Bacterial Extraction Research Articles

Correlation between chronic periodontitis and rheumatoid arthritis: A periodontist perspective

In this study, we evaluate the relationship between rheumatoid arthritis (RA) and chronic periodontitis on the basis of clinical attachment present and severity of attachment loss in both the cases. First of all Diagnosis of rheumatoid arthritis and chronic periodontitis was performed, thereafter bacterial DNA extraction from blood serum sample and subgingival dental plaque of each group through PCR and later DNA purification through Spin protocol was performed, oligonucleotide primer was used to detect t.forcythia and PCR amplification was done to detect T. Denticola for both the groups .PBDNA was detected in both SGP and serum samples of both the groups. In SGP samples, Tannerella forsythia was more frequently detected as compared to serum samples of both the groups. In result theclinical attachment Level (CAL) was observed to be higher in RA group as compared to CP group. Comparison of CAL according to severity was also observed in both the groups which suggested that RA group has mild periodontitis as compared to CP group in which moderate to severe periodontitis was seen, Detection of periodontal bacterial DNA by PCR assay PBDNA was detected in both SGP and serum samples. In SGP samples, Tannerella forsythia was more frequently detected as compared to serum samples of both the groups. So these are two common chronic inflammatory diseases with a similar host-mediated pathogenesis. Current evidence suggests that an association exists between periodontitis and RA. Well-designed multicenter longitudinal clinical trials and studies with sufficient sample sizes are needed to ascertain the relationship between these two diseases and whether periodontal treatment can reduce the severity of RA or prevent its onset.

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.

Localized surface plasmon-enhanced electrochemiluminescence biosensor for rapid, label-free, and single-step detection of broad-spectrum bacteria using urchin-like Au and Ag nanoparticles

In this study, localized surface plasmon-enhanced electrochemiluminescence (LSP-ECL) biosensor, a new ECL enhancement way for S2O82−/O2 system, was constructed based on urchin-like Au and Ag nanoparticles (AgNPs) for fast, one-step, and label-free detection of broad-spectrum bacteria. We found plasmonic metals with small diameters contributed to the ECL enhancement. Moreover, relying entirely on the physical puncture of urchin-like Au, the bacteria could be captured to the electrode surface without the need for target recognition agents. The single-step LSP-ECL biosensor exhibited high sensitivity in the range of 102–108 CFU/mL bacteria with the detection limit as low as 52 CFU/mL, meanwhile possessing a short detection time of 30 min. What’s more, the LSP-ECL biosensing strategy achieved detection of S.aureus present in plasma samples without involving bacterial culture and nucleic acid extraction. Considering the merits of label-free, short assay time, and one-step operation, the developed LSP-ECL strategy provides a versatile and culture-free platform for pathogenic bacteria detection.

Modified Spin Column-Based RNA Extraction Methods of Staphylococcus aureus using PureLink® RNA Mini Kit and Basic Laboratory Instrument

RNA extraction is an important process before gene expression assessment at the transcriptomic level. RNA is a sensitive material to environmental factors such as temperature and contaminants, so the RNA extraction process generally requires sophisticated and expensive laboratory instruments. In this study, we extract RNA from Staphylococcus aureus bacteria using the PureLink® RNA Mini Kit. The instruments used in this study are basic instruments such as a hand homogenizer and non-thermal centrifuge. The results of RNA extraction were visualized using agarose gel electrophoresis. These results indicate that bacterial RNA extraction can be performed using the PureLink® RNA Mini Kit even with inexpensive basic laboratory instruments.

OralDisk: A Chair-Side Compatible Molecular Platform Using Whole Saliva for Monitoring Oral Health at the Dental Practice.

Periodontitis and dental caries are two major bacterially induced, non-communicable diseases that cause the deterioration of oral health, with implications in patients’ general health. Early, precise diagnosis and personalized monitoring are essential for the efficient prevention and management of these diseases. Here, we present a disk-shaped microfluidic platform (OralDisk) compatible with chair-side use that enables analysis of non-invasively collected whole saliva samples and molecular-based detection of ten bacteria: seven periodontitis-associated (Aggregatibacter actinomycetemcomitans, Campylobacter rectus, Fusobacterium nucleatum, Prevotella intermedia, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola) and three caries-associated (oral Lactobacilli, Streptococcus mutans, Streptococcus sobrinus). Each OralDisk test required 400 µL of homogenized whole saliva. The automated workflow included bacterial DNA extraction, purification and hydrolysis probe real-time PCR detection of the target pathogens. All reagents were pre-stored within the disk and sample-to-answer processing took < 3 h using a compact, customized processing device. A technical feasibility study (25 OralDisks) was conducted using samples from healthy, periodontitis and caries patients. The comparison of the OralDisk with a lab-based reference method revealed a ~90% agreement amongst targets detected as positive and negative. This shows the OralDisk’s potential and suitability for inclusion in larger prospective implementation studies in dental care settings.

Simple direct DNA extraction protocols for efficient routine detection of plant pathogenic bacteria via conventional PCR

ABSTRACT Rapid, easy and direct DNA extraction protocols are required for use in routine analyses to detect plant pathogenic bacteria. For this purpose, five protocols for extracting bacterial DNA, which do not use toxic chemicals such as phenol and chloroform, were optimized for the detection of plant pathogenic bacteria quickly and directly from plant material using conventional polymerase chain reaction (PCR). The protocols are based on the use of different maceration and extraction buffers to ensure quality bacterial DNA extraction and to remove the PCR inhibitors. Results showed that the protocol #1 based on the use of Tris and TNPEE buffers and protocol #3 based on the use of 0.4% bovine serum albumin and 0.05% Tween 20 were able to detect Erwinia amylovora from apple and pear samples. Protocol #2 that used 2% polyvinyl pyrrolidone in maceration and extraction buffers was found to be efficient for detecting Erwinia amylovora, Agrobacterium tumefaciens, and Allorhizobium vitis directly from plant tissues. However, protocols #4 and #5 based on the use of 1% Triton X-100 and 0.1% Tween 20, respectively, were unable to detect the studied bacteria. Accordingly, the protocol #2 is proposed for efficient, direct and rapid detection of plant pathogenic bacteria in plant materials via PCR.

PM 7/121 (2) ‘Candidatus Liberibacter africanus’, ‘Candidatus Liberibacter americanus’ and ‘Candidatus Liberibacter asiaticus’

Specific scopeThis Standard describes a diagnostic protocol for ‘Candidatus Liberibacter africanus’, ‘Candidatus Liberibacter americanus’ and ‘Candidatus Liberibacter asiaticus’.1This Standard should be used in conjunction with PM 7/76 (5) Use of EPPO diagnostic protocols.Specific approval and amendmentFirst approved in 2014–09. Revised in 2021–04.

Elucidating biofilm diversity on water lily leaves through 16S rRNA amplicon analysis: Comparison of four DNA extraction kits.

PremiseWithin a broader study on leaf fossilization in freshwater environments, a long‐term study on the development and microbiome composition of biofilms on the foliage of aquatic plants has been initiated to understand how microbes and biofilms contribute to leaf decay and preservation. Here, water lily leaves are employed as a study model to investigate the relationship between bacterial microbiomes, biodegradation, and fossilization. We compare four DNA extraction kits to reduce biases in interpretation and to identify the most suitable kit for the extraction of DNA from bacteria associated with biofilms on decaying water lily leaves for 16S rRNA amplicon analysis.MethodsWe extracted surface‐associated DNA from Nymphaea leaves in early stages of decay at two water depth levels using four commercially available kits to identify the most suitable protocol for bacterial extraction, applying a mock microbial community standard to enable a reliable comparison of the kits.ResultsKit 4, the FastDNA Spin Kit for Soil, resulted in high DNA concentrations with better quality and yielded the most accurate depiction of the mock community. Comparison of the leaves at two water depths showed no significant differences in community composition.DiscussionThe success of Kit 4 may be attributed to its use of bead beating with a homogenizer, which was more efficient in the lysis of Gram‐positive bacteria than the manual vortexing protocols used by the other kits. Our results show that microbial composition on leaves during early decay remains comparable and may change only in later stages of decomposition.

POSSIBILITY OF SAP-FEEDING BEETLE, NITIDULIDAE, AS A SPREADING AGENT FOR BLOOD DISEASE BACTERIUM ON THE BANANA PLANT

Banana blood disease (BBD) caused by blood disease bacterium (BDB) is one of the major diseases of Indonesian banana plants. The foraging activity of flower-visiting insects was suspected as the vectors of the disease. This study aims at analyzing the potential of the sap-feeding beetle, Nitidulidae, as a vector of BDB. Beetle collection was done by using purposive sampling method on three cultivars of banana inflorescence: Nangka, Klutuk, and Siem. The bacterium was isolated from the outside and the inside of beetle tissues and was followed with bacterial DNA extraction. Confirmation of bacterial cell culture as BDB was conducted by biochemical assay and PCR using 16S rDNA oligonucleotide. The findings revealed that the beetle population in the three cultivars of banana cultivars was Nangka (66%), Siem (19%) and Klutuk (16%). According to the biochemical assay and PCR result, it has been found that the bacterial cell culture was found positive as BDB, which was putative 79% and homolog with BDB that is in accordance with the data provided by NCBI. Thereby, the discovery of BDB in sap-feeding beetle, Nitidulidae, showed that the foraging activity has the potential as an agent of spreader of BDB in banana plants.

Secondary metabolites from a marine sponge-derived actinomycete &amp;lt;italic&amp;gt;Streptomyces parvulus&amp;lt;/italic&amp;gt; MA1076

In recent years, researchers have discovered a large number of secondary metabolites with novel chemical structures and diverse activities from sponge-derived actinomycetes. Sponge-derived actinomycetes have become important sources of new drug lead compounds. The actinomycete <italic>Streptomyces parvulus</italic> MA1076 is derived from the sponge( <italic>Pseudoceratina</italic> sp.) on Yongxing Island in the South China Sea. In order to obtain the structural type and potential medicinal value of the secondary metabolites, strain identification and chemical composition analysis were carried out on the sponge-associated actinomycete <italic>Streptomyces parvulus</italic> MA1076. Bacterial genomic DNA rapid extraction kit was used to extract streptomyces genomic DNA. Streptomyces universal primers 27F and 1492R were used for PCR amplification, and the amplified products was sent for sequence testing. The sequencing result was used for strain identification through 16S rDNA sequence analysis and phylogenetic tree construction. Silica gel column chromatography, Sephadex LH-20 gel column chromatography, semi-preparative high performance liquid chromatography and other techniques were applied to separate and purify the ethyl acetate extract of the sponge-derived actinomycetes <italic>Streptomyces parvulus</italic> MA1076. The structure of the compound was identified by spectral data such as proton nuclear magnetic resonance spectroscopy (¹H-NMR), carbon nuclear magnetic resonance spectroscopy (¹³C-NMR), high-resolution mass spectrometry (HR-MS),and combined with the comparison with literature data. Six compounds were isolated and identified from the actinomycetes <italic>Streptomyces parvulus</italic> MA1076, which were actinomycin D (compound 1), daucosterol (compound 2), cyclo-((<italic>S</italic>)-Pro-(<italic>R</italic>)-Leu) (compound 3), thymidine (compound 4), 3′-<italic>O</italic>-acetylthymidine (compound 5), and indole-3-carboxyli acid (compound 6). Compounds 2-6 were isolated for the first time from <italic>Streptomyces parvulus</italic> MA1076. The six compounds were tested for their cell proliferation inhibitory activity on two pancreatic cancer cell lines (pc-12 and bc-16). Results showed that compound 1 had obvious proliferation inhibitory activity on both pancreatic cancer cell lines. Compounds 2-6 showed no obvious anti-tumor cell proliferation activity. The results of the study make full use of the abundant Chinese sponge resources and their associated microbial resources, exploring the structural diversity of sponge secondary metabolites to discover new chemical substances that may be contained in natural products, and to develop new antibacterial , antitumor drugs is of great theoretical significance and potential application value.

Impact of Staphylococci DNA extraction methodology on microsatellite-based PCR banding profile

Background: Microsatellite-based PCR has great utility in microbial typing by analysis of band profiles of PCR amplicons. Bacterial DNA extraction methods affect yield and quality of DNA extract, and consequently subsequent molecular-based studies. The study aimed to evaluate impact of bacterial DNA extraction methods on band profiles of microsatellite-based PCR. Methods: Genomic DNA was extracted from Staphylococcus epidermidis ATCC 12228 using QIAamp kit, tween and SDS methods, and submitted to microsatellite-based PCR using (GACA)4 primer. For each extraction method, 4 concentrations were tested: the first crude extracted DNA and other 3 concentrations obtained by 1:1, 1:3, and 1:5 dilutions of the crude samples. Results: Yield and purity of DNA extracted by QIAamp kit were higher than those of other two methods. The PCR amplified all tested DNAs with generation of band profiles ranged from 2 to 11 bands in number, and from 200 bp to 1500 bp in size. Marked inter-method differences were found due to variation in band numbers and sizes. Band patterns obtained for QIAamp kit and tween methods were more robust than for SDS method. Variations of PCR patterns of different DNA concentrations within each method were minimal. Conclusions: Microsatellite-based PCR band profiles vary with different genomic DNA extraction methods for the same bacterial strain. Quality of extracted DNA is more influencing than its concentration. Therefore, it is recommended to unify the extraction method of bacterial DNA for all tested bacterial strains that are submitted to such type of PCR.

Monosodium Glutamate Induces Changes in Hepatic and Renal Metabolic Profiles and Gut Microbiome of Wistar Rats.

The short- and long-term consumption of monosodium glutamate (MSG) increases urinary pH but the effects on the metabolic pathways in the liver, kidney and the gut microbiota remain unknown. To address this issue, we investigated adult male Wistar rats allocated to receive drinking water with or without 1 g% MSG for 2 weeks (n = 10, each). We performed a Nuclear Magnetic Resonance (NMR) spectroscopy-based metabolomic study of the jejunum, liver, and kidneys, while faecal samples were collected for bacterial DNA extraction to investigate the gut microbiota using 16S rRNA gene sequencing. We observed significant changes in the liver of MSG-treated rats compared to controls in the levels of glucose, pyridoxine, leucine, isoleucine, valine, alanine, kynurenate, and nicotinamide. Among kidney metabolites, the level of trimethylamine (TMA) was increased, and pyridoxine was decreased after MSG-treatment. Sequencing of the 16S rRNA gene revealed that MSG-treated rats had increased Firmicutes, the gut bacteria associated with TMA metabolism, along with decreased Bifidobacterium species. Our data support the impact of MSG consumption on liver and kidney metabolism. Based on the gut microbiome changes, we speculate that TMA and its metabolites such as trimethylamine-N-oxide (TMAO) may be mediators of the effects of MSG on the kidney health.

Novel perspective on a conventional technique: Impact of ultra-low temperature on bacterial viabilityand protein extraction.

Ultra-low temperature (ULT) storage of microbial biomass is routinely practiced in biological laboratories. However, there is very little insight regarding the effects of biomass storage at ULT and the structure of the cell envelope, on cell viability. Eventually, these aspects influence bacterial cell lysis which is one of the critical steps for biomolecular extraction, especially protein extraction. Therefore, we studied the effects of ULT-storage (-80°C) on three different bacterial platforms: Escherichia coli, Bacillus subtilis and the cyanobacterium Synechocystis sp. PCC 6803. By using a propidium iodide assay and a modified MTT assay we determined the impact of ULT storage on cellular viability. Subsequently, the protein extraction efficiency was determined by analyzing the amount of protein released following the storage. The results successfully established that longer the ULT-storage time lower is the cell viability and larger is the protein extraction efficiency. Interestingly, E. coli and B. subtilis exhibited significant reduction in cell viability over Synechocystis 6803. This indicates that the cell membrane structure and composition may play a major role on cell viability in ULT storage. Interestingly, E. coli exhibited concomitant increase in cell lysis efficiency resulting in a 4.5-fold increase (from 109 μg/ml of protein on day 0 to 464 μg/ml of protein on day 2) in the extracted protein titer following ULT storage. Furthermore, our investigations confirmed that the protein function, tested through the extraction of fluorescent proteins from cells stored at ULT, remained unaltered. These results established the plausibility of using ULT storage to improve protein extraction efficiency. Towards this, the impact of shorter ULT storage time was investigated to make the strategy more time efficient to be adopted into protocols. Interestingly, E. coli transformants expressing mCherry yielded 2.7-fold increase (93 μg/mL to 254 μg/mL) after 10 mins, while 4-fold increase (380 μg/mL) after 120 mins of ULT storage in the extracted soluble protein. We thereby substantiate that: (1) the storage time of bacterial cells in -80°C affect cell viability and can alter protein extraction efficiency; and (2) exercising a simple ULT-storage prior to bacterial cell lysis can improve the desired protein yield without impacting its function.

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.

Recent developments for antimicrobial applications of graphene-based polymeric composites: A review

Owing to its captivating properties, Graphene-based materials (GMBs) have appeared as a broad-spectrum antimicrobial material and attracting scientists and researchers’ attention. By focusing on the recent advances in the polymeric nanocomposite’s field, current study is positioned on the graphene-based polymeric composites having antibiofilm and antimicrobial activities to confront the current situations of microbial resistance against antibiotics and other drugs. Furthermore, this review summarizes the influence of different factors on the antimicrobial activities of graphene-based polymeric composites like size, shape, number of layers, concentration, dispersibility, and functionalization, and also shed the light on the possible mode of actions of these materials. Graphene-based polymeric composites exert its antimicrobial action by physical damages (e.g., direct sharp edges’ contact to cell membranes of bacteria, wrapping of the bacterial cell, phospholipid extraction, and photo-thermal ablation) and chemical damages (e.g. oxidative-stress and charge transfer). Besides, this review article thoroughly describes the antimicrobial applications of graphene-based polymeric composites, which are because of their good biocompatibility and superior antimicrobial properties such as wound bandages and dressings, antimicrobial coatings, drug delivery, food packaging, hydrogel formation, and water disinfection. Present review may stimulate major concerns, spur further developments, and provide valuable insight into the promising fields.

Evaluation of the orally administered calcium alginate aerogel on the changes of gut microbiota and hepatic and renal function of Wistar rats.

The present study evaluates the effect of calcium alginate aerogel as a potential drug carrier, on the liver and kidney functions, and on the gut microbiota of Wistar rats. The studied alginate aerogel was prepared in the form of nanoparticles using the jet cutting technique, and they were characterized in terms of specific surface areas, outer morphology and particle size distribution. For the in vivo study, calcium alginate aerogel was administered orally, and liver and kidney functions were tested for one week and for four weeks in two distinct studies. During the short-term in vivo study, feces samples were collected for bacterial DNA extraction followed by 16S rRNA gene sequencing analyses to detect changes in gut microbiota. Results showed that the prepared alginate aerogel has an average BET-specific surface area of around 540 m2/g, with a pore volume of 7.4 cc/g, and pore width of 30–50 nm. The in vivo study revealed that the levels of the studied kidney and liver enzymes didn’t exceed the highest level of the normal range. The study of gut microbiota showed different patterns; certain groups of bacteria, such as Clostridia and Bacteriodia, increased during the aerogels regime and continued to increase after the aerogel was stopped. While other groups such as Erysipelotrichia, and Candidatus saccharibacteria increased during aerogels treatment, and then decreased again after one month. Members of the Bacilli class showed a unique trend, that is, after being the most abundant group (63%) at time 0, their relative abundance decreased dramatically until it reached < 5%; which was the case even after stopping the aerogel treatment.

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.

No distinction in the gut microbiota between diarrhea predominant-irritable bowel syndrome and healthy subjects: matched case\u2013control study in Thailand

BackgroundAlteration in the gut microbiota has been proposed in irritable bowel syndrome (IBS) pathogenesis, especially in the diarrheal type (IBS-D). We conducted this study to evaluate the fecal microbiota in Thai IBS-D patients when compared with healthy subjects as well as to evaluate the effects of probiotics on changes in the gut microbiota correlated with symptoms.MethodsA matched case–control study was conducted on diagnosed IBS-D patients, based on the Rome IV criteria and healthy controls. Stool samples were collected in preservation tubes. Bacterial deoxyribonucleic acid extraction was performed and amplified. Next, 16S ribosomal ribonucleic acid genes sequencing was performed to identify the microbiome in both the groups. IBS-D patients were provided with a probiotic mixture that was rich in Lactobacillus acidophillus and Bifidobacterium bifidum over 8 weeks. Changes in the symptoms, stool characteristics, and fecal microbiota were evaluated and compared with the corresponding baseline values.ResultsTwenty IBS-D patients with 20 age and gender-matched controls were included in this study. The baseline characteristics were not significantly different between the groups, including the mode of birth and the history of breastfeeding in infancy. No significant difference was noted in the fecal microbiota between the IBS-D patients and controls. The IBS symptom severity scales (IBS-SSS) were not statistically different after probiotic prescription; although, the bowel movements, the sense of urgency to go to the toilet and passing of mucous stool had obviously decreased. No change was noted in the fecal microbiota after receiving the experimental probiotic, except for an increase in the proportion of B. bifidum.ConclusionAlteration in the gut microbiota composition was probably not the main pathogenic mechanism in the Thai IBS-D patients assessed in this study. However, modifying microbiomes with potentially protective bacteria seems to be a beneficial therapy.Thai Clinical trial registry: TCTR20191211006, Date of registration: 10 Dec 2019. Retrospectively registered, Clinical trial URL: www.clinicaltrials.in.th

Development of a rapid Salmonella detection method via phage-conjugated magnetic bead separation coupled with real-time PCR quantification

In this study, we used the lytic Siphovirus phage LPST10 to develop a rapid and accurate detection method for Salmonella. Salmonella cells were separated from complex food matrices through magnetic separation, then subjected to SYBR green-based real-time quantitative PCR detection. Bacterial concentrations of 102–106 (Colony forming unit per milliliter, CFU/mL), with 200 of μg LPST10 phage-MBs, and 15 min reactions at 37 °C were found to be optimal to allow the phage-magnetic beads to capture the Salmonella cells. The phage-magnetic beads specifically recognized Salmonella cells of different serotypes. Under optimal conditions, DNA was extracted from the resulting bacteria phage-magnetic bead complexes through either alkaline lysis with sodium dodecyl sulfate (SDS) or boiling. The detection assay was subsequently assessed in food matrices (Tryptic soy broth medium, milk, and lettuce). The detection limit reached <30 CFU/mL in the food matrices. The entire assay, including bacterial capture, DNA extraction, and real-time polymerase chain reaction (RT-PCR) detection, could be completed within 1.5 h. This approach using a lytic Siphovirus phage has potential for rapid, specific, and accurate detection of Salmonella enterica subsp.enterica in different food matrices.

The Gut Microbiota and Its Relevance to Peripheral Lymphocyte Subpopulations and Cytokines in Patients with Rheumatoid Arthritis.

Growing experimental and clinical evidence suggests that a chronic inflammatory response induced by gut microbiome critically contribute to the development of rheumatoid arthritis (RA). Previous studies demonstrated the disturbance of lymphocyte subpopulations in RA patients. The purpose of this study was to explore the characteristics of gut microbiome and the associations between bacterium and lymphocyte subpopulations as well as cytokines in patients with RA. Fecal samples from 205 RA patients and 199 healthy controls (HCs) were collected for bacterial DNA extraction and 16S ribosomal RNA (rRNA) gene sequencing. The levels of peripheral lymphocyte subpopulation such as T, B, CD4+T, CD8+T, NK, T helper 1 (Th1), Th2, Th17, and regulatory T cells (Tregs) of these subjects were detected by flow cytometry combined with standard absolute counting beads. The serum levels of cytokines interleukin-2 (IL-2), IL-4, IL-6, IL-10, IL-17, tumour necrosis factor-α (TNF-α), and interferon-γ (INF-γ) were tested by flow cytometric bead array (CBA). Alpha and beta diversity of gut microbiome were explored by bioinformatics analysis. Spearman rank correlation test was used to explore the relationships between gut microbiome and lymphocyte subsets as well as serum cytokines. The diversity and relative abundance of intestinal microbiota in patients with RA were significantly different from those in HCs. Detailly, the abundant of phylum Proteobacteria in RA patients was more than that in HCs, while Firmicutes was less than in HCs. There was increased relative abundance of genus Clostridium_XlVa as well as genus Blautia, more abundance of Ruminococcus2 in patients with lower levels of T, B, CD4+T, and Tregs. In addition, the relative abundances of Pelagibacterium, Oxalobacter, ClostridiumXlVb, and ClostridiumXVIII were correlated with cytokines. Gut microbiome of RA patients was clearly different from that of HCs. Abnormal bacteria communities are associated with the altered levels of lymphocyte subpopulation and cytokines, which might be one of the pathogenesis of RA.