IL1B, IL6, LGALS, and CATH1), antioxidant (SOD1, CAT, GPX1, GST, Nrf2, Keap1, HMOX1, and NQO1), and GIT health (CALB1, GT, and MUC2) genes in healthy and diarrheic lambs.ResultsVirulent genetic markers of pathogenic characteristics of E. coli (astA, Vt2e (Stx2e), CFA/I, groES and luxS) and Salmonella (invA, SopB, bcfC and avrA) were detected in all diarrheic lambs. PCR-DNA sequencing of immune, antioxidant and intestinal health genes found eleven single nucleotide polymorphisms (SNPs) linked to either diarrhea resistance or susceptibility in Barki lambs. Transcript levels of immune, antioxidant, and GIT health (CALB1, GT, and MUC2) genes varied between healthy and diarrheic lambs. Nucleotide sequence variation of the genes under inquiry between reference sequences in GenBank and those of the animals under investigation verified all identified SNPs. Significant (P = 0.001) erythrocytosis, neutrophilic leukocytosis, with lymphocytopenia were observed in diarrheic lambs. Significant (P = 0.001) increases in serum IL-1α, IL-1β, IL-6, TNF-α (90.5 ± 1.7, 101.8 ± 1.7, 72.3 ± 6.6, 71.26 ± 4.89 Pg/ml, respectively), serum Fb, Cp, Hp, SAA (230.7 ± 12.4 mg/dl, 6.5 ± 0.07 mg/dl, 2.5 ± 0.09 g/dl, 7.4 ± 0.4 mg/L, respectively), free radicals (MDA, NO), cortisol (6.91 ± 0.18 μg/dl) and growth hormone, with significant (P = 0.001) decreases in serum IL-10 (81.71 ± 1.05 Pg/ml), antioxidants (CAT, GPx), insulin, triiodothyronine (T3) and thyroxine (T4) in diarrheic lambs.ConclusionsThe study's findings provided credence to the theory that marker-assisted selection (MAS) could be used to predict and prevent diarrhea in Barki sheep by selecting lambs based on SNPs in genes linked to inflammation, antioxidants, and intestinal health. In order to establish an efficient management protocol and determine the most susceptible risk period for disease occurrence, gene expression profiles of the genes under investigation, pro-inflammatory cytokines and acute phase proteins may also be utilized as proxy biomarkers for lamb enteritis.

First report of Sclerotinia minor causing soft rot of Scabiosa atropurpurea in California.

In May of 2019, Scabiosa atropurpurea samples with brown discoloration, soft rot of the crown and lower stem, with presence of white mycelium and black sclerotia (Supp. Fig. 1A, B) were collected from a 0.10 ha open field diversified cut flower production in San Luis Obispo County, CA. Approximately 30 to 40% of the scabiosa crop planted in a quarter of the field, exhibited symptoms. Symptomatic crowns and lower stems from five plants were surface disinfested by rinsing in 0.1% Tween 20, soaking in 70% ethanol for 30 s, 1% sodium hypochlorite for 2 min and sterile water. Disinfested tissue was placed in 1/10 potato dextrose agar (PDA) and incubated at 20°C (12 h photoperiod). Resulting colonies (n = 5) formed abundant white mycelia, with black sclerotia formed on the outer edge of the plates after two weeks (Suppl. Fig. 1C). Sclerotia (n = 50) had an average size of 1.6 (± 0.19) mm in diameter. Morphological identification resulted in Sclerotinia sp. (Hao et al., 2003). The pathogen was further identified by DNA extraction of two hyphal tipped isolates, followed by amplification and sequencing of the rDNA internal transcribed spacer (ITS) region, ITS1/ITS4 (White et al. 1990), calmodulin (CaM), CAL-228F/CAL-737R (Carbone and Kohn, 1999), and DNA replication licensing factor Mcm7 Mcm7-709for/Mcm7-1348rev (Schmitt et al., 2009). NCBI BLAST searches with consensus sequences for each maker revealed 99 to 100% identity with S. minor ex-types for all loci (Supp. Table 1). A maximum parsimony multilocus phylogenetic analysis clustered Californian isolates with reference strains of S. minor (Supp. Fig. 1F). Sequences were deposited in GenBank (Supp. Table 1). Pathogenicity tests were conducted with isolate CS435, which was transferred onto PDA plates and incubated at 20°C for one week. Inoculum consisted of CS435 infested PDA plugs (1 cm3). In the greenhouse, the experiment was set as a complete randomized design and observed for six weeks. Fourteen-week-old scabiosa 'Merlot Red' grown in 3.78 L pots (n = 6), were inoculated by wounding plants at 0.5 cm above the crowns with a 1 mm probe. Inoculum was placed directly on top of the wound and was secured with parafilm. Negative control plants (n = 6) were wounded as above and inoculated with PDA plugs. In experiment 1(19.4 (± 3) °C, RH 46.9), 83% of plants exhibited yellowing of the lower leaves and wilting at one week post inoculation (wpi). Symptoms progressed over time until wilting, major leaf and stem necrosis, was observed in all inoculated plants (Supp. Fig 1E, D). Plant mortality incidence at five wpi was 83%. Pathogen signs including white mycelia and black sclerotia were also observed. In experiment 2 (20.0 (± 10) °C, RH 39.6), 66% of the total plants were symptomatic at five wpi: 33% exhibited yellowing of the lower leaves and wilting, and 33% of plants died. Disease did not develop in non-inoculated plants in either experiment. S. minor was successfully reisolated from surface disinfested tissue of at least 50% (Exp. 1) and 100% (Exp. 2) of inoculated plants, yielding identical sequences to those of the inoculated isolate. S. minor is a soil-borne pathogen that infects tomato, lettuce, brassica, and sunflower crops in California. This study stablishes S. minor as a new soil-borne pathogen of Scabiosa in California. Soil-borne pathogens are a recurrent issue in the cut flower industry, characterizing new pathogens in these crops can inform crop advisors and disease diagnostician to improve disease management in the ornamental industry.

Distribution of genotypes by the growth diferential factor 9 (GDF-9) gene locus in the romanov breed sheep population

The FecG gene, also known as GDF-9 (growth factor differentiation factor 9), plays an important role in regulating reproductive function in mammals. This paper presents the results of a study of a point mutation in the differential growth factor gene in Romanov breed sheep. Mutations in exon 1 of the GDF-9 gene can lead to disturbances in ovarian development and ovulation, resulting in infertility or reduced reproductive capacity in animals. The object of the study was Romanov breed sheep in a sample of 30 individuals. For DNA extraction, venous blood samples were taken from the jugular vein. In Romanov breed sheep for the GDF-9 gene, it was found that at the G1 locus of this gene there are two alleles – G and A. Three genotypes were also identified: GG (254 bp/117 bp), AG (410bp/254/117 bp) and AA (410 bp/410 bp). The observed χ2 criterion was less than expected, therefore, the empirical and theoretical frequencies do not differ significantly, and deviations from the distribution according to the Hardy-Weinberg law are not observed. The observed heterozygosity index (Ho) was 0.27. Allele frequencies were 0,8 for G and 0,2 for A. For genotypes the following were obtained: GG – 0,66, GA – 0,27 and AA – 0,07.It can be noted that the data on the frequencies of alleles and genotypes in the population of Romanov breed sheep differ from the results for the Kulunda fine-wool, Dagestan mountain, Salsk, Lacaune and Merino breeds. The research results can be used to monitor changes in allele frequencies and genotypes in sheep during the selection process.

Mycobacterium tuberculosis complex sample processing by mechanical lysis, an essential step for reliable whole genome sequencing

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.

Biodiversity of Aspergillus Species and Their Mycotoxin Production Potential in Dry Meat.

This study aimed to examine fungi diversity in dried beef meat sold in Ekiti State, characterize the isolated fungi, and determine the aflatoxin-producing ability of the Aspergillus fungi in the samples. Dried beef meat was collected from different markets in Ekiti State and screened for the presence of filamentous fungi using molecular methods. Samples were cultured aseptically on potato dextrose agar (PDA) for fungi isolation, and molecular identification was performed using DNA extraction, Polymerase chain Reaction (PCR), ITS-1/ITS-4 primer pair, and nucleotide sequencing. The results obtained indicated a range of filamentous fungi genera including Aspergillus, Rhizopus, Penicillium, Fusarium, Cladosporium, Alternaria, and other fungi species contaminating the dried meat at (43%), (42%), (3%), (2%), (2%), (1%), and (7%), respectively. High incidences were recorded for Aspergillus flavus, Aspergillus niger, and Aspergillus fumigatus in most of the screened samples. Aspergillus flavus accounted for (24.7%) of all the Aspergillus species isolated with the presence of the gene needed for aflatoxin production. The occurrences of these filamentous fungal species pose a cause for concern, as most of these fungal species are known producers of certain toxic substances. Maximum likelihood phylogenetic analysis showed a high similarity index score, which indicated a good relationship between isolated Aspergillus Species and the closely related strains from GenBank, isolated from different sources and countries. The implication of this study is that consumer health may be at risk through exposure to contaminated dried meat.

Clinical and genetic analysis of a Chinese pedigree affected with type 2 Long QT syndrome due to variant of KCNH2 gene

To explore the clinical and genetic etiology of a Chinese pedigree affected with type 2 Long QT syndrome (LQTS). A pedigree with type 2 LQTS presented at Fuwai Central China Cardiovascular Hospital on August 23, 2019 was selected as the study subject. Peripheral blood samples were collected from the proband and her parents. Following extraction of genomic DNA, whole exome sequencing (WES) was carried out for the proband, and candidate variant was screened through functional annotation and protein-protein interaction (PPI) analysis. Sanger sequencing was conducted to verify the pathogenicity of candidate variant. This study was approved by the Fuwai Central China Cardiovascular Hospital (Ethics No. 2019-15). WES revealed that the proband has harbored a missense variant of the KCNH2 gene, namely c.1478A>G (p.Tyr493Cys), which was confirmed by Sanger sequencing to have inherited from her father. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was classified as likely pathogenic (PM2_supporting+PM5+PP3+PP4). The KCNH2 gene c.1478A>G (p.Tyr493Cys) variant probably underlay the type 2 LQTS in this pedigree.

Barcode 100K Specimens: In a Single Nanopore Run.

It is a global priority to better manage the biosphere, but action must be informed by comprehensive data on the abundance and distribution of species. The acquisition of such information is currently constrained by high costs. DNA barcoding can speed the registration of unknown animal species, the most diverse kingdom of eukaryotes, as the BIN system automates their recognition. However, inexpensive sequencing protocols are critical as the census of all animal species is likely to require the analysis of a billion or more specimens. Barcoding involves DNA extraction followed by PCR and sequencing with the last step dominating costs until 2017. By enabling the sequencing of highly multiplexed samples, the Sequel platforms from Pacific BioSciences slashed costs by 90%, but these instruments are only deployed in core facilities because of their expense. Sequencers from Oxford Nanopore Technologies provide an escape from high capital and service costs, but their low sequence fidelity has, until recently, constrained adoption. However, the improved performance of its latest flow cells (R10.4.1) erases this barrier. This study demonstrates that a MinION flow cell can characterise an amplicon pool derived from 100,000 specimens while a Flongle flow cell can process one derived from several thousand. At $0.01 per specimen, DNA sequencing is now the least expensive step in the barcode workflow.

Oxygen exposure decreases the yield of high-molecular-weight DNA from some anaerobic bacteria and bacterial communities during DNA extraction

ObjectivesThe central challenge in third-generation sequencing lies in meeting the requirements for DNA quality (integrity and purity) and quantity. Therefore, novel improvements in DNA extraction methods are needed to satisfy these requirements. We reasoned that in anaerobic microbial communities, the presence of certain strict anaerobes containing oxygen-activated DNase activity might contribute substantially to the poor integrity of extracted metagenomic DNA (or genomic DNA from some pure cultures) if exposed to air. MethodsTo test this hypothesis, we developed an enhanced genomic and metagenomic DNA isolation technique that we applied to a specifically chosen set of both strict and aerotolerant anaerobes, as well as to the hindgut microbiota of a herbivorous marine fish. ResultsConsidering the quality (or degradation) of extracted DNA obtained under anaerobic versus aerobic conditions, we found that DNA extracted aerobically from cells of some strict anaerobes showed more degradation of high molecular weight DNA than analogous preparations under anaerobic conditions. In contrast, with the selected aerotolerant anaerobes, no discernible difference was found between the molecular sizes of DNA extracted aerobically and anaerobically. Metagenomic DNA extracted from the fish hindgut microbiota showed higher yields and better quality under anaerobic conditions compared to aerobic conditions. ConclusionOur study effectively demonstrates the advantages of our improved extraction protocol in anaerobic conditions. This is evident through the improved quality of extracted DNA. Such findings may be valuable for studies, especially metagenomic studies, where the quality and quantity of DNA are crucial for downstream analysis.

Demonstration of potential DNA contamination introduced by laboratory consumables using Fluorescein

The development of increasingly efficient DNA extraction and profiling kits has increased the amount of allelic information obtained from trace DNA samples, but also inadvertently, increased the detection of DNA contamination. This study aimed to evaluate the potential of DNA transfer using fluorescein, fluorescent under an alternate light source, in the use of a range of forensically relevant DNA profiling consumables. An evaluation of two pre-lysis methods adopting three different sample tubes, some with deliberate seal damage, showed the PrepFiler™ Automated Forensic DNA Extraction Kit caused leakage and crusting when the rim of the PrepFiler™ LySep column was compromised, but no leakage was observed under the same conditions using the Investigator STAR Lyse&Prep kit. The AutoLys tube showed minimal leakage using the PrepFiler™ chemistry. A DNA extract tube with an external thread, similar to the AutoLys tube, showed no leakage after fridge or freezer storage. However, it highlighted that a centrifugal spin does not guarantee all the DNA will pool at the base of the tube. A comparison of adhesive plate sealing films to 8-well strip caps for sealing 96-well PCR plates showed the adhesive plate sealing films presented a lower risk of DNA transfer, largely due to the adhesion of dispersed liquid on the sticky surface of the film. Overall, this study highlighted a number of variables that may be considered in the development of more refined contamination minimisation protocols in respect to increased sensitivities of DNA profiling.

Environmental monitoring of Listeria monocytogenes contamination in dairy processing facilities combining culturing technique and molecular methods

Objectiveof the present study was to investigate Listeria contamination in Sardinian sheep cheese-making plants using culture and DNA-based methods. Food and environmental samples were collected from different surfaces of 14 facilities. Samples were collected using pre-moistened sponge swabs. Each site was sampled using two sponges used one for microbiological detection and the other for extraction of total DNA. The DNA was submitted to real time PCR targeting the prs gene for Listeria spp., the gene inlA for Listeria monocytogenes and the gene iap for Listeria innocua. DNA metabarcoding was also conducted on the total DNA. Overall were collected 254 environmental and 36 food samples. The prevalence of Listeria spp. and Listeria monocytogenes was 18.6% and 10.0%, respectively. Samples enrichment coupled with quantitative PCR (qPCR) showed a greater sensitivity than the conventional culture method with an overall prevalence of 12.1% for L. monocytogenes. Both, qPCR and ribosomal DNA-metabarcoding conducted on total DNA extracted from the sponges performed poorly in comparison to the culture-based method detecting the presence of Listeria genus in as little as 7 (2.4%) and 4 (1.4%) samples respectively. The present study supports the use of a culture enrichment coupled with qPCR for routine monitoring of contamination in food premises.

New Refined Experimental Analysis of Fungal Growth in Degraded Bio-Based Materials

When exposed to different building environmental conditions, bio-composite materials, such as hemp mortars, represent a risk of mold proliferation. This later plays a critical role in the biodeterioration of the materials when their physical properties are locally modified by the natural aging process. The primary objectives of the present work are first to assess the evolution of the surface of contaminated mortar; second, to investigate an accurate DNA extraction method that could be used for both bio-composite mortars and their fiber sources collected in situ; then, to understand the process of the proliferation of mold strains on both hemp shives and hemp mortar; and finally, to compare mold strains present in these phases to show their relationship to mold contamination and their impact on human health. In situ hemp mortar contamination behavior was investigated in the region of Pau (France) two months after hemp mortar application in extreme conditions (high humidity, low temperature, no aeration), which did not match the standard conditions under which hemp mortar must be used. The SEM observations and FTIR and pH analyses highlighted the decrease in pH level and the presence of organic matter on the mortar surface. DNA sequencing results showed that hemp shives were the main source of fungal contamination of hemp mortar. A mold population analysis showed that the most dominant phylum was Ophistokonta, which represented 83.6% in hemp shives and 99.97% in hemp mortar. The Acrostalagmus genus representatives were the most abundant, with 42% in hemp shives and 96% in hemp mortar. The interconnection between the mold strain characteristics (particularly the ability to grow in extreme environments) and the presence of hemp mortar was emphasized.

Phage communities in household-related biofilms correlate with bacterial hosts

The average American spends 93% of their time in built environments, almost 70% of that is in their place of residence. Human health and well-being are intrinsically tied to the quality of our personal environments and the microbiomes that populate them. Conversely, the built environment microbiome is seeded, formed, and re-shaped by occupant behavior, cleaning, personal hygiene and food choices, as well as geographic location and variability in infrastructure. Here, we focus on the presence of viruses in household biofilms, specifically in showerheads and on toothbrushes. Bacteriophage, viruses that infect bacteria with high host specificity, have been shown to drive microbial community structure and function through host infection and horizontal gene transfer in environmental systems. Due to the dynamic environment, with extreme temperature changes, periods of wetting/drying and exposure to hygiene/cleaning products, in addition to low biomass and transient nature of indoor microbiomes, we hypothesize that phage host infection in these unique built environments are different from environmental biofilm interactions. We approach the hypothesis using metagenomics, querying 34 toothbrush and 92 showerhead metagenomes. Representative of biofilms in the built environment, these interfaces demonstrate distinct levels of occupant interaction. We identified 22 complete, 232 high quality, and 362 medium quality viral OTUs. Viral community richness correlated with bacterial richness but not Shannon or Simpson indices. Of quality viral OTUs with sufficient coverage (614), 532 were connected with 32 bacterial families, of which only Sphingomonadaceae, Burkholderiaceae, and Caulobacteraceae are found in both toothbrushes and showerheads. Low average nucleotide identity to reference sequences and a high proportion of open reading frames annotated as hypothetical or unknown indicate that these environments harbor many novel and uncharacterized phage. The results of this study reveal the paucity of information available on bacteriophage in indoor environments and indicate a need for more virus-focused methods for DNA extraction and specific sequencing aimed at understanding viral impact on the microbiome in the built environment.

Assessment of Skimmed Milk Flocculation for Bacterial Enrichment from Water Samples, and Benchmarking of DNA Extraction and 16S rRNA Databases for Metagenomics.

Water samples for bacterial microbiome studies undergo biomass concentration, DNA extraction, and taxonomic identification steps. Through benchmarking, we studied the applicability of skimmed milk flocculation (SMF) for bacterial enrichment, an adapted in-house DNA extraction protocol, and six 16S rRNA databases (16S-DBs). Surface water samples from two rivers were treated with SMF and vacuum filtration (VF) and subjected to amplicon or shotgun metagenomics. A microbial community standard underwent five DNA extraction protocols, taxonomical identification with six different 16S-DBs, and evaluation by the Measurement Integrity Quotient (MIQ) score. In SMF samples, the skimmed milk was metabolized by members of lactic acid bacteria or genera such as Polaromonas, Macrococcus, and Agitococcus, resulting in increased relative abundance (p < 0.5) up to 5.0 log fold change compared to VF, rendering SMF inapplicable for bacterial microbiome studies. The best-performing DNA extraction protocols were FastSpin Soil, the in-house method, and EurX. All 16S-DBs yielded comparable MIQ scores within each DNA extraction kit, ranging from 61-66 (ZymoBIOMICs) up to 80-82 (FastSpin). DNA extraction kits exert more bias toward the composition than 16S-DBs. This benchmarking study provided valuable information to inform future water metagenomic study designs.

Hildenbrandia ardreae sp. nov. (Rhodophyta), recorded before as Hildenbrandia dawsonii (nom. inval.)

Four species of the red algal genus Hildenbrandia have been recorded from Pacific Mexico: H. dawsonii reported on the west coast of Baja California and Jalisco; H. occidentalis with one record from the west coast of Baja California; H. prototypus occurring all along the coast, including the Gulf of California; and the most common and abundant species, H. rubra from Pacific Baja California to Oaxaca, in tropical Pacific Mexico. All four species have been studied morphologically and molecularly using representative specimens. The molecular status regarding the phylogenetic position of H. dawsonii in relation to other species is unclear. H. dawsonii nomenclatural status was analyzed, and additional material from the University Herbaria at UC-Berkekley (UC) was reviewed. Hildenbrandia dawsonii is an invalid name because its alleged basionym (Hildenbrandia canariensis var. dawsonii) is invalid. Therefore, a new species is described: Hildenbrandia ardreae sp. nov., selecting a Dawson specimen from Punta Norte, Cedros Island, Baja California, as the holotype. Furthermore, morphological characters, once used as diagnostic at the species level, are considered irrelevant. The new holotype proposed in this study shows the same characteristics stated by F. Ardré in her original publication (1959) and could be a potential source for DNA extraction for comparative studies.