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Body fluid identification is an essential step in the forensic biology workflow that can assist DNA analysts in determining where to collect DNA evidence. Current presumptive tests lack the specificity that molecular techniques can achieve; therefore, molecular methods, including microRNA (miRNA) and microbial signature characterization, have been extensively researched in the forensic community. Limitations of each method suggest combining molecular markers to increase the discrimination efficiency of multiple body fluids from a single assay. While microbial signatures have been successful in identifying fluids with high bacterial abundances, microRNAs have shown promise in fluids with low microbial abundance (blood and semen). This project synergized the benefits of microRNAs and microbial DNA to identify multiple body fluids using DNA extracts. A reverse transcription (RT)‐qPCR duplex targeting miR‐891a and let‐7g was validated, and miR‐891a differential expression was significantly different between blood and semen. The miRNA duplex was incorporated into a previously reported qPCR multiplex targeting 16S rRNA genes of Lactobacillus crispatus, Bacteroides uniformis, and Streptococcus salivarius to presumptively identify vaginal/menstrual secretions, feces, and saliva, respectively. The combined classification regression tree model resulted in the presumptive classification of five body fluids with 94.6% overall accuracy, now including blood and semen identification. These results provide proof of concept that microRNAs and microbial DNA can classify multiple body fluids simultaneously at the quantification step of the current forensic DNA workflow. [ABSTRACT FROM AUTHOR]

Molecular methods for body fluid identification have been extensively researched in the forensic community over the last decade, mostly focusing on RNA‐based methods. Microbial DNA analysis has long been used for forensic applications, such as postmortem interval estimations, but only recently has it been applied to body fluid identification. High‐throughput sequencing of the 16S ribosomal RNA gene by previous research groups revealed that microbial signatures and abundances vary across human body fluids at the genus and/or species taxonomic level. Since quantitative PCR is still the current technique used in forensic DNA analysis, the purpose of this study was to design a qPCR multiplex targeting the 16S gene of Bacteroides uniformis, Streptococcus salivarius, and Lactobacillus crispatus that can distinguish between feces, saliva, and vaginal/menstrual secretions, respectively. Primers and probes were designed at the species level because these bacteria are highly abundant within their respective fluid. The validated 16S triplex was evaluated in DNA extracts from thirty donors of each body fluid. A classification regression tree model resulted in 96.5% classification accuracy of the population data, which demonstrates the ability of this 16S triplex to presumptively identify these fluids with high confidence at the quantification step of the forensic workflow using minimal input volume of DNA extracted from evidentiary samples. [ABSTRACT FROM AUTHOR]

Introduction: Bacterial pleuritis is one of the most important pleural and respiratory infectious diseases, in addition, there have been no reports describing the clinical characteristics of patients with bacterial pleuritis according to molecular methods. An accurate understanding of the clinical characteristics and etiology of bacterial pleuritis is an issue that must be addressed. Objectives: The aim of this study was to clarify the clinical characteristics of the bacterial species in bacterial pleuritis. Methods: Pleural effusion samples were obtained from 29 patients with bacterial pleuritis. The microbiota of pleural effusion samples was analyzed by clone library analysis using the 16S ribosomal RNA gene. Results: The phylotypes of Fusobacterium spp. (24.1%) were most frequently the predominant phylotypes, followed by those of Streptococcus anginosus group (SAG) (20.7%) and S. aureus (17.2%). The predominant phylotypes of obligate anaerobes, including the Fusobacterium spp., were detected in 11 of 29 patients (37.9%). Patients in the SAG group were significantly older and presented lower serum albumin levels than those in the obligate anaerobe and other bacterial groups. Patients from the obligate anaerobe group took longer to present symptoms, and therefore the diagnosis of pleuritis was also delayed, in comparison to patients in the other bacterial groups. Conclusions: Our results demonstrated that there were characteristic differences between patients in SAG, obligate anaerobe and other bacterial groups. Physicians may need to consider treatment strategy options based on the clinical characteristics of patients with bacterial pleuritis. [ABSTRACT FROM AUTHOR]

Background and objective Recent advances in cultivation-independent molecular biological modalities for detecting bacterial species have indicated that several bacterial species may play a role in the pathogenesis of certain infectious diseases. The aim of this study was to evaluate the role of bacterial flora in the pathogenesis of nontuberculous mycobacteriosis ( NTM) using a bacterial floral analysis of bronchoalveolar lavage fluid ( BALF) with 16S rRNA gene sequencing in patients with bronchiectasis. Methods Fifty-eight patients with bronchiectasis evaluated using chest computed tomography were enrolled. BALF obtained from the most affected lung lesions was evaluated using culture and culture-independent methodologies. Approximately 600 bp of the bacterial 16S rRNA gene ( E341F-E907R) was amplified via polymerase chain reaction using universal primers, and clone libraries were constructed. Nucleotide sequences of 96 randomly chosen clones for each specimen were determined, and the homology was searched using a basic local alignment search tool to determine the bacterial phylotypes and their proportions (bacterial floral analysis) in each specimen. Results Twenty-nine patients with bronchiectasis were diagnosed with NTM based on culture-based methods using Ogawa medium. The molecular method showed a significantly high rate of anaerobes among the patients with NTM compared with that observed in the bronchiectasis patients without NTM. In addition, findings of collapse/consolidation were significantly related to the proportion of Prevotella species in the BALF samples determined using the molecular method ( P < 0.001). Conclusion Given the results of the present study, anaerobes may play an important role in the pathogenesis of bronchiectasis in patients with NTM. [ABSTRACT FROM AUTHOR]

Faveri M, Gonçalves LFH, Feres M, Figueiredo LC, Gouveia LA, Shibli JA, Mayer MPA. Prevalence and microbiological diversity of Archaea in peri-implantitis subjects by 16S ribosomal RNA clonal analysis. J Periodont Res 2011; 46: 338-344. © 2011 John Wiley & Sons A/S This study evaluated the prevalence and the molecular diversity of Archaea in the subgingival biofilm samples of subjects with peri-implantitis. Fifty subjects were assigned into two groups: Control ( n = 25), consisting of subjects with healthy implants; and Test ( n = 25), consisting of subjects with peri-implantitis sites, as well as a healthy implant. In the Test group, subgingival biofilm samples were taken from the deepest sites of the diseased implant. In both groups, subgingival biofilm was collected from one site with a healthy implant and from one site with a periodontally healthy tooth. DNA was extracted and the 16S ribosomal RNA gene was amplified with universal primer pairs for Archaea. Amplified genes were cloned and sequenced, and the phylotypes were identified by comparison with known 16S ribosomal RNA sequences. In the Control group, Archaea were detected in two and three sites of the implant and the tooth, respectively. In the Test group, Archaea were detected in 12, 4 and 2 sites of diseased implants, healthy implants and teeth, respectively. Diseased implants presented a significantly higher prevalence of Archaea in comparison with healthy implants and natural teeth, irrespective of group. Over 90% of the clone libraries were formed by Methanobrevibacter oralis, which was detected in both groups. Methanobacterium congelense/curvum was detected in four subjects from the Test group and in two subjects from the Control group. Although M. oralis was the main species of Archaea associated with both healthy and diseased implant sites, the data indicated an increased prevalence of Archaea in peri-implantitis sites, and their role in pathogenesis should be further investigated. [ABSTRACT FROM AUTHOR]

Bacterial content may be helpful in differentiating forensic soil samples; however, the effectiveness of bacterial profiling depends on several factors, including uniqueness among different habitat types, the level of heterogeneity within a habitat, and changes in bacterial communities over time. To examine these, soils from five diverse habitats were tested over a 1 year period using terminal restriction fragment length polymorphism (TRFLP) analysis. Soil samples were collected at central locations monthly, and 10 feet in cardinal directions quarterly. Similarity indices were found to be least related among habitats, while the greatest bacterial similarities existed among collection locations within a habitat. Temporally, however, bacterial content varied considerably, and there was substantial overlap in similarity indices among habitats during different parts of the year. Taken together, the results indicate that while bacterial DNA profiling may be useful for forensic soil analysis, certain variables, particularly time, must be considered. [ABSTRACT FROM AUTHOR]

We used high-throughput DNA sequencing methods combined with bio-geochemical profiles to characterize the internal environment and community structure of the microbiome of the basidiomycete fungus Pisolithus arhizus (Scop.) Rauschert from soils within a geothermal feature of Yellowstone National Park. Pisolithus arhizus is unique in that it forms closed fruiting bodies that sequester visible sulfur within. Fourier transform infrared spectroscopy (FTIR) analysis demonstrates that the P. arhizus fruiting body also concentrates copper, manganese, nickel, and zinc and contains pure granular silica. Gas chromatography-mass spectrometry (GC-MS) analysis indicates an environment rich in hydrocarbons. Oxygen probe analysis reveals that zones of up to 4× atmospheric oxygen exist within nanometers of zones of near anoxia. Analysis of microbial community structure using high-throughput DNA sequencing methods shows that the fruiting body supports a microbiome that reflects the physiochemical environment of the fruiting body. Diversity and richness measures indicate a microbiome that is significantly richer and more diverse than that of the soils in which P. arhizus grows. Further, P. arhizus sporocarps are enriched significantly in Proteobacteria (primarily Burkholderia) Gemmatimonadetes, Bacteroidetes, Verrucomicrobia, Nitrospirae, Elusimicrobia, and Latescibacteria (WS3) while soils are enriched in Actinobacteria (primarily Mycobacterium), Dormibacteraeota (AD3), and Eremiobacteraeota (WPS-2). Finally, pairwise % similarity comparisons indicate that P. arhizus harbors two lineages that may represent new groups in the candidate phylum radiation (CPR). Together, these results demonstrate that P. arhizus provides a novel environment for microbiome studies and provides for interesting hypotheses regarding the evolution, origins, and functions of symbioses and novel microbes., (© 2020 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)