Search Results (37)

Microbiota have emerged as a promising tool for estimating the post-mortem interval (PMI) in forensic investigations. The role of oral and nasal microbiota in cadaver decomposition is crucial; however, their distribution across human cadavers at different PMIs remains underexplored. In this study, we collected 88 swab samples from the oral and nasal cavities of 10 healthy volunteers and 34 human cadavers. Using 16S rRNA gene sequencing, we conducted comprehensive analyses of the alpha diversity, beta diversity, and relative abundance distribution to characterize the microbial communities in both healthy individuals and cadavers at varying PMIs and under different freezing conditions. Random forest models identified Firmicutes, Proteobacteria, Bacteroidota, Actinobacteriota, and Fusobacteriota as potential PMI-associated biomarkers at the phylum level for both the oral and nasal groups, along with genus-level biomarkers specific to each group. These biomarkers exhibited nonlinear changes over increasing PMI, with turning points observed on days 5, 12, and 22. The random forest inference models demonstrated that oral biomarkers at both the genus and phylum levels achieved the lowest mean absolute error (MAE) values in the training dataset (MAE = 2.16 days) and the testing dataset (MAE = 5.14 days). Additionally, freezing had minimal impact on the overall phylum-level microbial composition, although it did affect the relative abundance of certain phyla. At the genus level, significant differences in microbial biomarkers were observed between frozen and unfrozen cadavers, with the oral group showing greater stability compared to the nasal group. These findings suggest that the influence of freezing should be considered when using genus-level microbial data to estimate PMIs. Overall, our results highlight the potential of oral and nasal microbiota as robust tools for PMI estimation and emphasize the need for further research to refine predictive models and explore the environmental factors shaping microbial dynamics. Full article

Frozen shoulder (FS) is a complex and multifactorial condition characterized by persistent inflammation, fibrosis, and metabolic dysregulation. Despite extensive research, the underlying drivers of FS remain poorly understood. Recent findings indicate the coexistence of pro-inflammatory and fibrosis-resolving macrophages within affected tissues, suggesting a dysregulated immune response influenced by metabolic and neuroendocrine factors. This review proposes that leptin resistance, a hallmark of metabolic syndrome and chronic inflammation, may play a central role in FS pathogenesis by impairing macrophage polarization, perpetuating inflammation, and disrupting fibrosis resolution. The JAK-STAT signaling pathway, critically modulated by leptin resistance, may further contribute to immune dysregulation by sustaining inflammatory macrophage activation and interfering with tissue remodeling. Additionally, FS shares pathogenic features with fibrotic diseases driven by TGF-β signaling, mitochondrial dysfunction, and circadian disruption, further linking systemic metabolic dysfunction to localized fibrotic pathology. Beyond immune and metabolic regulation, alterations in gut microbiota, bacterial translocation, and chronic psychosocial stress may further exacerbate systemic inflammation and neuroendocrine imbalances, intensifying JAK-STAT dysregulation and leptin resistance. By examining the intricate interplay between metabolism, immune function, and fibrotic remodeling, this review highlights targeting leptin sensitivity, JAK-STAT modulation, and mitochondrial restoration as novel therapeutic strategies for FS treatment. Future research should explore these interconnections to develop integrative interventions that address both the metabolic and immune dysregulation underlying FS, ultimately improving clinical outcomes. Full article

Fecal microbiota transplantation (FMT) is the most effective therapy for preventing recurrent Clostridioides difficile infection (rCDI). However, the impact of FMT formulations and storage conditions on bacterial viability, community structure, functionality, and clinical efficacy remains under-investigated. We studied the effect of different storage conditions on the bacterial viability (live/dead staining and cell sorting), community structure (16S rDNA analysis), and metabolic functionality (fermentation) of frozen and lyophilized FMT formulations. The clinical success rates of rCDI patients were correlated retrospectively with FMT formulations, storage durations, and host factors using the Edmonton FMT program database. Bacterial viability remained at 10–20% across various storage conditions and formulations and was comparable to that of fresh FMT. Live and dead bacterial fractions in both frozen and lyophilized FMT preparations exhibited distinct community structures. Storage durations, but not temperatures, negatively affected bacterial diversity. More short-chain fatty acids were found in the metabolomic profiling of in vitro fermentation products using lyophilized than frozen FMT. Clinical success rates in 537 rCDI patients receiving a single dose of FMT were not significantly different among the three formulations. However, longer storage durations and advanced recipient age negatively impacted clinical efficacy. Together, our findings suggest that FMT formulations and storage durations should be considered when establishing guidelines for product shelf life for optimal treatment outcomes. Full article

During an Antarctic expedition that took place in December 2010–January 2011 in the East Antarctic coastal region, soil samples were collected in aseptic conditions and stored for over a decade in freezers at −20 °C. Due to the shortly afterward passing of the Antarctic researcher in charge, Teodor Negoiță, the samples remained unintentionally frozen for a long period and were made available for research 13 years later. A chemical analysis of soil as well as screening for viable microbial presence was performed; soil analysis was conducted via inductively coupled plasma atomic emission spectroscopy (ICP-AES) and Fourier-transform infrared spectroscopy coupled with attenuated total reflection (FTIR-ATR). The presence of aerobic and facultative aerobic microbiotas was evaluated through a Biolog Ecoplates assay, and isolated strains were 16S sequenced for final taxonomic identification. The results obtained new insights into Antarctic soil characteristics from both chemical and microbiological aspects, even after over a decade of conservation. Full article

This study explored the effects of replacing live fish feed with artificial compound feed on the growth performance, nutritional composition, digestive performance, and gut microbiota of Chinese giant salamander (Andrias davidianus). The experiment was divided into three groups, each with three biological replicates. The salamander juveniles were fed artificial compound feed (S), live fish (H), and frozen fish (D) for 184 days. Results showed no significant differences in growth performance among the three groups (p > 0.05). The water content in the S group was significantly higher than that in the H group (p < 0.05), and the crude fat content was slightly higher, whereas other nutritional components showed no significant differences (p > 0.05). The analysis of amino acid composition found no significant differences among the three groups of Chinese giant salamanders (p > 0.05). The analysis of fatty acid composition revealed that the feed group had significantly lower levels of polyunsaturated fatty acids compared with the other two groups (p < 0.05), and significantly higher levels of saturated fatty acids (p < 0.05). There were no significant changes in digestive enzyme activity among the three salamander groups (p > 0.05). There were also no significant differences in the remaining antioxidant indicators in the intestine and liver among the three groups of salamanders (p > 0.05). The S group had significantly higher Pielou_e and Shannon indices (p < 0.05), thereby indicating a greater diversity and evenness of the gut microbiota. In conclusion, replacing live feed with artificial compound feed does not affect the growth performance, nutritional composition, antioxidant capacity, and digestive function of Chinese giant salamander juveniles. Moreover, it enhances the diversity and richness of the gut microbiota, providing effective data to support the development of artificial feeds for giant salamanders. Full article

Frozen meat is an important source of protein in pet food, and has attracted much attention in recent years. In this study, we compared the effect of meat meal (MM), frozen meat (FM), and hydrolyzed frozen meat (HFM) as ingredients in extruded pet food on its palatability and apparent digestibility, as well as its effects on the immune response, fecal microbiota, and metabolome of British shorthair cats. A total of 24 British shorthair cats were allocated to the MM, FM, and HFM groups according to body weight and gender. The palatability test lasted 4 days and the feeding test lasted 45 days. The results showed that the FM and HFM diets had better palatability than the MM diet (p < 0.05) and significantly improved dry matter and crude protein digestibility (p < 0.05). The serum IL-10 level was significantly higher in the HFM group compared to the MM and FM groups (p < 0.05). The serum IgM levels were also found to be significantly higher in the FM group compared to the MM and HFM groups (p < 0.05). The blood urea nitrogen/creatinine ratio was significantly lower in the HFM and FM groups than in the MM group (p < 0.05). Cats fed HFM had a higher abundance of fecal Actinobacteria and Bifidobacterium and a lower content of Bacteroidota (p < 0.05). Furthermore, serum metabolomics analysis revealed that the tryptophan (Trp) metabolism and bile acid metabolism were affected by HFM. Overall, FM and HFM were better for the cat’s health than meat meal, but they also have some potential risks. Full article

Human health and the human microbiome are inevitably intertwined, increasing their relevance in clinical research. However, the collection, transportation and storage of faecal samples may introduce bias due to methodological differences, especially since postal shipping is a common practise in large-scale clinical cohort studies. Using four different Omics layer, we determined the structural (16S rRNA sequencing, cytometric microbiota profiling) and functional integrity (SCFAs, global metabolome) of the microbiota in relation to different easy-to-handle conditions. These conditions were storage at −20 °C, −20 °C as glycerol stock, 4 °C and room temperature with and without oxygen exposure for a maximum of one week. Storage time affected the microbiota on all Omics levels. However, the magnitude was donor-dependent, highlighting the need for purpose-optimized sample collection in clinical multi-donor studies. The effects of oxygen exposure were negligible for all analyses. At ambient temperature, SCFA and compositional profiles were stable for 24 h and 48 h, respectively, while at 4 °C, SCFA profiles were maintained for 48 h. The global metabolome was highly susceptible, already changing at 24 h in non-frozen conditions. Thus, faecal microbiota was best preserved on all levels when transported as a native sample frozen within 24 h, leading to the least biased outcomes in the analysis. We conclude that the immediate freezing of native stool samples for transportation to the lab is best suited for planned multi-Omics analyses that include metabolomics to extend standard sequencing approaches. Full article

Head and neck paragangliomas (HNPGLs) are rare neoplasms arising from paraganglia of the parasympathetic nervous system. HNPGLs are characterized by high vascularity and are located in proximity to major vessels and nerves, which may be potential sources of microbial invasion in these tumors. There have been no studies in the literature on the microbiota in HNPGLs. Investigation of the microbiome associated with paragangliomas is important for understanding tumor pathogenesis. In this study, we investigated the microbiome composition in two sets of HNPGLs. First, 29 fresh frozen (FF) tissues were subjected to 16S rRNA gene sequencing; concurrently, a panel of candidate laboratory-derived contaminants was investigated. Second, we analyzed microbial reads from whole transcriptome sequencing data obtained for 82 formalin-fixed paraffin-embedded (FFPE) HNPGLs. The bacterial diversity in FF tumors was found to be significantly lower than that observed in FFPE HNPGLs. Based on 16S rRNA gene sequencing, only seven bacterial families were identified as potential tumor inhabitants: Bryobacteraceae, Enterococcaceae, Neisseriaceae, Legionellaceae, Vibrionaceae, Obscuribacteraceae, and Mycobacteriaceae. However, RNA-Seq demonstrated higher sensitivity for identifying microbiome composition and revealed abundant bacterial families that partially correlated with those previously described in pheochromocytomas and extra-adrenal paragangliomas. No viruses were found in HNPGLs. In summary, our findings indicated the presence of a microbiome in HNPGLs, comprising a number of bacterial families that overlap with those observed in pheochromocytomas/paragangliomas and glioblastomas. Full article

Fecal Microbiota Transplantation (FMT) represents a promising therapeutic tool under study for several purposes and is currently applied to the treatment of recurrent Clostridioides difficile infection. However, since the use of fresh stool was affected by several issues linked to donor screening, the development of a frozen stool bank is a reliable option to standardize FMT procedures. Nevertheless, different environmental factors impact microbial viability. Herein, we report the effect of different thawing temperatures and storage conditions on bacterial suspensions in the FMT procedure. In total, 20 stool samples were divided into aliquots and tested across a combination of different storing periods (15, 30; 90 days) and thawing procedures (4 °C overnight, room temperature for 1 h; 37 °C for 5 min). Focusing on storage time, our data showed a significant reduction in viability for aerobic and anaerobic bacteria after thawing for 15 days, while no further reductions were observed until after 90 days. Instead, among the different thawing procedures, no significant differences were observed for aerobic bacteria, while for anaerobes, thawing at 37 °C for 5 min was more effective in preserving the bacterial viability. In conclusion, the frozen fecal microbiota remained viable for at least three months, with an excellent recovery rate in all three thawing conditions. Full article

Objectives: The aim of this study was to confirm the effectiveness of FMT on the basis of optimum composition of the faecal microbiota of the donor for support therapy in patients with ulcerative colitis, and to observe the viability of the microbiota in frozen and lyophilised administration forms of FMT under various storage conditions. Methods: The bacterial microbiota composition of the FMT samples was assessed using amplicon sequencing via next-generation sequencing (NGS) technology, conducted on the Illumina MiSeq platform. The BD FACS Canto flow cytometer was used to analyse the metabolic activity of FMT samples. Results: FMT analysis confirmed the presence of key butyrate-producing organisms, specifically highlighting species such as Bifidobacterium adolescentis, Faecalibacterium prausnitzi, Coprococcus catus, Eubacterium rectale, alongside contributions from genera Roseburia and Blautia. These organisms play a crucial role in maintaining intestinal health in humans. The viable microorganism counts were significantly higher (p < 0.001) in the frozen form of FMT (−70 °C) in comparison to lyophilised forms (−70 °C, 4 °C and 20 °C) throughout the storage period. Conclusion: The conducted NGS analyses allowed us to confirm the suitability of our FMT donor as a potential candidate for the target group of patients diagnosed with ulcerative colitis. From the point of view of optimum utilisation of FMT at its highest metabolic activity for the purpose of transplantation, its storage for a maximum of 2 months under specified conditions was confirmed as the most suitable for the frozen and all lyophilised FMT forms. Full article

The calf’s gastrointestinal tract (GIT) microbiome undergoes rapid shifts during early post-natal life, which can directly affect calf performance. The objectives of this study were to characterise and compare differences in the establishment and succession of GIT microbiota, GIT morphological changes, and the growth of dairy calves from birth until weaned. Forty-four newborn Holstein-Friesian calves were randomly selected and assigned to Treatment (TRT) and Control (CON) groups. The TRT group calves received a once-daily dose of a direct-fed microbial (DFM) liquid product containing Lacticaseibacillus paracasei, Lentilactobacillus buchneri, and Lacticaseibacillus casei, all formerly known as Lactobacillus. Fresh faecal samples were manually taken from the rectum of all calves, and gross necropsy was performed on the forestomachs and gastrointestinal tracts. Bacterial DNA was extracted from frozen faecal samples for 16S rRNA gene amplicon sequencing. Calves in the TRT group had greater live weights (p = 0.02) at weaning compared with calves in the CON group (mean = 69.18 kg, SD = 13.37 kg). The average daily live weight gain (ADG) and total feed intake were similar between the two groups. Calves in the TRT group had greater duodenum, abomasum, and reticulum weights (p = 0.05). Rumen and intestinal development (p < 0.05) and faecal microbial diversity (p < 0.05) were more pronounced in the TRT group. The relative abundances of eight genera differed (p < 0.001) between the groups. Supplementing calves with the LAB-based DFM increased live weight at weaning and had a more pronounced effect on the development of rumen and the gastrointestinal tract and on microbiota diversity and evenness. Future work is needed to better understand the potential association of LAB-DFM products on gut mucosa-associated microbiota. Full article

Natural starter cultures, characterised by undefined microbiota, can contribute to the technological process, giving peculiar characteristics to artisanal fermented foods. Several species have a long history of safe use and have obtained Qualified Presumption of Safety (QPS) status from the European Food Safety Authority (EFSA), whereas others (non-QPS) could represent a potential risk for consumers’ health and must undergo a safety assessment. In this work, the biodiversity, at species and strain level, by pulsed-field gel electrophoresis (PFGE) and (GTG)₅ rep-PCR, of an undefined natural starter culture, in frozen and lyophilized form, obtained from ewe’s raw milk avoiding thermal treatment or microbial selection, was investigated. The culture was constituted by different biotypes of Enterococcus durans, Enterococcus faecium, Enterococcus faecalis, and Lacticaseibacillus paracasei. Streptococcus oralis and Streptococcus salivarius were also found, over species belonging to the Streptococcus bovis–Streptococcus equinus complex (SBSEC), like Streptococcus gallolyticus subsp. macedonicus, Streptococcus lutetiensis, and Streptococcus equinus. Molecular investigation on virulence and antibiotic resistance genes, as well as minimum inhibitory concentration (MIC) determination, revealed that all the non-QPS strains can be considered safe in the perspective of using this culture for cheesemaking. The obtainment of a natural culture directly from ewe’s raw milk bypassing thermal treatment and selection of pro-technological bacteria can be advantageous in terms of biodiversity preservation, but non-QPS microorganisms can be included in the natural starter and also in cheeses, especially in traditional ones obtained from fermenting raw milk. Following EFSA guidelines, artisanal factories should not be allowed to produce starter cultures by themselves from raw milk, running the risk of including some non-QPS species in their culture, and only selected starters could be used for cheesemaking. A revision of the criteria of QPS guidelines should be necessary. Full article

The bacterial community of the intestinal microbiota influences many host functions, and similar effects have been recently reported for the fungal community (mycobiota). Cobia is a tropical fish that has been studied for its potential in marine aquaculture. However, the study of its bacterial community has been underreported and the mycobiota has not been investigated. We analyzed the gut bacterial and fungal profile present in the intestinal mucosa of reared adult cobias fed two diets (frozen fish pieces (FFPs) and formulated feed (FF)) for 4 months by sequencing the 16S rRNA (V3-V4) and internal transcribed spacer-2 (ITS2) regions using Illumina NovaSeq 6000. No significant differences in the alpha diversity of the bacterial community were observed, which was dominated by the phyla Proteobacteria (~96%) and Firmicutes (~1%). Cobia fed FF showed higher abundance of 10 genera, mainly UCG-002 (Family Oscillospiraceae) and Faecalibacterium, compared to cobia fed FFPs, which showed higher abundance of 7 genera, mainly Methylobacterium-Methylorubrum and Cutibacterium. The inferred bacterial functions were related to metabolism, environmental information processing and cellular processes; and no differences were found between diets. In mycobiota, no differences were observed in the diversity and composition of cobia fed the two diets. The mycobiota was dominated by the phyla Ascomycota (~88%) and Basidiomycota (~11%). This is the first study to describe the gut bacterial and fungal communities in cobia reared under captive conditions and fed on different diets and to identify the genus Ascobulus as a new member of the core fish mycobiota. Full article

The aquatic environment is a reservoir of many species that have not yet been exploited at a global level and have not been extensively investigated. The aim of the present work was to study the microbial populations, the bacterial communities and physicochemical parameters (pH, water activity, humidity, salinity) in raw, frozen, boiled, dehydrated and salted final products of two sea cucumber species (Holothuria polii and Holothuria tubulosa) originating from the Hellenic seawaters. The results indicated that all products were found at acceptable levels from a microbiological point of view. The metabarcoding analysis of the 16S rRNA gene revealed the existence of several different bacterial groups, the presence and abundance of which were mainly dependent on product type, even though some moderate differences in the microbiota profile between the two sea cucumber species were also detected in some of the products. Overall, the present work deals with an underexplored aquatic product and provides novel and useful information for the aquatic food industry, consumers and other stakeholders, increasing, in parallel, the need for further scientific attention in the near future. Our findings could be exploited as a baseline to highlight a promising aquatic food product provided to the international market. Full article

Fecal microbiota transplantation (FMT) is under investigation for several indications, including ulcerative colitis (UC). The clinical success of FMT depends partly on the engraftment of viable bacteria. Because the vast majority of human gut microbiota consists of anaerobes, the currently used aerobic processing protocols of donor stool may diminish the bacterial viability of transplanted material. This study assessed the effect of four processing techniques for donor stool (i.e., anaerobic and aerobic, both direct processing and after temporary cool storage) on bacterial viability. By combining anaerobic culturing on customized media for anaerobes with 16S rRNA sequencing, we could successfully culture and identify the majority of the bacteria present in raw fecal suspensions. We show that direct anaerobic processing of donor stool is superior to aerobic processing conditions for preserving the bacterial viability of obligate anaerobes and butyrate-producing bacteria related to the clinical response to FMT in ulcerative colitis patients, including Faecalibacterium, Eubacterium hallii, and Blautia. The effect of oxygen exposure during stool processing decreased when the samples were stored long-term. Our results confirm the importance of sample conditioning to preserve the bacterial viability of oxygen-sensitive gut bacteria. Anaerobic processing of donor stool may lead to increased clinical success of FMT, which should further be investigated in clinical trials. Full article