Proteomic characterization and biological activities of the mucus produced by the zoanthid Palythoa caribaeorum (Duchassaing & Michelotti, 1860).

Mucus, produced by Palythoa caribaeorum has been popularly reported due to healing, anti-inflammatory, and analgesic effects. However, biochemical and pharmacological properties of this mucus remains unexplored. Therefore, the present study aimed to study its proteome profile by 2DE electrophoresis and MALDI-TOF. Furthermore, it was evaluated the cytotoxic, antibacterial, and antioxidant activities of the mucus and from its protein extract (PE). Proteomics study identified14 proteins including proteins involved in the process of tissue regeneration and death of tumor cells. The PE exhibited cell viability below 50% in the MCF-7 and S-180 strains. It showed IC50 of 6.9 µg/mL for the J774 lineage, and also, favored the cellular growth of fibroblasts. Furthermore, PE revealed activity against Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Staphylococcus epidermidis (MIC of 250 µg/mL). These findings revealed the mucus produced by Palythoa caribaeorum with biological activities, offering alternative therapies for the treatment of cancer and as a potential antibacterial agent.

Development of an In Vitro Model of the Gut Microbiota Enriched in Mucus-Adhering Bacteria.

Culturing the gut microbiota in in vitro models that mimic the intestinal environment is increasingly becoming a promising alternative approach to study microbial dynamics and the effect of perturbations on the gut community. Since the mucus-associated microbial populations in the human intestine differ in composition and functions from their luminal counterpart, we attempted to reproduce in vitro the microbial consortia adhering to mucus using an already established three-dimensional model of the human gut microbiota. Electrospun gelatin structures supplemented or not with mucins were inoculated with fecal samples and compared for their ability to support microbial adhesion and growth over time, as well as to shape the composition of the colonizing communities. Both scaffolds allowed the establishment of long-term stable biofilms with comparable total bacterial loads and biodiversity. However, mucin-coated structures harbored microbial consortia especially enriched in Akkermansia, Lactobacillus, and Faecalibacterium, being therefore able to select for microorganisms commonly considered mucosa-associated in vivo. IMPORTANCE These findings highlight the importance of mucins in shaping intestinal microbial communities, even those in artificial gut microbiota systems. We propose our in vitro model based on mucin-coated electrospun gelatin structures as a valid device for studies evaluating the effects of exogenous factors (nutrients, probiotics, infectious agents, and drugs) on mucus-adhering microbial communities.

Mimicking the Gastrointestinal Mucus Barrier: Laboratory-Based Approaches to Facilitate an Enhanced Understanding of Mucus Permeation.

The gastrointestinal mucus layer plays a significant role in maintaining gut homeostasis and health, offering protective capacities against the absorption of harmful pathogens as well as commensal gut bacteria and buffering stomach acid to protect the underlying epithelium. Despite this, the mucus barrier is often overlooked during preclinical pharmaceutical development and may pose a significant absorption barrier to high molecular weight or lipophilic drug species. The complex chemical and physical nature of the dynamic mucus layer has proven problematic to reliably replicate in a laboratory setting, leading to the development of multiple mucus models with varying complexity and predictive capacity. This, coupled with the wide range of analysis methods available, has led to a plethora of possible approaches to quantifying mucus permeation; however, the field remains significantly under-represented in biomedical research. For this reason, the development of a concise collation of the available approaches to mucus permeation is essential. In this review, we explore widely utilized mucus mimics ranging in complexity from simple mucin solutions to native mucus preparations for their predictive capacity in mucus permeation analysis. Furthermore, we highlight the diverse range of laboratory-based models available for the analysis of mucus interaction and permeability with a specific focus on in vitro, ex vivo, and in situ models. Finally, we highlight the predictive capacity of these models in correlation with in vivo pharmacokinetic data. This review provides a comprehensive and critical overview of the available technologies to analyze mucus permeation, facilitating the efficient selection of appropriate tools for further advancement in oral drug delivery.

Differential analysis of microbiomes in mucus and tissues obtained from colorectal cancer patients.

The outer mucus layer of the colorectal epithelium is easily removable and colonized by commensal microbiota, while the inner mucus layer is firmly attached to the epithelium and devoid of bacteria. Although the specific bacteria penetrating the inner mucus layer can contact epithelial cells and trigger cancer development, most studies ignore the degree of mucus adhesion at sampling. Therefore, we evaluated whether bacteria adhering to tissues could be identified by removing the outer mucus layer. Our 16S rRNA gene sequencing analysis of 18 surgical specimens of human colorectal cancer revealed that Sutterella (P = 0.045) and Enterobacteriaceae (P = 0.045) were significantly enriched in the mucus covering the mucosa relative to the mucosa. Rikenellaceae (P = 0.026) was significantly enriched in the mucus covering cancer tissues compared with those same cancer tissues. Ruminococcaceae (P = 0.015), Enterobacteriaceae (P = 0.030), and Erysipelotrichaceae (P = 0.028) were significantly enriched in the mucus covering the mucosa compared with the mucus covering cancers. Fusobacterium (P = 0.038) was significantly enriched in the mucus covering cancers compared with the mucus covering the mucosa. Comparing the microbiomes of mucus and tissues with mucus removed may facilitate identifying bacteria that genuinely invade tissues and affect tumorigenesis.

Colon mucus in colorectal neoplasia and beyond.

Little was known about mammalian colon mucus (CM) until the beginning of the 21st century. Since that time considerable progress has been made in basic research addressing CM structure and functions. Human CM is formed by two distinct layers composed of gel-forming glycosylated mucins that are permanently secreted by goblet cells of the colonic epithelium. The inner layer is dense and impenetrable for bacteria, whereas the loose outer layer provides a habitat for abundant commensal microbiota. Mucus barrier integrity is essential for preventing bacterial contact with the mucosal epithelium and maintaining homeostasis in the gut, but it can be impaired by a variety of factors, including CM-damaging switch of commensal bacteria to mucin glycan consumption due to dietary fiber deficiency. It is proven that impairments in CM structure and function can lead to colonic barrier deterioration that opens direct bacterial access to the epithelium. Bacteria-induced damage dysregulates epithelial proliferation and causes mucosal inflammatory responses that may expand to the loosened CM and eventually result in severe disorders, including colitis and neoplastic growth. Recently described formation of bacterial biofilms within the inner CM layer was shown to be associated with both inflammation and cancer. Although obvious gaps in our knowledge of human CM remain, its importance for the pathogenesis of major colorectal diseases, comprising inflammatory bowel disease and colorectal cancer, is already recognized. Continuing progress in CM exploration is likely to result in the development of a range of new useful clinical applications addressing colorectal disease diagnosis, prevention and therapy.

Mucus, commensals, and the immune system.

The immune system in the large intestine is separated from commensal microbes and comparatively rare enteric pathogens by a monolayer of diverse epithelial cells overlaid with a compact and adherent inner mucus layer and a looser outer mucus layer. Microorganisms, collectively referred to as the mucus-associated (MA) microbiota, physically inhabit this mucus barrier, resulting in a dynamic and incessant dialog to maintain both spatial segregation and immune tolerance. Recent major findings reveal novel features of the crosstalk between the immune system and mucus-associated bacteria in health and disease, as well as disease-related peripheral immune signatures indicative of host responses to these organisms. In this brief review, we integrate these novel observations into our overall understanding of host-microbiota mutualism at the colonic mucosal border and speculate on the significance of this emerging knowledge for our understanding of the prevention, development, and progression of chronic intestinal inflammation.

Preliminary Comparison of Endoscopic Brush and Net Catheters as the Sampling Tool to Analyze the Intestinal Mucus in the Rectum with Ulcerative Colitis Patients.

BACKGROUND: The pathophysiology of ulcerative colitis (UC) remains unclear, but early lesions on the colorectal mucosal surface may play an important role in its etiology. Intestinal mucus samples, including inner and outer layers, are collected by net or brush catheters, but the quality of the samples obtained by each method has not been fully investigated. OBJECTIVE: The purpose of this study was to compare the microbiome and protein content of intestinal mucus collected by net and brush catheters during colonoscopy. METHODS: Intestinal mucus samples from the lower rectum of 4 patients with UC were collected using a net catheter, a brush catheter, and intestinal fluid suction. Microbiome and protein content were analyzed using 16S rRNA gene sequencing and mass spectrometry. RESULTS: The patients demonstrated significant differences in microbiome alpha diversity (p < 0.05), but this difference was not observed between the sampling methods. Net catheter samples demonstrated higher total protein concentrations than brush catheter samples. The brush catheter group had more Lachnospira, a butyrate-producing bacterium, when compared to the net group. The brush catheter group also had more oral bacteria of Staphylococcus and Dialister in those with active phase when compared to the net group. CONCLUSIONS: Brush catheters are more likely to collect the intestinal mucus inner layer, whereas net catheters are more likely to collect larger samples that include the outer mucus layer, as well as the intestinal fluid. Two sampling methods with different types of collection of the mucosa may lead to different results among patients with mucosal vulnerabilities.

Microbial diversity of garden snail mucus.

The search for new natural compounds for application in medicine and cosmetics is a trend in biotechnology. One of the sources of such active compounds is the snail mucus. Snail physiology and the biological activity of their fluids (especially the mucus) are still poorly studied. Only a few previous studies explored the relationship between snails and their microbiome. The present study was focused on the biodiversity of the snail mucus used in the creation of cosmetic products, therapeutics, and nutraceuticals. The commonly used cultivation techniques were applied for the determination of the number of major bacterial groups. Fluorescence in situ hybridization for key taxa was performed. The obtained images were subjected to digital image analysis. Sequencing of the 16S rRNA gene was also done. The results showed that the mucus harbors a rich bacterial community (10.78 × 1010 CFU/ml). Among the dominant bacteria, some are known for their ability to metabolize complex polysaccharides or are usually found in soil and plants (Rhizobiaceae, Shewanella, Pedobacter, Acinetobacter, Alcaligenes). The obtained data demonstrated that the snail mucus creates a unique environment for the development of the microbial community that differs from other parts of the animal and which resulted from the combined contribution of the microbiomes derived from the soil, plants, and the snails.

Adhesion properties of cell surface proteins in Lactobacillus strains in the GIT environment.

As a broadly defined member of lactic acid bacteria (LAB), the Lactobacillus strain is well characterized in food fermentation and specific strains can enhance the intestinal barrier function and be recognized as the probiotic strain. In recent years, many molecules of the cell surface are thought to be related to the adhesion property in the gastrointestinal mucosa. Mucus layer-related proteins, extracellular matrix proteins, and immunoglobulins also exhibit immunity regulation and protection of the intestinal epithelial barrier function. Meanwhile, the effects of bile and the low pH of the gastrointestinal tract (GIT) on Lactobacillus colonization are also needed to be considered. Furthermore, LAB can adhere and aggregate in the GIT to promote the maturity of biofilm and the extracellular matrix secreting through the signal molecules in the quorum sensing (QS) system. Therefore, it is of great interest to use the QS system to regulate the initial adhesion ability of Lactobacillus and further enhance the probiotic effect of the biofilm formation of beneficial bacteria. This review summarizes the adhesion properties of cell surface proteins derived from Lactobacillus strains in recent studies and provides valuable information on the QS effect on the adhesion property of Lactobacillus strains in the GIT environment.

A Lateral Flow Test for Staphylococcus aureus in Nasal Mucus Using a New DNAzyme as the Recognition Element.

Detection of pathogenic bacteria in complex biological matrices remains a major challenge. Herein, we report the selection and optimization of a new DNAzyme for Staphylococcus aureus (SA) and the use of the DNAzyme to develop a simple lateral flow device (LFD) for detection of SA in nasal mucus. The DNAzyme was generated by in vitro selection using a crude extra/intracellular mixture derived from SA, which could be used directly for simple solution or paper-based fluorescence assays for SA. The DNAzyme was further modified to produce a DNA cleavage fragment that acted as a bridging element to bind DNA-modified gold nanoparticles to the test line of a LFD, producing a simple colorimetric dipstick test. The LFD was evaluated with nasal mucus samples spiked with SA, and demonstrated that SA detection was possible in minutes with minimal sample processing.

Intestinal mucus barrier: a missing piece of the puzzle in food allergy.

The prevalence of food allergies has reached epidemic levels but the cause remains largely unknown. We discuss the clinical relevance of the gut mucosal barrier as a site for allergic sensitization to food. In this context, we focus on an important but overlooked part of the mucosal barrier in pathogenesis, the glycoprotein-rich mucus layer, and call attention to both beneficial and detrimental aspects of mucus-gut microbiome interactions. Studying the intricate links between the mucus barrier, the associated bacteria, and the mucosal immune system may advance our understanding of the mechanisms and inform prevention and treatment strategies in food allergy.

Arcobacter vandammei sp. nov., isolated from the rectal mucus of a healthy pig.

A study on the polyphasic taxonomic classification of an Arcobacter strain, R-73987T, isolated from the rectal mucus of a porcine intestinal tract, was performed. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the strain could be assigned to the genus Arcobacter and suggested that strain R-73987T belongs to a novel undescribed species. Comparative analysis of the rpoB gene sequence confirmed the findings. Arcobacter faecis LMG 28519T was identified as its closest neighbour in a multigene analysis based on 107 protein- encoding genes. Further, whole-genome sequence comparisons by means of average nucleotide identity and in silico DNA-DNA hybridization between the genome of strain R-73987T and the genomes of validly named Arcobacter species resulted in values below 95-96 and 70  %, respectively. In addition, a phenotypic analysis further corroborated the conclusion that strain R-73987T represents a novel Arcobacter species, for which the name Arcobacter vandammei sp. nov. is proposed. The type strain is R-73987T (=LMG 31429T=CCUG 75005T). This appears to be the first Arcobacter species recovered from porcine intestinal mucus.

Gut Microbiota and Dietary Factors as Modulators of the Mucus Layer in Inflammatory Bowel Disease.

The gastrointestinal tract is optimized to efficiently absorb nutrients and provide a competent barrier against a variety of lumen environmental compounds. Different regulatory mechanisms jointly collaborate to maintain intestinal homeostasis, but alterations in these mechanisms lead to a dysfunctional gastrointestinal barrier and are associated to several inflammatory conditions usually found in chronic pathologies such as inflammatory bowel disease (IBD). The gastrointestinal mucus, mostly composed of mucin glycoproteins, covers the epithelium and plays an essential role in digestive and barrier functions. However, its regulation is very dynamic and is still poorly understood. This review presents some aspects concerning the role of mucus in gut health and its alterations in IBD. In addition, the impact of gut microbiota and dietary compounds as environmental factors modulating the mucus layer is addressed. To date, studies have evidenced the impact of the three-way interplay between the microbiome, diet and the mucus layer on the gut barrier, host immune system and IBD. This review emphasizes the need to address current limitations on this topic, especially regarding the design of robust human trials and highlights the potential interest of improving our understanding of the regulation of the intestinal mucus barrier in IBD.

Elucidating bacterial adhesion to mucosal surface by an original AFM approach.

BACKGROUND: Fish skin represents an ancient vertebrate mucosal surface, sharing characteristics with other mucosal surfaces including those of the intestine. The skin mucosa is continuously exposed to microbes in the surrounding water and is therefore important in the first line defense against environmental pathogens by preventing bacteria from accessing the underlying surfaces. Understanding the microbe-host interactions at the fish skin mucosa is highly relevant in order to understand and control infection, commensalism, colonization, persistence, infection, and disease. Here we investigate the interactions between the pathogenic bacteria Aeromonas salmonicida (A. salmonicida) and Yersinia ruckeri (Y. ruckeri), respectively, and the skin mucosal surface of Atlantic salmon fry using AFM force spectroscopy. RESULTS: The results obtained revealed that when retracting probes functionalized with bacteria from surfaces coated with immobilized mucins, isolated from salmon mucosal surfaces, rupture events reflecting the disruption of adhesive interactions were observed, with rupture strengths centered around 200 pN. However, when retracting probes functionalized with bacteria from the intact mucosal surface of salmon fish fry no adhesive interactions could be detected. Furthermore, rheological measurements revealed a near fluid-like behavior for the fish fry skin mucus. Taken together, the experimental data indicate that the adhesion between the mucin molecules within the mucous layer may be significantly weaker than the interaction between the bacteria and the mucin molecules. The bacteria, immobilized on the AFM probe, do bind to individual mucins in the mucosal layer, but are released from the near fluid mucus with little resistance upon retraction of the AFM probe, to which they are immobilized. CONCLUSION: The data provided in the current paper reveal that A. salmonicida and Y. ruckeri do bind to the immobilized mucins. However, when retracting the bacteria from intact mucosal surfaces, no adhesive interactions are detected. These observations suggest a mechanism underlying the protective function of the mucosal surface based on the clearing of potential threats by adhering them to loosely attached mucus that is subsequently released from the fish skin.

Impact of dietary porcine blood by-products in meagre (Argyrosomus regius) physiology, evaluated by welfare biomarkers and the antibacterial properties of the skin mucus.

Tools are required for quick and easy preliminary evaluation of functional feeds efficiency on fisheries. The analysis of skin mucus biomarkers is a recent alternative approach providing a faster feed-back from the laboratory which is characterized by being less invasive, more rapid and with reduced costs. The effect of replacing fishmeal and fish protein hydrolysates by means of two porcine by-products, the porcine spray-dried plasma (SDPP) and pig protein hydrolysate (PPH), in compound diets (50.4% crude protein, 16.2% crude protein, 22.1 MJ/kg feed) was evaluated in juvenile meagre (Argyrosomus regius) during a two-months period. To determine the impact of these dietary replacements, growth and food performance were measured together with digestive enzymes activities and filet proximal composition. Additionally, skin mucus was collected and characterized by determining main mucus biomarkers (protein, glucose, lactate, cortisol, and antioxidant capacity) and its antibacterial properties, measured by the quick in vitro co-culture challenges. In comparison to the control group, the inclusion of PPH and SDPP, in meagre diets reduced growth (7.4-8.8% in body weight), increased feed conversion ratios (9.0-10.0%), results that were attributed to a reduction in feed intake values (24.2-33.0%) (P < 0.05). Porcine blood by-products did not modify the activity of gastric and pancreatic digestive enzymes as well as those involved in nutrient absorption (alkaline phosphatase) nor liver oxidative stress condition (P > 0.05). In contrast, a reduction in fillet lipid content associated to an increase in fillet protein levels were found in fish fed SDPP and PPH diets (P < 0.05). As compared to the control diet, the dietary replacement did not alter the levels of the skin mucus biomarkers related to stress (cortisol and antioxidant capacity) or nutritional status (soluble protein, glucose and lactate) (P > 0.05). Interestingly, regardless of the worst performance in somatic growth, meagre fed diets containing both tested porcine by-products showed a significantly improved antibacterial capacity of their skin mucus. This enhancement was more prominent for fish fed with the PPH diet, which may be attributed to a higher content of immunomodulatory bioactive compounds in PPH. Further research will be necessary to provide insights on how the inclusion of SDPP and PPH, at the expense of dietary fishmeal and fish protein hydrolysates, affects feed intake and growth performance in meagre. However, the use of skin mucus biomarkers has been demonstrated to be an excellent methodology for a preliminary characterization of the functional feeds, in particular for their prophylactic properties by the study of mucus antibacterial activity.

Cohousing-mediated microbiota transfer from milk bioactive components-dosed mice ameliorate colitis by remodeling colonic mucus barrier and lamina propria macrophages.

Human milk oligosaccharides (HMOs) and milk fat globule membrane (MFGM) are highly abundant in breast milk, and have been shown to exhibit potent immunomodulatory effects. Yet, their role in the gut microbiota modulation in relation to colitis remains understudied. Since the mixtures of fructo-oligosaccharides (FOS) and galacto-oligosaccharides (GOS) perfectly mimic the properties and functions of HMOs, the combination of MFGM, FOS, and GOS (CMFG) has therefore been developed and used in this study. Here, CMFG were pre-fed to mice for three weeks to investigate its preventive effect on dextran sodium sulfate (DSS) induced colitis. Moreover, CMFG-treated and vehicle-treated mice were cohoused to further elucidate the preventive role of the gut microbiota transfer in colitis. At the end of the study, 16S rDNA gene amplicon sequencing, short-chain fatty acids (SCFAs) profiling, transcriptome sequencing, histological analysis, immunofluorescence staining and flow cytometry analysis were conducted. Our results showed that CMFG pre-supplementation alleviated DSS-induced colitis as evidenced by decreased disease activity index (DAI) score, reduced body weight loss, increased colon length and mucin secretion, and ameliorated intestinal damage. Moreover, CMFG reduced macrophages in the colon, resulting in decreased levels of IL-1ß, IL-6, IL-8, TNF-α, and MPO in the colon and circulation. Furthermore, CMFG altered the gut microbiota composition and promoted SCFAs production in DSS-induced colitis. Markedly, the cohousing study revealed that transfer of gut microbiota from CMFG-treated mice largely improved the DSS-induced colitis as evidenced by reduced intestinal damage and decreased macrophages infiltration in the colon. Moreover, transfer of the gut microbiota from CMFG-treated mice protected against DSS-induced gut microbiota dysbiosis and promotes SCFAs production, which showed to be associated with colitis amelioration. Collectively, these findings demonstrate the beneficial role of CMFG in the gastrointestinal diseases, and further provide evidence for the rational design of effective prophylactic functional diets in both animals and humans.

Carvacrol, Thymol, and Garlic Essential Oil Promote Skin Innate Immunity in Gilthead Seabream (Sparus aurata) Through the Multifactorial Modulation of the Secretory Pathway and Enhancement of Mucus Protective Capacity.

One of the main targets for the use of phytogenics in aquafeeds is the mucosal tissues as they constitute a physical and biochemical shield against environmental and pathogenic threats, comprising elements from both the innate and acquired immunity. In the present study, the modulation of the skin transcriptional immune response, the bacterial growth capacity in skin mucus, and the overall health condition of gilthead seabream (Sparus aurata) juveniles fed a dietary supplementation of garlic essential oil, carvacrol, and thymol were assessed. The enrichment analysis of the skin transcriptional profile of fish fed the phytogenic-supplemented diet revealed the regulation of genes associated to cellular components involved in the secretory pathway, suggesting the stimulation, and recruitment of phagocytic cells. Genes recognized by their involvement in non-specific immune response were also identified in the analysis. The promotion of the secretion of non-specific immune molecules into the skin mucus was proposed to be involved in the in vitro decreased growth capacity of pathogenic bacteria in the mucus of fish fed the phytogenic-supplemented diet. Although the mucus antioxidant capacity was not affected by the phytogenics supplementation, the regulation of genes coding for oxidative stress enzymes suggested the reduction of the skin oxidative stress. Additionally, the decreased levels of cortisol in mucus indicated a reduction in the fish allostatic load due to the properties of the tested additive. Altogether, the dietary garlic, carvacrol, and thymol appear to promote the gilthead seabream skin innate immunity and the mucus protective capacity, decreasing its susceptibility to be colonized by pathogenic bacteria.

Spatial analysis of gut microbiome reveals a distinct ecological niche associated with the mucus layer.

Mucus-associated bacterial communities are critical for determining disease pathology and promoting colonization resistance. Yet the key ecological properties of mucus resident communities remain poorly defined. Using an approach that combines in situ hybridization, laser microdissection and 16s rRNA sequencing of spatially distinct regions of the mouse gut lumen, we discovered that a dense microbial community resembling a biofilm is embedded in the mucus layer. The mucus-associated biofilm-like community excluded bacteria belonging to phylum Proteobacteria. Additionally, it was significantly more diverse and consisted of bacterial species that were unique to it. By employing germ-free mice deficient in T and B lymphocytes we found that formation of biofilm-like structure was independent of adaptive immunity. Instead the integrity of biofilm-like community depended on Gram-positive commensals such as Clostridia. Additionally, biofilm-like community in the mucus lost fewer Clostridia and showed smaller bloom of Proteobacteria compared to the lumen upon antibiotic treatment. When subjected to time-restricted feeding biofilm-like structure significantly enhanced in size and showed enrichment of Clostridia. Taken together our work discloses that mucus-associated biofilm-like community represents a specialized community that is structurally and compositionally distinct that excludes aerobic bacteria while enriching for anaerobic bacteria such as Clostridia, exhibits enhanced stability to antibiotic treatment and that can be modulated by dietary changes.

Detection of associated bacteria in aspiration pneumonia and lung abscesses using partial 16S rRNA gene amplicon sequencing.

OBJECTIVES: Lower respiratory tract infections (LRTIs) are often caused by the patient's own oral commensal bacteria. Causative bacteria must be identified to select the appropriate antimicrobial agents; however, the pathogens are identified via routine culture methods in only approximately half of LRTI cases. METHODS: To investigate LRTI-associated bacteria, we conducted culture testing under aerobic and anaerobic conditions using culture-independent partial 16S rRNA gene amplicon sequencing analysis using a high-throughput sequencer in cases of aspiration pneumonia and lung abscesses. RESULTS: Culture testing of 17 aspiration pneumonia cases revealed Streptococcus spp. (n = 13), Prevotella spp. (n = 9), and Veillonella spp. (n = 8); 16S rRNA analysis of these cases yielded Streptococcus spp. (n = 16), Veillonella spp. (n = 12), Haemophilus spp. (n = 12), Prevotella spp. (n = 11), and Rothia spp. (n = 11). Culture testing of 8 lung abscess cases revealed Streptococcus spp. (n = 7) and Fusobacterium spp. (n = 4); 16S rRNA analysis of these cases yielded Fusobacterium spp. (n = 8), Prevotella spp. (n = 7), Streptococcus spp. (n = 6), and Porphyromonas spp. (n = 5). All taxa with abundance ratios of ≥50% on the 16S rRNA analysis were also detected in the cultures. However, several taxa were either undetected in the cultures despite relatively high abundance ratios on the 16S rRNA analysis or negative on the 16S rRNA analysis and isolated only by culturing. CONCLUSION: Our data provide a comprehensive list of bacterial taxa that may be associated with aspiration pneumonia and lung abscesses. In empirically treating LRTIs, this information will help determine the best treatment against the targeted anaerobes.

Proteomic profile and protease activity in the skin mucus of greater amberjack (Seriola dumerili) infected with the ectoparasite Neobenedenia girellae - An immunological approach.

Skin mucus is considered the first barrier against diseases in fish. The skin mucus protein profile of the greater amberjack (Seriola dumerili) and its changes due to experimental infection with Neobenedenia girellae were studied by combining 2-DE-MS/MS and gel-free LC-MS/MS proteomic approaches. The 2-DE results led to the identification of 69 and 55 proteins in noninfected and infected fish, respectively, and revealed that keratins were specifically cleaved in parasitized fish. Therefore, the skin mucus of the infected fish showed a higher protease activity due to, at least in part, an increase of metal-dependent protease and serine-type protease activities. Additionally, through a gel-free LC-MS/MS analysis, 1377 and 1251 different proteins were identified in the skin mucus of healthy and parasitized fish, respectively. The functional analysis of these proteins demonstrated a statistical overrepresentation of ribosomal proteins (a well-known source of antimicrobial peptides) in N. girellae-infected fish. In contrast, the components of membranes and protein transport GO categories were underrepresented after infection. Immune system process-related proteins constituted 2.5% of the total skin mucosal proteins. Among these skin mucosal proteins, 14 and 15 proteins exclusive to non-parasitized and parasitized fish were found, respectively, including specific serine-type proteases and metalloproteases in the parasitized fish. Moreover, the finding of tryptic peptides exclusive to some bacterial genera, obtained by gel-free LC-MS/MS, allowed us to construct a preliminary map of the microbiota living in the mucus of S. dumerili, with Pseudomonas and Paracoccus the most represented genera in both noninfected and infected fish.