Freeze-Thaw Pretreatment Can Improve Efficiency of Bacterial DNA Extraction From Meconium

Although the presence of live microbes in utero remains under debate, newborn gastrointestinal bacteria are undoubtedly important to infant health. Measuring bacteria in meconium is an ideal strategy to understand this issue; however, the low efficiency of bacterial DNA extraction from meconium has limited its utilization. This study aims to improve the efficiency of bacterial DNA extraction from meconium, which generally has low levels of microflora but high levels of PCR inhibitors in the viscous matrix. The research was approved by the ethical committee of the Xiamen Maternity and Child Health Care Hospital, Xiamen, China. All the mothers delivered naturally, and their newborns were healthy. Meconium samples passed by the newborns within 24 h were collected. Each sample was scraped off of a sterile diaper, transferred to a 5-ml sterile tube, and stored at −80°C. For the assay, a freeze-thawing sample preparation protocol was designed, in which a meconium-InhibitEX buffer mixture was intentionally frozen 1–3 times at −20°C, −80°C, and (or) in liquid nitrogen. Then, DNA was extracted using a commercial kit and sequenced by 16S rDNA to verify the enhanced bacterial DNA extraction efficiency. Ultimately, we observed the following: (1) About 30 mg lyophilized meconium was the optimal amount for DNA extraction. (2) Freezing treatment for 6 h improved DNA extraction at −20°C. (3) DNA extraction efficiency was significantly higher with the immediate thaw strategy than with gradient thawing at −20°C, −80°C, and in liquid nitrogen. (4) Among the conditions of −20°C, −80°C, and liquid nitrogen, −20°C was the best freezing condition for both improving DNA extraction efficiency and preserving microbial species diversity in meconium, while liquid nitrogen was the worst condition. (5) Three freeze-thaw cycles could markedly enhance DNA extraction efficiency and preserve the species diversity of meconium microflora. We developed a feasible freeze-thaw pretreatment protocol to improve the extraction of microbial DNA from meconium, which may be beneficial for newborn bacterial colonization studies.

Oral Microbiota and Salivary Levels of Oral Pathogens in Gastro-Intestinal Diseases: Current Knowledge and Exploratory Study

Various bi-directional associations exist between oral health and gastro-intestinal diseases. The oral microbiome plays a role in the gastro-intestinal carcinogenesis and fusobacteria are the most investigated bacteria involved. This paper aims to review the current knowledge and report the preliminary data on salivary levels of Fusobacterium nucleatum, Porphyromonas gingivalis and Candida albicans in subjects with different gastro-intestinal conditions or pathologies, in order to determine any differences. The null hypothesis was “subjects with different gastro-intestinal diseases do not show significant differences in the composition of the oral microbiota”. Twenty-one subjects undergoing esophagastroduodenoscopy or colonscopy were recruited. For each subject, a salivary sample was collected before the endoscopy procedure, immediately stored at -20°C and subsequently used for genomic bacterial DNA extraction by real-time PCR. Low levels of F. nucleatum and P. gingivalis were peculiar in the oral microbiota in subjects affected by Helicobater pylori-negative chronic gastritis without cancerization and future studies will elucidate this association. The level of C. albicans did not statistically differ among groups. This preliminary study could be used in the future, following further investigation, as a non-invasive method for the search of gastrointestinal diseases and associated markers.

Characterization of biliary microbiota dysbiosis in extrahepatic cholangiocarcinoma

Extrahepatic cholangiocarcinoma (CCA) accounts for 3% of digestive cancers. The role of biliary microbiota as an environment-related modulator has been scarcely investigated in CCA, and the putative impact of associated diseases has not been yet assessed. We characterized the biliary microbiota in CCA patients in order to identify a specific CCA-related dysbiosis. The biliary effluents were collected through an endoscopic retrograde pancreatic cholangiography (ERCP) examination involving 28 CCA and 47 patients with gallstones, herein considered as controls. The biliary effluents were submitted to bacterial DNA extraction and 16S rRNA sequencing, using Illumina technology. Overall, 32% of CCA and 22% of controls displayed another associated disease, such as diabetes, pancreatitis, inflammatory bowel disease, or primary sclerosing cholangitis. Such associated diseases were considered in the comparisons that were made. Principal coordinate analysis (PCoA) detected a significant disparity of biliary microbiota composition between CCA patients and controls without an associated disease. Amongst the most abundant phyla, Proteobacteria did not significantly differ between CCA patients and controls, whereas Firmicutes levels were lower and Bacteroidetes higher in CCAs’ biliary microbiota than in the controls’ microbiota. The most abundant genera were Enterococcus, Streptococcus, Bacteroides, Klebsiella, and Pyramidobacter in CCA’s biliary microbiota. Additionally, levels of Bacteroides, Geobacillus, Meiothermus, and Anoxybacillus genera were significantly higher in CCA patients’ biliary microbiota, without an associated disease, in comparison with controls. A specific CCA-related dysbiosis was identified as compared to controls independently from associated diseases. This suggests that a microorganism community may be involved in CCA pathogenesis.

Efficiency of chemical versus mechanical disruption methods of DNA extraction for the identification of oral Gram-positive and Gram-negative bacteria

Objective Clinical diagnostics often requires the detection of multiple bacterial species in limited clinical samples with a single DNA extraction method. This study aimed to compare the bacterial DNA extraction efficiency of two lysis methods automated with the MagNA-Pure LC instrument. The samples included five oral bacterial species (three Gram-positive and two Gram-negative) with or without human saliva background. Methods Genomic DNA (gDNA) was extracted from bacterial cultures by bead-beating lysis (BMP) or chemical lysis (MP), followed by automated purification and measurement by quantitative PCR. Results For pure bacterial cultures, the MP method yielded higher quantities of extracted DNA and a lower detection limit than the BMP method, except where the samples contained high numbers of Gram-positive bacteria. For bacterial cultures with a saliva background, no difference in gDNA extraction efficacy was observed between the two methods. Conclusions The efficiency of a bacterial DNA extraction method is not only affected by the bacterial cell wall structure but also by the sample milieu. The MP method provided superior gDNA extraction efficiency when the samples contained a single bacterial species, whereas either of the BMP and MP methods could be applied with similar efficiencies to samples containing multiple species of bacteria.

Microbiota Profile with Stunting Children in West Sumatera Province, Indonesia

BACKGROUND: Microbiota profile plays an important role in the growth of children. Recently, a number of microbiota profile studies have illustrated association with child stunting. AIM: Here, this study applied microbiota profile for stunting children in Indonesia to know a framework for future activities toward further characterization of microbiota profile contribution to stunting. METHODS: In this case–control study, we collected 96 samples with 48 stunting children and 48 non-stunting children in Pasaman and West Pasaman district as stunting locus areas in West Sumatra Province, Indonesia. All study subjects met the inclusion criteria: Children ≤3 years of age and they did not suffer from gastrointestinal disorders. Samples collected were then carried out by intestinal bacterial DNA extraction. All sequences were obtained from the bacterial 16S rRNA gene, which was amplified from microbial DNA extracted from a child fecal sample. Bioinformatic analysis of microbiota DNA sequencing results compared with the intestinal microbiota profile of infants. RESULTS: This study found in intestinal of stunting children identified 61 species of bacteria which were only found in the intestines of stunting children and not found in non-stunting children. The dominant bacteria in intestinal microbiota profile of Pasaman and West Pasaman district, West Sumatera Province, Indonesia, among stunting children were Firmicutes (47.52%), Proteobacteria (21.12%), and Bacteroidetes (16.15%). The high number of these microbiota associated with high amount of carbohydrate intake among stunting children than dietary protein. CONCLUSION: This study confirmed the role of microbiota profile in the incidence of stunting children.

BACTERIAL DNA EXTRACTION METHOD FOR THE DETECTION OF HELICOBACTER PYLORI FROM HUMAN FECAL SAMPLES

Objective: The work proposed the implementation of a method of DNA extraction for the detection of the pathogen from 50 stool samples. Methods: A method of DNA extraction with Chelex resin was applied and then detected by conventional polymerase chain reaction (PCR). Results: By PCR, 11 samples were positive for Helicobacter pylori. Conclusions: The Chelex extraction methodology allows obtaining DNA with quality necessary to be detected by PCR, making it a fast methodology for its diagnostic application.

High Quality of Bacterial DNA Extraction from Corbicula fluminea Tissue in Kelantan

Corbicula fluminea served as a popular traditional food in Kelantan, Malaysia. This clam is highly consumed by the local people from the last decade. However, there is lack of the study that obtain high quality of bacterial metagenome deoxyribonucleic acid (DNA) extraction from C. fluminea. Improper DNA extraction protocol always fails to extract a high integrity of bacterial DNA bands. This study compares the effectiveness and efficiency of conventional cetyltrimethylammonium bromide (CTAB) protocol, a commercial kit and modified CTAB protocol for bacterial DNA extraction from the soft tissue surface of raw C. fluminea. The instruments used to examine the quality of the extracted bacterial DNA were DeNovix DS-11 spectrophotometer, gel electrophoresis machine and UV transilluminator. The results showed that the bacterial DNA extracted from modified CTAB protocol the highest purity and integrity with the A₂₆₀/A₂₈₀ ratio of 1.92 ± 0.01 as well as the DNA band with minimum smear. This conclude that modified CTAB protocol is the best among the three extraction protocols for the bacterial extraction from the C. fluminea.

Development of a bacterial DNA extraction modular chip using a magnetic particle and portable vibration motor

The bacterial DNA was simply purified by magnetic particles with a portable vibration motor. To effectively extract DNA in the field, the 3D-printed device was employed with low electric power system.