Application of 16S rDNA Sequencing Technology in the Analysis of Pathogenic Bacteria in Sputum of Severe Pneumonia

The diagnosis of pathogenic bacteria in severe pneumonia is difficult and the prognosis is poor. Its outcome is closely related to bacterial pathogenicity and the timeliness and pertinence of antibiotic treatment. Therefore, early diagnosis is of great significance to the prognosis of patients. Sputum examination and culture is the gold standard for the diagnosis of pathogens of severe pneumonia. However, due to the long time of bacterial culture, the early use of antibiotics, the change of bacteria species, mixed infection and other problems, the results of bacterial culture in sputum are often false negative. With the continuous application of new molecular biology techniques in clinical detection, the classification of bacteria and microorganisms has deepened from the identification of phenotypic characteristics to the classification of gene characteristics. Sequencing analysis with 16S rDNA sequencing technology has the characteristics of high sequencing flux, large amount of data obtained, short cycle, and can more comprehensively reflect the species composition of microbial community, real species distribution and abundance information. In this paper, 16S rDNA sequencing technology was used to analyze the bacterial population composition in the sputum of severe pneumonia, and to explore a new method of etiological diagnosis.

Application of 16s rDNA Sequencing in the Analysis of Pathogenic Bacteria in Sputum of Severe Bacterial Pneumonia

Objective: 120 patients with severe pneumonia who were kept in the comprehensive ICU of our hospital in 2018 were selected, and 16s rDNA sequencing was performed to analyze the composition of pathogenic bacteria in the sputum of severe pneumonia. Methods: The sputum samples of patients with severe bacterial pneumonia were collected, and the diversity of pathogens in the samples was analyzed by polymerase chain reaction (PCR) amplification and high-throughput sequencing (16s rDNA PCR-DGGE). Results: Sequence showed that sputum samples contained a relatively large number of species, and there were many species that were not detected by sequencing. The dominant bacteria were Streptococcus, Sphingomonas, Corynebacterium, Denatobacteria, Aquobacteria, Acinetobacteria, Prevotella, Klebsiella, Pseudomonas, etc. Conclusion: Bacteria caused by sputum of severe bacterial pneumonia are complex and diverse, which provides new methods and ideas for individualized treatment of patients with severe pneumonia.

Association between childhood obesity and gut microbiota:16S rRNA gene sequencing-based cohort study

BACKGROUND This study aimed to identify characteristic gut genera in obese and normal-weight children(8-12 years old)using 16S rDNA sequencing.The research aimed to provide insights for mechanistic studies and prevention strategies for childhood obesity.Thirty normal-weight and thirty age-and sex-matched obese children were included.Questionnaires and body measurements were collected,and fecal samples underwent 16S rDNA sequencing.Significant differences in body mass index(BMI)and body-fat percentage were observed between the groups.Analysis of gut microbiota diversity revealed lowerα-diversity in obese children.Differences in gut microbiota composition were found between the two groups.Prevotella and Firmicutes were more abundant in the obese group,while Bacteroides and Sanguibacteroides were more prevalent in the control group.AIM To identify the characteristic gut genera in obese and normal-weight children(8-12-year-old)using 16S rDNA sequencing,and provide a basis for subsequent mechanistic studies and prevention strategies for childhood obesity.METHODS Thirty each normal-weight,1:1 matched for age and sex,and obese children,with an obese status from 2020 to 2022,were included in the control and obese groups,respectively.Basic information was collected through questionnaires and body measurements were obtained from both obese and normal-weight children.Fecal samples were collected from both groups and subjected to 16S rDNA sequencing using an Illumina MiSeq sequencing platform for gut microbiota diversity analysis.RESULTS Significant differences in BMI and body-fat percentage were observed between the two groups.The Ace and Chao1 indices were significantly lower in the obese group than those in the control group,whereas differences were not significant in the Shannon and Simpson indices.Kruskal-Wallis tests indicated significant differences in unweighted and weighted UniFrac distances between the gut microbiota of normal-weight and obese children(P<0.01),suggesting substantial disparities in both the species and quantity of gut microbiota between the two groups.Prevotella,Firmicutes,Bacteroides,and Sanguibacteroides were more abundant in the obese and control groups,respectively.Heatmap results demonstrated significant differences in the gut microbiota composition between obese and normal-weight children.CONCLUSION Obese children exhibited lowerα-diversity in their gut microbiota than did the normal-weight children.Significant differences were observed in the composition of gut microbiota between obese and normal-weight children.

Integrating 16S sequencing and metabolomics study on anti-rheumatic mechanisms against collagen-induced arthritis of Wantong Jingu Tablet

Wantong Jingu Tablet(WJT),a mixture of traditional Chinese medicine,was reported to relieve the symptoms of rheumatoid arthritis(RA),but its pharmacological mechanism was not completely understood.The aim of this study was to investigate the therapeutic mechanisms of WJT for RA in vivo.The effects of WJT on joint pathology,as well as the levels of Bax,Bcl-2,caspase-3,cleaved-caspase-3,ERK1/2,pERK1/2,TNF-α,IL-1β,and IL-6 were measured using collagen-induced arthritis(CIA)rats.The intestinal flora composition and the metabolites alteration were analyzed by 16S rDNA sequencing and metabolomics method,respectively.We found that WJT ameliorated the severity of the CIA rats which might be mediated by inducing apoptosis,inactivating the MEK/ERK signals and reducing the production of pro-inflammatory cytokines.WJT,in part,relieved the gut microbiota dysbiosis,especially bacterial phylum Bacteroidetes,Tenericutes and Deferribacteres,as well as bacterial genus Vibrio,Macrococcus and Vagococcus.3’-N-debenzoyl-2’-deoxytaxol,tubulysin B,and magnoline were significantly associated with the specific genera.We identified serotonin,glutathione disulfide,N-acetylneuraminic acid,naphthalene and thromboxane B2 as targeted molecules via metabolomics.Our findings contributed to the understanding of RA pathogenesis,and WJT played essential roles in gut microbiota health and metabolite modulation in the CIA rats.

Effect of Lycopus lucidus Turcz.supplementation on gut microflora and short chain fatty acid composition in Crj:CD-1 mice

We investigated the diversity and composition of microflora in feces of Lycopus lucidus Turcz.-fed mice.In addition,we evaluated the production of major cytokines(Interleukin-6 and-10)which are related to inflammation and fatty acid composition of several tissues.16S ribosomal DNA sequencing-based microbiome taxonomic profiling analysis was performed utilizing the EzBioCloud data base.Male mice fed on L.lucidus showed a significantly reduced number of lactic acid bacteria and coliform in the feces compared with the control group(p<0.05).16S rDNA sequencing analysis of fecal samples showed that L.lucidus supplementation decreased the community of harmful microflora(Enterobacteriaceae including Escherichia coli and Bacteroides sp.)in feces compared with the control group(p<0.05).There were no significant differences in mRNA expression of cytokine IL-6 and IL-10 between the control and L.lucidus fed groups.The fecal fatty acid composition in the L.lucidus group had percentages of 4:0,6:0,8:0 and 10:0 in the intestine but those short chain fatty acids were not detected in the control group.Our results showed that L.lucidus supplementation influenced gut environment by decreasing harmful microflora and increased the percentages of several short fatty acids.

Structural and functional characteristics of microbiota in oropharynx of sub-healthy children with gastrointestinal heat retention syndrome differentiated by traditional Chinese medicine

Objective:To explore the structural and functional characteristics of microbiota in oropharynx of subhealthy children with gastrointestinal heat retention syndrome(GHRS)differentiated by traditional Chinese medicine,and screen the biometric operational taxonomic units(OTUs)to assist the clinical diagnosis.Methods:We recruited children according to the“GHRS diagnostic scale”,collected their oropharyngeal swabs,and sequenced the 16 SrDNA V4 region.We described the bacterial structure with alpha-indexes,beta-distances,and relative abundances;moreover,we screened the differential genera/OTUs with Wilcoxon rank-sum test,Metagenome Seq analysis,and linear discriminant analysis effect size(LEf Se)analysis,in which biometric OTUs were selected to construct the receiver operating characteristic curve to verify the diagnostic value.The bacterial function was predicted with Kyoto Encyclopedia of Genes and Genomes pathways according to 16S rDNA gene by using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States.Results:The study population was composed of 10 children with GHRS and 10 healthy control children.GHRS children were more likely to overeat(gluttony,P=.033).Alpha-indexes,such as Sobs,abundancebased coverage estimator,Bootstrap,and Qstat,were significantly higher in the GHRS group,while betadistances did not exhibit any significant intergroup differences.There were 9 differently distributed nonpredominant genera between the groups in Wilcoxon rank-sum test,as well as 13 non-predominant genera in Metagenome Seq analysis and 3 non-predominant OTUs in LEfSe analysis.OTU44 and OTU196 were used to construct the receiver operating characteristic curve,and the area under curve was 0.92.Predicted functions showed that pathways related to oxidative phosphorylation and carbon metabolism were enriched in healthy control samples,while the pathway related to renin secretion was remarkably enriched in GHRS samples.Conclusion:Unique oropharyngeal microbial structure and function were identified in GHRS children.OTU44 and OTU196 were specific OTUs,which could be used as biomarkers of GHRS to assist clinical diagnosis.

Isolation and characterization of Acidithiobacillus caldus from several typical environments in China

Six strains of moderately thermophilic sulfur-oxidizing bacteria were isolated from several different typical environments in China. The identities of the isolates were confirmed by analyses of their 16S rRNA genes, and some key physiological traits. The isolates are Gram negative, rod-shaped bacteria, their optimal temperature and pH value for growth are 45-50℃ and 2.5-3.5 respectively. They are autotrophic and used elemental sulfur, sodium thiosulfate and potassium tetrathionate as electron donor, while a little glucose stimulated their growth. 16S rDNA sequences analysis reveals that the strains are phylogenetically clustered to Acidithiobacillus caldus.

Isolation and identification of a sulfate reducing bacteria and sequence analysis of its dissimilatory sulfite reductase gene

A sulfate reducing bacteria was isolated from mining sewage of Daqing Oilfield by Hungate anaerobic technology. Physiological-biochemical analysis showed that the strain could utilize polyacrylamide as sole carbon and nitrogen source. The sequence analysis of 16S rDNA illustrated that the similarity of F8 and Desulfovibrio desulfuricans (AF192153) was 99%, and the similarity sequence of dissimilatory sulfite reductase gene (DSR) cloned from the strain and Desulfovibrio desulfuricans (AF273034) was 98%. Their phylogenitic analysis was basically anastomosed, and thus temporarily named as Desulfovibrio desulfuricans F8. The DSR cloned from F8 strain was 2740 bp in length consisting of three ORF, DSRA, DSRB and DSRD as a single operon (DSRABD) regulated by the same operator. DSRA contained typical conservative box of sulfate—sulfite reducing enzyme (SiteⅠand SiteⅡ), which could bind siroheme and [Fe4S4]. DSRB retained a [Fe4S4] binding site, with an uncomplimentary structure for siroheme binding. There was no conservative box in DSRD. Sequence analysis of DSR will provide a theoretical basis for quantitative detection, metabolic pathway modification through gene engineering, and sulfate reducing bacteria (SRB) suppression.

Isolation and Genetic Analysis of Halophilous Bacteria from Marine Sediment Collected at Tianjin Port, China

[Objective] Marine sediment from Tianjin Port has a extremely high salinity.The bacteria which live in such habitats have evolved distinct physiological,metabolic,and morphological characteristics to survive.The objective of this study is to identify all the specific salt-tolerant characteristics and the genetic evolution of the bacteria in the sediment.[Methods] In this study,the total DNA of sediment from Tianjin Port was extracted,and 16S rDNA was used to conduct an analysis of the fauna of sediment bacteria. We also isolated sediment bacteria using beef extract-peptone media with seven different NaCl concentrations (0,0.5%,2%,5%,10%,15%,and 20%),aiming to analyze the dominant species of halophilous bacteria under different salinities.[Results] 1) With each stepwise increase of salinity from 0.5% to 20%,the total number of isolated bacterial colonies decreased.14 strains of bacteria were identified and classified by the16S rDNA sequencing analysis.Of these,four could tolerate 0~2% salinity,four could tolerate 0~5% salinity,one could tolerate 0~15% salinity,and one tolerated within the full 0~20% salinity range.Further four strains were only able to tolerate within a few narrow salinity ranges.such as 5%~10%,10%~15%,10%~20% and 15%~20%;2) The quantity of bacteria strains that can be isolated from the marine sediment decreased with the increase of salinity. Also, the Shannon wiener index and species richness index of marine sediment bacteria decreased significantly from 5% salinity.However,there were no significant differences in the species evenness index;3) When the salinity was 0~10%,the dominant species was Bacillus.When the salinity was 15%, Halomonas was the dominant species.When the salinity was 20%,there were no significant differences in the proportions of these species.[Conclusion] Our results showed that some bacteria could tolerate living conditions with high salinity,and we even found a species which can tolerate a wide range of salinities (0~20%).In further study,it would be valuable to analyze these bacteria's unique physiological and biochemical functions that allow them to adapt to environments with high salinity.It can provide theories to promote the development of microbial population resources in marine sediment and the reclaimation of salinized soil by salt tolerant microorganisms.

Partial function prediction of sulfate-reducing bacterial community from the rhizospheres of two typical coastal wetland plants in China

Sulfate-reducing bacteria(SRB)are ubiquitous anaerobic microorganisms that play signifi cant roles in the global biogeochemical cycle.Coastal wetlands,one of the major habitats of SRB,exhibit high sulfate-reducing activity and thus play signifi cant roles in organic carbon remineralization,benthic geochemical action,and plant-microbe interactions.Recent studies have provided credible evidence that the functional rather than the taxonomic composition of microbes responds more closely to environmental factors.Therefore,in this study,functional gene prediction based on PacBio single molecular real-time sequencing of 16S rDNA was applied to determine the sulfate-reducing and organic substrate-decomposing activities of SRB in the rhizospheres of two typical coastal wetland plants in North and South China:Zostera japonica and Scirpus mariqueter.To this end,some physicochemical characteristics of the sediments as well as the phylogenetic structure,community composition,diversity,and proportions of several functional genes of the SRB in the two plant rhizospheres were analyzed.The Z.japonic a meadow had a higher dissimilatory sulfate reduction capability than the S.mariqueter-comprising saltmarsh,owing to its larger proportion of SRB in the microbial community,larger proportions of functional genes involved in dissimilatory sulfate reduction,and the stronger ability of the SRB to degrade organic substrates completely.This study confi rmed the feasibility of applying microbial community function prediction in research on the metabolic features of SRB,which will be helpful for gaining new knowledge of the biogeochemical and ecological roles of these bacteria in coastal wetlands.

The molecular biological characterization of a strain of biohydrogen-producing anaerobe in Clostridium Genus

The anaerobic process of biohydrogen production was developed recently. The isolation and identification of biohydrogen producing anaerobic bacteria with high evolution rate and yield is an important foundation of the fermented biohydrogen production process through which anaerobic bacteria digest organic wastewater. By considering physiological and biochemical traits, morphological characteristics and a 16S rDNA sequence, the isolated Rennanqilyf33 is shown to be a new species.

Characterization of Fe(Ⅲ)-Reducing Enrichment Cultures and Isolation of Enterobacter sp. Nan-1 from the Deep-Sea Sediment, South China Sea

To characterize the Fe(III)-reducing bacteria,enrichment cultures were initiated by inoculating deep-sea sediment from the South China Sea(SCS)into the media with hydrous ferric oxide(HFO)as the sole electron acceptor.As indicated by Meta 16S rDNA Amplicon Sequencing,the microorganisms related to Fe(III)-reduction in the enrichment cultures were mainly Shewanella and Enterobacter.A new facultative Fe(III)-reducing bacterium was obtained and identified as Enterobacter sp.Nan-1 based on its 16S rRNA gene sequence and physiological characterizations.Enterobacter sp.Nan-1 was not only a mesophilic bacterium capable of reducing HFO with a wide range of salinity(4,34,40,50 and 60 g L−1)efficiently,but also a piezotolerant bacterium that can proceed Fe(III)-reduction sustainedly at hydrostatic pressures between 0.1 and 50 MPa using glucose and pyruvate as carbon source.Furthermore,the geochemical characteristics of deep-sea sediment indicated that the microbial metabolism and iron reduction both remain active in the well-developed Fe(III)-reducing zone where the strain Nan-1 was obtained.To our knowledge,Enterobacter sp.Nan-1 could serve as a new applicative Fe(III)-reducing bacterium for future investigation on the iron biogeochemical cycle and diagenetic process of organic matter in the deep-sea environment.

Bioleaching of spent Ni-Cd batteries and phylogenetic analysis of an acidophilic strain in acidified sludge

Biohydrometallurgy is a novel method to recycle discarded batteries,in which sewage sludge is used as microorganisms and culture due to the presence of indigenous Thiobacilli.A two-step continuous flow leaching system consisting of an acidifying reactor and a leaching reactor was introduced to achieve the bioleaching of spent nickel-cadmium(Ni-Cd)batteries.The acid supernatant produced in the acidifying reactor by the microorganisms with ferrous ions as the substrate was conducted into the leaching reactor to dissolve electrode materials.The efficiency of a batch treatment of batteries was examined.The results showed that the complete dissolution of two AA-sized Ni-Cd batteries with 0.6 L/d acid supernatant took about 30,20,and 35 days for Ni,Cd,and Co,respectively.But the dissolution ability of the three metals was different.Cd and Co can be leached mostly for pH below 4.0 while the complete dissolution of Ni can be achieved for pH below 2.5.Meanwhile,a strain(named Thiooxidans.WL)accounting for the reduction of pH in the acidified sludge was isolated and sequenced.It was identified to be 100%similar to Acidithiobacillus ferrooxidans strain Tf-49 based on 16S rDNA sequence analysis.The relevant phylogenetic tree constructed indicates that the strain should be classified into genus Acidithiobacillus ferrooxidans.

Screening and Denitrification Characteristics of a Slight Halophilic Heterotrophic Nitrobacteria

A slight halophilic heterotrophic nitrobacteria named gs1 was separated from the matured activated sludge. According to the morphological observation,physiological biochemical tests and sequence analysis of the 16S rDNA,strain gs1 was identified to be as Pseudomonas sp. Sodium acetate and ammonium chloride were used as carbon and nitrogen sources,respectively,to investi-gate the characteristics of the bacterium. When cultured for 24 h under aerobic conditions,with the removal rates of the NH4+-N and COD being 82.2% and 74.73%,respectively,strain gs1 will have a nitrification function of producing NO2--N. When cultured for 24 h under aerobic conditions in nitrite medium,the removal rate of the NO2--N became 100%,and when cultured for 24 h under aerobic conditions in nitrate medium,the removal rate of the NO3--N became 97%. The result shows that this strain functions for either nitrification or denitrification,i.e.,it can complete the full process of biological deoxidation.

Isolation of perchlorate-reducing Azospira suillum strain JB524 from tidal flats of the Yellow Sea

Objective:To isolate and identify perchlorate^(-)reducing bacterium from an enriched consortium from tidal flats of the Yellow Sea.Methods:A perchlorate^(-)enriched consortium from tidal flats of the Yellow Sea was used to isolate Azospira suillum(A.suillum)strain JB524.The strain was identified based on partial 16S rDNA sequencing.Perchlorate reduction by the strain was tested with acetate as the e^(-)donor in the presence of NaCl,nitrate and at different growth temperatures using standard anaerobic techniques.The complete enzymatic destruction of perchlorate was confirmed as evolution of O2 by chlorite dismutase in the absence of acetate.Results:Strain JB524 shared 100%16S rDNA sequence similarity with the type strain A.suillum PST isolated from a swine waste treatment lagoon.Perchlorate reduction coincided with concomitant increase in cell density.Although,acclimatization of the strain PST at suboptimal temperature for perchlorate reduction is not reported,the newly isolated strain could rapidly reduce perchlorate at 22°C after brief acclimatization.Conclusions:Reduction of perchlorate by A.suillum strain JB524 was negatively affected in the presence of NaCl,suboptimal temperature,presence of nitrate,and limiting amount of acetate as the e^(-)donor.