Recent molecular techniques to strengthen ecological studies in echinoderms / Técnicas moleculares recientes que fortalecen los estudios ecológicos de equinodermos

Abstract Introduction: Echinoderms, an integral component of marine ecosystems worldwide, have captivated scientific interest for centuries. Despite this longstanding attention, comprehending key facets such as trophic relationships, diet composition, and host-microbiota relationships still represents a challenge using traditional techniques. Recent years, however, have witnessed a transformative shift, thanks to the emergence of advanced molecular techniques, offering new approaches to strengthen ecological studies in echinoderms. Objective: Explore how recent advancements in molecular tools have impacted ecological research on echinoderms. Specifically, we aim to investigate the potential of these tools to shed light on trophic interactions, diet composition, and the characterization of gut microbial communities in these organisms. Methods: Available literature was used to clarify how novel molecular techniques can improve ecological studies. The focus is diet, trophic relationships, and gut microbiota. Results: Traditionally, studies of stomach contents using compound microscopy have provided an idea of ingested material; nevertheless, sometimes a simple magnified visualization of dietary content does not allow exhaustive identification of the entire food spectrum, as it is limited due to the rapid digestion and maceration of food items within the echinoderm's digestive tract. The use of DNA-metabarcoding, targeting specific DNA regions, such as the mitochondrial COI gene, has allowed us to enhance the accuracy and precision of diet characterization by enabling the identification of prey items down to the species or even genetic variant level, providing valuable insights into specific dietary preferences. Another approach is the use of stable isotopes, particularly carbon and nitrogen, which provide a powerful tool to trace the origin and flow of nutrients through food webs. By analyzing the isotopic signatures in muscular tissues and food items, we can discern the sources of their primary food items and gain insights into their trophic position within the ecosystem. Lastly, a third new technique used to elucidate the characterization of the prokaryotic community is 16S rRNA sequencing. This method allows us to explore the composition and dynamics of the digestive tract microbial communities. Conclusions: This is a promising era for ecological research on echinoderms, where advances of molecular tools have enabled an unprecedented level of detail, resolving longstanding challenges in comprehending their trophic interactions, diet composition, and host-microbiota relationships, and opening new avenues of investigation in ecological studies.

Resumen Introducción: Los equinodermos, un componente integral de los ecosistemas marinos en todo el mundo, han captado el interés científico durante siglos. A pesar de esta prolongada atención, el comprender facetas clave como las relaciones tróficas, la composición de la dieta y las relaciones huésped-microbiota todavía representa un desafío utilizando técnicas tradicionales. Sin embargo, los últimos años han sido testigos de un cambio transformador, gracias a la aparición de técnicas moleculares avanzadas, que ofrecen nuevos enfoques para fortalecer los estudios ecológicos en equinodermos. Objetivo: Explorar cómo los avances recientes en herramientas moleculares han impactado la investigación ecológica sobre equinodermos. Específicamente, nuestro objetivo es investigar el potencial de estas herramientas para arrojar luz sobre las interacciones tróficas, la composición de la dieta y la caracterización de las comunidades microbianas intestinales en estos organismos. Métodos: Se utilizó la literatura disponible para aclarar cómo las nuevas técnicas moleculares pueden mejorar los estudios ecológicos. La atención se centra en la dieta, las relaciones tróficas y la microbiota intestinal. Resultados: Tradicionalmente, los estudios del contenido estomacal mediante microscopía compuesta han proporcionado una idea del material ingerido; Sin embargo, a veces una simple visualización ampliada del contenido dietético no permite una identificación exhaustiva de todo el espectro alimentario, ya que está limitado debido a la rápida digestión y maceración de los alimentos dentro del tracto digestivo del equinodermo. El uso de metabarcoding de ADN, dirigidos a regiones específicas del ADN, como el gen COI mitocondrial, nos ha permitido mejorar la exactitud y precisión de la caracterización de la dieta al permitir la identificación de presas hasta el nivel de especie o incluso de variante genética, lo que proporciona valiosos resultados sobre preferencias dietéticas específicas. Otro enfoque es el uso de isótopos estables, en particular carbono y nitrógeno, que proporcionan una poderosa herramienta para rastrear el origen y el flujo de nutrientes a través de las redes alimentarias. Al analizar las firmas isotópicas en los tejidos musculares y los alimentos, podemos discernir las fuentes de sus alimentos primarios y obtener información sobre su posición trófica dentro del ecosistema. Por último, una tercera técnica nueva utilizada para dilucidar la caracterización de la comunidad procariótica es la secuenciación del ARNr 16S. Este método nos permite explorar la composición y dinámica de las comunidades microbianas del tracto digestivo. Conclusiones: Esta es una era prometedora para la investigación ecológica sobre equinodermos, donde los avances de las herramientas moleculares han permitido un nivel de detalle sin precedentes, resolviendo desafíos de larga data en la comprensión de sus interacciones tróficas, composición de la dieta y relaciones huésped-microbiota, y abriendo nuevas vías de investigación. en estudios ecológicos.

Interaction Between Bruceoside B and Intestinal Flora and Its Inhibitory Effect on Human Lung Cancer A549 Cells / 中国实验方剂学杂志

ObjectiveTo explore the interaction between bruceoside B and gut microbiota and the inhibitory activity of its metabolites on human lung cancer A549 cells， and to explore the value of bruceoside B in the treatment of non-small cell lung cancer（NSCLC）. MethodBruceoside B was co-incubated with the human gut microbiota under anoxic conditions in vitro， and ultra high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS） was used to analyze the metabolic transformation products. Cell counting kit-8（CCK-8） assay was performed to determine the effects of bruceoside B and its metabolites on the proliferation of human lung cancer A549 cells and the half inhibitory concentration（IC50） was calculated. Five healthy male rats were gavaged with bruceoside B（2 mg·kg-1） for 7 days after adaptive feeding. The feces of rats were collected before and after administration. 16S rRNA sequencing was used to assess gut microbiota. ResultBruceoside B was mainly metabolized to brusatol by human gut microbiota， the IC50 of bruceoside B and the conversion product to A549 cells were 1 755.50， 19.57 μmol·L-1， respectively， and the conversion product had a better activity at inhibiting A549 cells proliferation than bruceoside B. Additionally， The results of intestinal flora analysis showed no significant differences in α diversity and β diversity of gut microbiota after administration. In terms of species abundance， at the phylum level， bruceoside B decreased the relative abundance of Actinobacteriota and Proteobacteria， increased the relative abundance of Firmicutes， Patescibacteria and Cyanobacteria. At the genus level， bruceoside B decreased the relative abundance of Staphylococcus， Aerococcus and Psychrobacter， increased the relative abundance of Romboutsia， Lactobacillus， Clostridium sensu stricto 1， Norank-f-norank-o-Clostridia-UCG-014， Turicibacter， Allobaculum and Candidatus Saccharimonas. The results of functional prediction showed that the gut microbiota functional compositions were relatively stable. ConclusionBruceoside B can be deglycosylated by intestinal flora and converted into brusatol， with a significant increase in antitumor activity. The administration of bruceoside B will not cause significant changes in the structure and function of the intestinal flora， resulting in intestinal microecological balance disorders， and the administration appears to be beneficial to the intestinal flora of NSCLC patients.

Effects of acupuncture combined with Buyang Huanwu Decoction on the cerebral blood flow hypo perfusion model rats with carotid artery stenosis based on high-throughput sequencing / 国际中医中药杂志

Objective:To explore the effects of acupuncture combined with Buyang Huanwu Decoction on intestinal flora in cerebral blood flow hypo perfusion model rats with carotid artery stenosis.Methods:Totally 40 rats were randomly divided into sham-operation group, model group, TCM treatment group and acupuncture and drug combination treatment group, with 10 rats in each group. Except the sham-operation group, the other groups were prepared cerebral ischemia model by needle control and thread embolism method. TCM treatment group received Buyang Huanwu Decoction 100 mg/kg for gavage, once a day, and the intervention lasted for 2 weeks. In the acupuncture and drug combination group, based on the TCM treatment group, Baihui and its left and right sides of 2 mm were selected for acupuncture, once a day, and continuous intervention was performed for 2 weeks. Neurological function evaluation and behavioral function score were performed 7 and 14 days after administration, respectively. 16S rRNA sequencing was used to comprehensively characterize the structure and composition of fecal microflora of rats in each group. Linear discriminant analysis Effect Size (LEfSe) was used to analyze the difference of intestinal bacteria among groups.Result:On the 7th and 14th day after administration, compared with the model group, the neurological function score in the TCM treatment group and the acupuncture and drug combination group decreased ( P<0.05), and the behavioral function score increased ( P<0.05). Compared with model group, the Shannon index of TCM treatment group and acupuncture and drug combination group increased ( P<0.05). The abundance of Firmicutes increased ( P<0.05), and the abundance of Bacteroidetes and Proteobacteria decreased ( P<0.05); the abundance of Clostridia increased ( P<0.05), and the abundance of Gammaproteobacteria decreased ( P<0.05). The abundance of Escherichia-Shigella and Bacteroides decreased ( P<0.05); the abundance of lactobacillus significantly increased ( P<0.05). Conclusion:Acupuncture combined with Buyang Huanwu Decoction can improve the symptoms of cerebral hypoperfusion model rats with carotid artery stenosis, and the mechanism may be to increase the abundance of probiotics.

Identification of skin bacterial profiles of early deceased bodies and the relation to post-mortem interval

@#Post-mortem microbiology (PMM) is an important tool in identifying possible causes of sudden unexpected death, as an infectious cause is highly suspected. However, contamination is a major problem in microbiology, and this has increased the difficulty determining the true pathogen that contributes to death in post-mortem cases. Skin commensals are common contaminants in blood cultures. This study was conducted to investigate the skin flora on early deceased bodies and observe the bacteria detected at different post-mortem intervals (PMIs). As blood is usually drawn from the neck and femoral sites for PMM examination, the two body sites were chosen as the sampling sites. Skin swab samples from the neck and femoral (n=80) of each early deceased body were collected by sterile cotton swabs. DNA was extracted from the swabs and then subjected to high-throughput 16S rRNA sequencing by using the Illumina MiSeq platform. Staphylococcus was found to be the most dominant genus in both neck and femoral sites. LEfSe results showed that Cutibacterium is significantly different at the neck site while Corynebacterium is more abundant at femoral site. There are significant differences at genus level between PMI<5H and PMI>5H at both neck and femoral sites. The findings of the present study may act as a reference for microbiologists and forensic pathologists when mixed growth or contamination occurs in post-mortem blood cultures.

Effects of Bisphenol A and Its Substitute, Bisphenol F, on the Gut Microbiota in Mice / 生物医学与环境科学（英文）

OBJECTIVE@#The aim of this study was to assess the impact of bisphenol A (BPA) and its substitute, bisphenol F (BPF), on the colonic fecal community structure and function of mice.@*METHODS@#We exposed 6-8-week-old male C57BL/6 mice to 5 mg/(kg∙day) and 50 μg/(kg∙day) of BPA or BPF for 14 days. Fecal samples from the colon were analyzed using 16S rRNA sequencing.@*RESULTS@#Gut microbiome community richness and diversity, species composition, and function were significantly altered in mice exposed to BPA or BPF. This change was characterized by elevated levels of Ruminococcaceae UCG-010 and Oscillibacter and decreased levels of Prevotella 9 and Streptococcus. Additionally, pathways related to carbohydrate and amino acid metabolism showed substantial enrichment.@*CONCLUSION@#Mice exposed to different BP analogs exhibited distinct gut bacterial community richness, composition, and related metabolic pathways. Considering the essential role of gut bacteria in maintaining intestinal homeostasis, our study highlights the intestinal toxicity of BPs in vertebrates.

Tracing probiotic producing bacterial species from gut of buffalo (Bubalus bubalis), South-East-Asia

Abstract Due to extensive application of antibiotics as growth promoters in animal feed, antimicrobial resistance has been increased. To overcome this challenge, rumen microbiologists search for new probiotics to improve the rate of livestock production. The present study was aimed to isolate and evaluate breed-specific lactic acid bacteria (LAB) as potential animal probiotics. The current study was conducted during 10 months from July 2020 to April 2021, in which a total of n=12 strains were isolated from different samples including milk, rumen, and feces of Nilli Ravi Buffaloes. These isolates were evaluated for their antimicrobial potential against common animal pathogens (Bacillus spp., E. coli, Staphylococcus aureus, Salmonella spp., Listeria spp.). All the isolates were identified using 16S rRNA gene sequencing and the phylogenetic analyses inferred that these strains showed close relations to the species of various genera; Enterococcus lactis, Pediococcus pentosaceus, Bacillus subtilis Weissella cibaria, Weissella soli, Bacillus tequilensis, Weissella bombi, Bacillus licheniformis, Lactococcus lactis, Bacillus megaterium, Lactobacillus ruminis, and Lactococcus lactis. NMCC-Ru2 has exhibited the enormous potential of antimicrobial activity, 28 mm, for Salmonella typhimurium;23 mm for Listeria monocytogenes 21 mm for E.coil. Highest resistance was seen in NMCC-Ru2 agasint test antbiotic, like 25.5 mm for Tetracycline. Overall results revesl that the probiotic profile of isolates was achieved using standard criteria, particularly with animal probiotic properties

Resumo Devido à extensa aplicação de antibióticos como promotores de crescimento na alimentação animal, a resistência aos antimicrobianos aumentou. Para superar esse desafio, os microbiologistas do rúmen buscam novos probióticos para melhorar a produtividade do gado. O presente estudo teve como objetivo isolar e avaliar bactérias lácticas específicas de raças (BAL) como potenciais probióticos animais. 12 cepas foram isoladas de diferentes amostras, incluindo leite, rúmen e fezes de búfalos Nilli Ravi. Esses isolados foram avaliados quanto ao seu potencial antimicrobiano contra patógenos animais comuns (Bacillus spp., E. coli, Staphylococcus aureus, Salmonella spp., Listeria spp.). Todos os isolados foram identificados por meio do sequenciamento do gene 16S rRNA e as análises filogenéticas inferiram que essas cepas apresentaram estreita relação com as espécies de vários gêneros; Enterococcus lactis, Pediococcus pentosaceus, Bacillus subtilis, Weissella cibaria, Weissella soli, Bacillus tequilensis, Weissella bombi, Bacillus licheniformis, Lactococcus lactis, Bacillus megaterium, Lactobacillus ruminis e Lactococcus lactis. O perfil probiótico dos isolados foi obtido usando critérios padrão, particularmente com propriedades probióticas animais.

Tracing probiotic producing bacterial species from gut of buffalo (Bubalus bubalis), South-East-Asia / Rastreando espécies bacterianas produtoras de probióticos de intestino de búfalo (Bubalus bubalis), sudeste da Ásia

Due to extensive application of antibiotics as growth promoters in animal feed, antimicrobial resistance has been increased. To overcome this challenge, rumen microbiologists search for new probiotics to improve the rate of livestock production. The present study was aimed to isolate and evaluate breed-specific lactic acid bacteria (LAB) as potential animal probiotics. The current study was conducted during 10 months from July 2020 to April 2021, in which a total of n=12 strains were isolated from different samples including milk, rumen, and feces of Nilli Ravi Buffaloes. These isolates were evaluated for their antimicrobial potential against common animal pathogens (Bacillus spp., E. coli, Staphylococcus aureus, Salmonella spp., Listeria spp.). All the isolates were identified using 16S rRNA gene sequencing and the phylogenetic analyses inferred that these strains showed close relations to the species of various genera; Enterococcus lactis, Pediococcus pentosaceus, Bacillus subtilis Weissella cibaria, Weissella soli, Bacillus tequilensis, Weissella bombi, Bacillus licheniformis, Lactococcus lactis, Bacillus megaterium, Lactobacillus ruminis, and Lactococcus lactis. NMCC-Ru2 has exhibited the enormous potential of antimicrobial activity, 28 mm, for Salmonella typhimurium;23 mm for Listeria monocytogenes 21 mm for E.coil. Highest resistance was seen in NMCC-Ru2 agasint test antbiotic, like 25.5 mm for Tetracycline. Overall results revesl that the probiotic profile of isolates was achieved using standard criteria, particularly with animal probiotic properties

Devido à extensa aplicação de antibióticos como promotores de crescimento na alimentação animal, a resistência aos antimicrobianos aumentou. Para superar esse desafio, os microbiologistas do rúmen buscam novos probióticos para melhorar a produtividade do gado. O presente estudo teve como objetivo isolar e avaliar bactérias lácticas específicas de raças (BAL) como potenciais probióticos animais. 12 cepas foram isoladas de diferentes amostras, incluindo leite, rúmen e fezes de búfalos Nilli Ravi. Esses isolados foram avaliados quanto ao seu potencial antimicrobiano contra patógenos animais comuns (Bacillus spp., E. coli, Staphylococcus aureus, Salmonella spp., Listeria spp.). Todos os isolados foram identificados por meio do sequenciamento do gene 16S rRNA e as análises filogenéticas inferiram que essas cepas apresentaram estreita relação com as espécies de vários gêneros; Enterococcus lactis, Pediococcus pentosaceus, Bacillus subtilis, Weissella cibaria, Weissella soli, Bacillus tequilensis, Weissella bombi, Bacillus licheniformis, Lactococcus lactis, Bacillus megaterium, Lactobacillus ruminis e Lactococcus lactis. O perfil probiótico dos isolados foi obtido usando critérios padrão, particularmente com propriedades probióticas animais.

Ocular conjunctival microbiome profiling in dry eye disease: A case control pilot study

Purpose: Keratoconjunctivitis sicca (KCS) or dry eye disease (DED) is a multifactorial disease that results in discomfort, visual disturbance, and tear film instability with potential damage to the ocular surface. A pilot study was undertaken to determine if there were any major substantial differences in the ocular microbiome in DED patients versus healthy controls. Methods: The bacterial communities residing in the conjunctiva of patients with DED (n = 4) and healthy controls (n = 4) were assessed by 16S ribosomal RNA (rRNA) gene sequencing of the V4–V5 region. Results: The phyla Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes were most dominant and accounted for 97% and 94.5% of all bacterial sequences in patients and controls, respectively. At the genus level, 27 bacterial genera were found with more than two?fold difference between patients and controls. Four of these – Acinetobacter, Corynebacterium, Lactobacillus, and Pseudomonas spp. – dominated the ocular microbiome of all subjects, but were proportionately lower in DED (16.5%) compared to controls (37.7%). Several bacterial genera were found to be unique in DED (34) and controls (24). Conclusion: This pilot study is an attempt to profile the ocular microbiome in patients with DED that demonstrated a higher concentration of microbial DNA compared to controls, with Firmicutes phyla dominating the bacterial population in patients with DED.

High microbiome variability in pediatric tracheostomy cannulas in patients with similar clinical characteristics

Abstract Objectives: To evaluate the bacterial microbiome found in tracheostomy cannulas of a group of children diagnosed with glossoptosis secondary to Robin Sequence (RS), and its clinical implications. Methods: Pediatric patients were enrolled in the study at the time of the cannula change in the hospital. During this procedure, the removed cannula was collected and stored for amplicon sequencing of 16s rRNA. DNA extraction was performed using DNeasy PowerBiofilm Kit (QIAGEN® - Cat nº 24000-50) while sequencing was performed with the S5 (Ion S5™ System, Thermo Fisher Scientific), following Brazilian Microbiome Project (BMP) protocol. Results: All 12 patients included in the study were using tracheostomy uncuffed cannulas of the same brand, had tracheostomy performed for over 1-year and had used the removed cannula for approximately 3-months. Most abundant genera found were Aggregatibacter, Pseudomonas, Haemophilus, Neisseria, Staphylococcus, Fusobacterium, Moraxella, Streptococcus, Alloiococcus, and Capnocytophaga. Individual microbiome of each individual was highly variable, not correlating to any particular clinical characteristic. Conclusion: The microbiome of tracheostomy cannulas is highly variable, even among patients with similar clinical characteristics, making it challenging to determine a standard for normality. © 2022 Associa¸c˜ao Brasileira de Otorrinolaringologia e Cirurgia C´ervico-Facial. Published by Elsevier Editora Ltda. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).

Novel primer designing and PCR-AFLP approach for an expeditious detection of coliforms in potable waters

Molecular techniques involving 16S rRNA gene have long been proved to be a mainstay of sequence-based bacterial analysis and enhance the competence of bacterial removal in drinking water and food. The main goal of this analysis was to reduce the time of detection of total coliforms by developing 16S rRNA based DNA markers by targeting variable region in the 16S rRNA gene position of V2 and V9. Coliform specific primers (189F and 1447R) were designed to amplify total coliform with an amplicon size of 1300 bp. The PCR product was later digested with Hind III and BseRI (restriction enzymes) to differentiate the type of contamination caused by fecal and non-fecal coliforms respectively. The digested amplicons were run on agarose gel electrophoresis and contamination levels were estimated based on the respective band pattern. This method can be applicable to know the coliform contamination levels of potable waters, in food and beverage industries within a short period of time. To our knowledge, this is the first report on newly designed primers which not only amplify coliform bacteria, followed by various restriction digestions of these amplicons but also provides unique band patterns to identify coliforms at genus level.

Identification of 15 bird remains using the 16S rRNA gene sequence / 中国法医学杂志

Objective The mitochondrial 16S rRNA gene PCR sequencing method was applied to identify the bird species involved in the case of bird remains.Methods Using frozen muscle tissue samples from 15 unknown bird remains,the PCR amplification of the 16S rRNA DNA barcode fragment was performed.Results Through sequence alignment and phylogenetic analysis,it was determined that the 15 samples were associated with six bird species,including four Streptopelia chinensis,one Turdus eunomus,five Passer montanus,two Chloris sinica,two Fringilla montifringilla,and one Phoenicurus auroreus.These species belong to 2 orders,6 families,and 6 genera,all of which are protected as listed species under the wildlife conservation regulations.Conclusion The 16S rRNA gene segment can be regarded as a reliable approach for accurately identifying bird species from remains,providing a dependable basis for qualitative and sentencing determinations in judicial cases.

Efficacy of acidified aliphatic ester in the treatment of atopic dermatitis in mouse models and preliminary exploration of its mechanisms of action / 中华皮肤科杂志

Objective:To investigate the efficacy of acidified aliphatic ester in the treatment of atopic dermatitis (AD) in mouse models, and to preliminarily explore its mechanisms of action.Methods:Twenty female BALB/c mice aged 6 to 8 weeks were randomly divided into 2 groups: 5 mice in the blank control group were topically treated with absolute ethanol on both ears (14.3 μl per ear) every day, and 15 mice in the model group were topically treated with calcipotriol liniment (14.3 μl per ear) and 20 g/L ovalbumin (25 μl per ear) on both ears every day for 10 consecutive days to establish AD-like mouse models. From day 11, 15 mice in the model group were randomly divided into 3 groups (5 mice in each group), including AD model group, aliphatic ester group, and acidified aliphatic ester group; in the forenoon, all the 3 groups continued to be topically treated with calcipotriol liniment and ovalbumin to maintain AD-like models; in the afternoon, the aliphatic ester group and acidified aliphatic ester group were topically treated with aliphatic ester and acidified aliphatic ester respectively (10 μl per ear), and no treatment was given to the AD model group. Changes in body weight, ear thickness, ear skin lesion scores, and scratching frequency were observed. Ear skin swabs were obtained from the mice on days 10 and 14 for 16S rRNA gene - based microbial diversity tests. On day 14, mice were sacrificed after reflectance confocal microscopy examinations of the ear skin, ear tissues were resected for hematoxylin and eosin staining, mast cell staining, and real-time fluorescence-based quantitative PCR (RT-qPCR), and blood samples were collected for detection of serum IgE levels. One-way analysis of variance was used for analysis of data that met homogeneity of variance criteria, and least significant difference- t test for multiple comparisons. Results:On day 14, the severity of mouse ear lesions was the highest in the AD model group, followed in turn by the aliphatic ester group, acidified aliphatic ester group, and blank control group; compared with the AD model group, the acidified aliphatic ester group showed significantly decreased mouse ear thickness ( F = 897.50, P < 0.001), skin lesion scores ( F = 268.80, P < 0.001), scratching frequency ( F = 64.36, P < 0.001), and epidermal thickness ( F = 256.20, P < 0.001). In addition, RT-qPCR indicated that the expression of inflammatory factors such as interleukin (IL) -33, thymic stromal lymphopoietin, IL-4, and tumor necrosis factor-α in lesional areas, and the degree of mast-cell infiltration were all significantly lower in the acidified aliphatic ester group than in the AD model group ( F = 3.38, 8.70, 41.73, 44.30, 134.30, P = 0.049, = 0.001, < 0.001, < 0.001, <0.001, respectively). Microbial diversity tests showed that the acidified aliphatic ester treatment could inhibit the colonization of Staphylococcus spp. in the ears of AD-like mouse models, and the Shannon index and Simpson index significantly differed among the 4 groups ( F = 9.00, 7.92, P = 0.001, 0.002, respectively) . Conclusion:Acidified aliphatic ester could improve skin lesions of AD-like mouse models, possibly by regulating immunity, suppressing inflammation, and restoring skin microecological diversity.

Effects of Huzhang Qingmai decoction on cognitive function and intestinal flora in mice with chronic cerebral ischemia / 药学实践与服务

Objective To investigate the mechanism of action of Huzhang Qingmai decoction (HZQMY) on the improvement of cognitive function in mice with chronic cerebral ischemia from the perspective of intestinal flora. Methods A mouse model of chronic cerebral ischemia was established by placing microcoils around the bilateral common carotid arteries to induce bilateral carotid artery stenosis (BCAS). After 12 weeks of intragastric administration, the cognitive function of the mice was measured by the Morris water maze; the myelin damage was analyzed by LFB staining; The contents of the cecum of the mice in each group were extracted and analyzed by 16S rRNA sequencing. Results The results of the water maze experiment showed that the mice in the HZQMY group had a significantly shorter escape latency, increased the number of crossings platform and the percentage of target quadrants. LFB staining showed that the white matter damage in the model group was severe; the white matter damage in the HZQMY group was milder. The results of 16S rRNA sequencing showed that compared with the model group, the abundance of Verrucomicrobiota, Akkermansia, and ErysiPelatoclostridium capsulatum in the intestinal flora in HZQMY group was significantly reduced (P<0.05), while the abundances of Eubacterium_xylanoPhilum and Allobaculum were significantly increased (P<0.05). Conclusion The protective effect of HZQMY, which has the effect of improving cognitive function in mice with chronic cerebral ischemia, may be related to the regulation of intestinal flora in mice with chronic cerebral ischemia.

Traceability of Geographic Origin Using Human Skin and Oral Microbiota / 法医学杂志

OBJECTIVES@#To explore the possibility of using human skin and oral microorganisms to estimate the geographic origin of an individual through the sequencing analysis of bacterial 16S rRNA gene.@*METHODS@#Microbial DNA was extracted from the palm and oral microorganisms of the Han population in Shanghai and Chifeng, Inner Mongolia, and the composition and diversity of the microbiota were analyzed by full-length 16S rRNA gene sequencing. Then, differential species were screened and a geographic location prediction model was constructed.@*RESULTS@#The compositions of palm and oral microorganisms between Shanghai and Chifeng samples were both different. The abundance and uniformity of palm side skin microorganisms were higher in Chifeng samples than in Shanghai samples, while there was no significant difference in oral microorganisms. Permutational multivariate analysis of variance (PERMANOVA) confirmed that the β-diversity between the samples from the two places were statistically significant, and the coefficients of determination (R2) for skin and oral samples were 0.129 and 0.102, respectively. Through principal co-ordinates analysis (PCoA), the samples from the two places could be preliminarily distinguished. The predictive model had the accuracies of 0.90 and 0.83 for the geographic origin using the skin and oral samples, respectively.@*CONCLUSIONS@#There are differences in the compositions of palm and oral microbiota between Han populations in Shanghai and Chifeng. The prediction model constructed by the random forest algorithm can trace the unknown individuals from the above two places.

Isolation and molecular characterization of bacterial species from Sikog waterfall, Padawan, Sarawak

Aims@#Bacteria are microorganisms that are commonly distributed in any environment. They are also found abundantly in marine environments such as waterfalls and rivers. Some bacteria participate in various biological activities and possess no health risk; however, other species could be pathogenic and have been directly associated with various diseases in animals and humans. Therefore, it is crucial to analyze the antibiotic resistance profiles of bacteria in the research area based on regularly used antibiotics in clinical and agricultural contexts to establish a data baseline for health providers and public usage.@*Methodology and results@#Water samplings were done twice and collected from upstream, midstream, and downstream of the Sikog waterfall. A total of ninety isolates were isolated and analyzed using (GTG)5 genetic fingerprinting to determine the genetic similarities. Based on the dendrogram generated using Gelj Version 2.0 software, 41 bacterial isolates were subjected to 16S rRNA gene sequencing for species identification. The Kirby-Bauer disk diffusion method was implemented to determine the level of susceptibility toward certain antibiotics. Sequence analysis was performed using BLAST, revealing that the isolates constitute 17 genera, including Pseudomonas, Alcaligenes, Stenotrophomonas, Staphylococcus, Bacillus, Lysinibacillus, Rossellomorea, Citrobacter, Enterobacter, Kosakonia, Klebsiella, Escherichia, Serratia, Cronobacter, Aeromonas, Chromobacterium and Kocuria. According to the overall antibiotic susceptibility analysis, streptomycin (10 µg) exhibited the highest rate of resistance among bacterial isolates, with 36.84%, followed by penicillin (10 units) (36.36%), rifampicin (5 µg) (27.27%) and ampicillin (10 µg) (26.32%).@*Conclusion, significance and impact of study@#The research findings revealed the predominant bacteria found in the recreational water of Sikog waterfall and their antibiotic susceptibility, which could be helpful in the treatment of bacterial infections for future clinical reference. Simultaneously, the public, particularly communities in the study area, should be informed about the potential health risk associated with diverse resistant enteric bacteria in the recreational water.

Isolation and molecular identification of ethanol-tolerant Acetobacter species from Lansium domesticum (Dokong) and Nephelium lappaceum (Rambutan) vinegar

Aims@#Acetic acid bacteria (AAB) are a group of Gram-negative or Gram-variable bacteria that oxidise ethanol during the production of vinegar. The aim of this study was to isolate the AAB from both Lansium domesticum (Dokong) and Nephelium lappaceum (Rambutan), mother of vinegars (MV) and vinegars, as a potential starter culture for vinegar production.@*Methodology and results @#The MV and vinegar samples were collected from six to eight weeks of fermented Dokong and Rambutan vinegar from the Food Laboratory of Universiti Malaysia Kelantan (UMK), Jeli. The enriched samples were inoculated on selective Carr and GYC solid media. From the Carr medium, thirty-seven isolates that showed a yellow clear zone and seventy-eight isolates that showed a halo clear zone on the GYC medium were selected. Sixty isolates that produced higher total acidity (>60%) were characterized by Gram staining. Sixteen Gram-negative and fourteen Gram-variable isolates were subjected to 2.0% ethanol Carr medium to select for ethanol tolerance. Five ethanol-tolerant isolates were suitable for 16S rRNA gene sequence analysis and molecular identification because they had 4% to 10% ethanol tolerance level utilisation on Carr solid medium. Based on the morphological and biochemical characteristics, isolates DV1 and RMV30 were Gram-variable. Meanwhile, RMV2, RMV19 and RMV37 were Gram-negative bacteria. RMV2, RMV19, RMV30 and RMV37 isolates were catalase-positive and oxidase negative. Only DV1 was catalase and oxidase positive. From the BLAST analysis, the obtained nucleotide sequences showed 100% homology, with RMV2, identified as Acetobacter fabarum, and DV1, RMV19, RMV30 and RMV37 were identified as A. pasteurianus. @*Conclusion, significance and impact of study @#Based on 16S rRNA gene sequences, five isolates were identified as Acetobacter species: Four isolates, DV1, RMV19, RMV30 and RMV37 strains, were identified as A. pasteurianus and RMV2 was identified as A. fabarum. DV1, RMV2, RMV19, RMV30 and RMV37 showed significant differences at (p<0.05) for ethanol utilisation at 4% and the highest toleration up to an ethanol concentration of 10%. The ability to tolerate high ethanol concentration during vinegar fermentation is a desirable in producing high acetic acid for vinegar production.

Evaluation of different methods for the effective isolation of high-quality DNA from rodent faecal samples

Aims@#The inefficient lysis of recalcitrant bacterial cell wall and subsequent isolation of DNA from environmental samples can lead to a bias in the qualitative and quantitative assessment of bacteria present in the sample. Thus, the selection of an optimum DNA isolation method is the important first step for biosurveillance and metagenomic analyses. This study aims to determine the optimal DNA isolation method out of four commercial DNA isolation kits (A, B, C and D) and two conventional methods (E and F), for rodent faecal droppings. The key selection criterion is the general bacterial diversity contained in the isolated DNA, as evaluated by the Shannon-Weaver index based on the maximal number of PCR-amplicons of partial 16S rRNA gene, derived from each method. The amplicons were separated in accordance with their difference in nucleotide sequences via DGGE. @*Methodology and results@#Five faecal samples of wild rodents were collected from different sites and preserved in DNA/RNA shield reagent (Zymo Research). Each sample was extracted, and the DNA extracts were then subjected to amplification of the bacterial 16S rRNA and DGGE separation of the amplicons. Method E showed a higher yield of DNA (average 324.22 ng/µL) as compared to the other methods. However, the majority of the DNA extracts showed partial degradation. The DGGE profiles showed the highest number of amplicons were generated from DNA extracted from Method A and B with a total of 168 and 167 respectively. This is indicated by the Shannon-Weaver index which were 0.306 and 0.305, respectively. @*Conclusion, significance and impact of study@#Method A is the optimum DNA isolation method for rodent faecal samples as its isolated DNA contains the most diverse bacteria. The isolated DNA can then be used for PCR-biosurveillance or metagenomic sequencing and analyses.

Study of"Butuyajie"Compound on Iproving Antibiotic-associated Diarrhea / 世界科学技术-中医药现代化

Objective To explore the intestinal barrier regulation effect of Butuyajie recipe on antibiotic-associated diarrhea rats.Methods 60 SD male rats were randomly divided into blank group,model group,positive drug group(1 g·kg-1),and high-dose,medium-dose and low-dose groups of Butuyajie recipe(40.5,20.25,10.125 g·kg-1).The model was replicated by intragastric administration of lincomycin hydrochloride(5 g·kg-1)for 7 consecutive days.After successful modeling,the materials were obtained after drug intervention for 7 days.Intestinal pathological morphology was observed by HE staining.ELISA kit to detect DAO,MPO,LPS.Take each organ tissue to detect bacterial translocation.Feces were collected for 16S rRNA gene high-throughput sequencing analysis.Results Compared with the normal group,the serum levels of DAO,MPO and LPS in the model group were significantly increased(P<0.001,P<0.01),and the sIgA content in the intestinal mucosa was significantly decreased(P<0.001).Promote intestinal bacterial translocation(P<0.001,P<0.01).The diversity of intestinal flora was significantly reduced,and the levels of intestinal microflora and genera were significantly changed.Butuyajie recipe can reduce the content of DAO,MPO and LPS(P<0.001,P<0.01,P<0.05),significantly increase the content of sIgA(P<0.01,P<0.05),and effectively inhibit the translocation of intestinal bacteria(P<0.001,P<0.01,P<0.05).At the same time,it corrects the intestinal microecological structure by increasing Firmicutes,inhibiting the proportion of Bacteroidetes and Proteobacteria,and regulating Lactobacillus,Sphingomonas,Pseudomonas,and Enterobacter.Conclusion Butuyajie recipe can reduce the permeability of intestinal mucosa,reduce the translocation of intestinal flora,protect the intestinal immune barrier,regulate the diversity of intestinal flora structure,improve the intestinal microecological disorder,and can effectively treat antibiotic-associated diarrhea.

Identification and phylogenetic analysis of two clinical isolates of Chryseobacterium / 中华微生物学和免疫学杂志

Objective:To analyze the biological characteristics, phylogenic features and clinical significance of SQ219 and SQ220 isolated from clinical sputum and midstream urine specimens.Methods:The culture and biochemical characteristics of the two strains were observed. VITEK2 System, drug sensitivity testing and MALDI-TOF mass spectrometry were used for bacterial identification. Phylogenetic analysis based on 16S rRNA and core genome was performed. The average nucleotide identity (ANI) based on whole genome sequences was calculated.Results:SQ219 and SQ220 were Gram-stain-negative, aerobic, catalase- and oxidase-positive, and non-motile bacteria. Their optimum growth was observed in NaCl-free medium at 30℃ and pH7. Flexirubin-type pigments were produced by SQ220 on Colombia blood agar, but not by SQ219. Both SQ219 and SQ220 were resistant to aztreonam, amikacin, tobramycin and colistin, which was consistent with the drug resistance phenotype of genus Chryseobacterium. The genome sequences of SQ219 and SQ220 were 5.08 Mb and 4.80 Mb in length, and the G+ C contents were 36.72% and 36.36%, respectively. Both strains carried β-lactam resistance gene ( blaCGA). 16S rRNA phylogenetic analysis showed that SQ219 and SQ220 were closely related to Chryseobacterium gambrini DSM18014 T with the similarities of 98.93% and 98.36%, respectively. Core genome phylogenetic analysis revealed that SQ219 and SQ220 were highly homologous to Chryseobacterium gambrini DSM18014 T. However, the ANI values between the two strains and Chryseobacterium gambrini DSM18014 T were 92.49% and 93.27%, respectively, below the threshold for prokaryotic species identification. Conclusions:Based on the phenotypic and phylogenetic data, SQ219 and SQ220 represent a novel species of the genus Chryseobacterium. This study would help promote the understanding of the evolution of Chrysobacterium and provide reference for the identification of new species of Chrysobacterium.

Identification and biological characterization of a Streptococcus parasuis strain / 中华微生物学和免疫学杂志

Objective:To identify a strain isolated from the cerebrospinal fluid of a patient and to investigate its biological characteristics.Methods:The strain was analyzed by several methods including Gram staining, biochemical identification, 16S rRNA and recN gene sequencing, average nucleotide identity (ANI), antibiotic susceptibility testing and detection of drug resistance and virulence genes. Results:The strain was Gram-positive cocci and formed α-hemolytic colonies on the blood plate. It was identified as Streptococcus parasuis by 16S rRNA, recN gene and whole-genome sequencing. It was sensitive to multiple antibiotics and carried the genes encoding a variety of virulence factors such as adhesion. Conclusions:Streptococcus parasuis could cause human infection and be identified by whole-genome sequencing.