Perspectives in Searching Antimicrobial Peptides (AMPs) Produced by the Microbiota.

Changes in the structure and function of the microbiota are associated with various human diseases. These microbial changes can be mediated by antimicrobial peptides (AMPs), small peptides produced by the host and their microbiota, which play a crucial role in host-bacteria co-evolution. Thus, by studying AMPs produced by the microbiota (microbial AMPs), we can better understand the interactions between host and bacteria in microbiome homeostasis. Additionally, microbial AMPs are a new source of compounds against pathogenic and multi-resistant bacteria. Further, the growing accessibility to metagenomic and metatranscriptomic datasets presents an opportunity to discover new microbial AMPs. This review examines the structural properties of microbiota-derived AMPs, their molecular action mechanisms, genomic organization, and strategies for their identification in any microbiome data as well as experimental testing. Overall, we provided a comprehensive overview of this important topic from the microbial perspective.

Complete genome sequence and characterization of a novel Enterococcus faecium with probiotic potential isolated from the gut of Litopenaeus vannamei.

Litopenaeus vannamei, the Pacific whiteleg shrimp, is one of the most marketable species in aquaculture worldwide. However, it is susceptible to different infections causing considerable losses in production each year. Consequently, using prebiotics that promotes the proliferation of beneficial bacteria and strengthen the immune system is a current strategy for disease control. In this study, we isolated two strains of E. faecium from the gut of L. vannamei fed with agavin-supplemented diets. These isolates showed antibacterial activity against Vibrio parahaemolyticus, Vibrio harveyi and Vibrio alginolyticus, most likely due to peptidoglycan hydrolase (PGH) activity. Furthermore, we sequenced the genome of one isolate. As a result, we observed three proteins related to the production of bacteriocins, a relevant trait for selecting probiotic strains since they can inhibit the invasion of potential pathogens. Additionally, the genome annotation showed genes related to the production of essential nutrients for the host. It lacked two of the most common factors associated with virulence in Enterococcus pathogenic strains (esp and hyl). Thus, this host-probiotic-derived strain has potential application not only in shrimp health but also in alternative aquatic environments, as it is adapted to coexist within the gut shrimp microbiota, independently of the diet.

The Two-Faced Role of crAssphage Subfamilies in Obesity and Metabolic Syndrome: Between Good and Evil.

Viral metagenomic studies of the human gut microbiota have unraveled the differences in phage populations between health and disease, stimulating interest in phages' role on bacterial ecosystem regulation. CrAssphage is a common and abundant family in the gut virome across human populations. Therefore, we explored its role in obesity (O) and obesity with metabolic syndrome (OMS) in a children's cohort. We found a significantly decreased prevalence, diversity, and richness of the crAssphage Alpha subfamily in OMS mainly driven by a decrease in the Alpha_1 and Alpha_4 genera. On the contrary, there was a significant increase in the Beta subfamily in OMS, mainly driven by an increase in Beta_6. Additionally, an overabundance of the Delta_8 genus was observed in OMS. Notably, a decreased abundance of crAssphages was significantly correlated with the overabundance of Bacilli in the same group. The Bacilli class is a robust taxonomical biomarker of O and was also significantly abundant in our OMS cohort. Our results suggest that a loss of stability in the Alpha subfamily of crAssphages is associated with O and OMS. Contrary, an overabundance of the Delta subfamily was found in OMS. Our study advises the importance of considering the dual role (good and evil) of crAssphage subfamilies and their participation in conditions such as O, where we suggest that Alpha loss and Delta gain are associated with obese individuals.

White spot syndrome virus impact on the expression of immune genes and gut microbiome of black tiger shrimp Penaeus monodon.

The gut microbiome plays an essential role in the immune system of invertebrates and vertebrates. Pre and pro-biotics could enhance the shrimp immune system by increasing the phenoloxidase (PO), prophenoloxidase (ProPO), and superoxide dismutase activities. During viral infection, the host immune system alteration could influence the gut microbiome composition and probably lead to other pathogenic infections. Since the JAK/STAT pathway is involved in white spot syndrome virus (WSSV) infection, we investigated the intestine immune genes of STAT-silenced shrimp. During WSSV infection, expression levels of PmVago1, PmDoral, and PmSpätzle in PmSTAT-silenced shrimp were higher than normal. In addition, the transcription levels of antimicrobial peptides, including crustinPm1, crustinPm7, and PmPEN3, were higher in WSSV-challenged PmSTAT-silenced shrimp than the WSSV-infected normal shrimp. Meanwhile, PmSTAT silencing suppressed PmProPO1, PmProPO2, and PmPPAE1 expressions during WSSV infection. The microbiota from four shrimp tested groups (control group, WSSV-infected, PmSTAT-silenced, and PmSTAT-silenced infected by WSSV) was significantly different, with decreasing richness and diversity due to WSSV infection. The relative abundance of Bacteroidetes, Actinobacteria, and Planctomycetes was reduced in WSSV-challenged shrimp. However, at the species level, P. damselae, a pathogen to human and marine animals, significantly increased in WSSV-challenged shrimp. In constrast, Shewanella algae, a shrimp probiotic, was decreased in WSSV groups. In addition, the microbiota structure between control and PmSTAT-silenced shrimp was significantly different, suggesting the importance of STAT to maintain the homeostasis interaction with the microbiota.

Transcriptome Analysis of Soursop (Annona muricata L.) Fruit under Postharvest Storage Identifies Genes Families Involved in Ripening.

Soursop (Annona muricata L.) is climacteric fruit with a short ripening period and postharvest shelf life, leading to a rapid softening. In this study, transcriptome analysis of soursop fruits was performed to identify key gene families involved in ripening under postharvest storage conditions (Day 0, Day 3 stored at 28 ± 2 °C, Day 6 at 28 ± 2 °C, Day 3 at 15 ± 2 °C, Day 6 at 15 ± 2 °C, Day 9 at 15 ± 2 °C). The transcriptome analysis showed 224,074 transcripts assembled clustering into 95, 832 unigenes, of which 21, 494 had ORF. RNA-seq analysis showed the highest number of differentially expressed genes on Day 9 at 15 ± 2 °C with 9291 genes (4772 up-regulated and 4519 down-regulated), recording the highest logarithmic fold change in pectin-related genes. Enrichment analysis presented significantly represented GO terms and KEGG pathways associated with molecular function, metabolic process, catalytic activity, biological process terms, as well as biosynthesis of secondary metabolites, plant hormone signal, starch, and sucrose metabolism, plant-pathogen interaction, plant-hormone signal transduction, and MAPK-signaling pathways, among others. Network analysis revealed that pectinesterase genes directly regulate the loss of firmness in fruits stored at 15 ± 2 °C.

Agavin induces beneficial microbes in the shrimp microbiota under farming conditions.

Prebiotics and probiotics have shown a number of beneficial impacts preventing diseases in cultured shrimps. Complex soluble carbohydrates are considered ideal for fostering microbiota biodiversity by fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPS). Here we evaluated the growth performance and microbiota composition of the white shrimp Litopenaeus vannamei after dietary intervention using agavin as a FODMAP prebiotic under farming conditions. Adult L. vannamei were raised at a shrimp farm and the effect of agavin supplemented at 2% (AG2) or 10% (AG10) levels were compared to an agavin-free basal diet (BD). After 28 days-trial, the feed conversion ratio, total feed ingested, and protein efficiency ratio was significantly improved on animals fed with AG2. At the same time, no effect on growth performance was observed in AG10. Surprisingly, after sequencing the V3-V4 regions of the 16S rRNA gene a higher microbial richness and diversity in the hepatopancreas and intestine was found only in those animals receiving the AG10 diet, while those receiving the AG2 diet had a decreased richness and diversity, both diets compared to the BD. The beta diversity analysis showed a clear significant microbiota clustering by agavin diets only in the hepatopancreas, suggesting that agavin supplementation had a more substantial deterministic effect on the microbiota of hepatopancreas than on the intestine. We analyzed the literature to search beneficial microbes for shrimp's health and found sequences for 42 species in our 16S data, being significantly increased Lactobacillus pentosus, Pseudomonas putida and Pseudomonas synxantha in the hepatopancreas of the AG10 and Rodopseudomonas palustris and Streptococcus thermophiles th1435 in the hepatopancreas of the AG2, both compared to BD. Interestingly, when we analyzed the abundance of 42 beneficial microbes as a single microbial community "meta-community," found an increase in their abundance as agavin concentration increases in the hepatopancreas. In addition, we also sequenced the DNA of agavin and found 9 of the 42 beneficial microbes. From those, Lactobacillus lactis and Lactobacillus delbrueckii were found in shrimps fed with agavin (both AG2 and AG10), and Lysinibacillus fusiformis in AG10 and they were absent the BD diet, suggesting these three species could be introduced with the agavin to the diet. Our work provides evidence that agavin supplementation is associated with an increase of beneficial microbes for the shrimp microbiota at farming conditions. Our study provides the first evidence that a shrimp prebiotic may selectively modify the microbiota in an organ-dependent effect.

Protocol for the isolation, sequencing, and analysis of the gut phageome from human fecal samples.

The phage-bacteria interactions in the gut microbiome are critical for health and disease, but viruses of the human gut microbiome are poorly understood. Here, we present a simple and cost-efficient protocol for collecting viral-like particles (VLPs) from human fecal samples. We describe VLPs quantification using epifluorescence and TEM microscopy, followed by DNA sequencing and bioinformatics analysis. This protocol characterizes the gut phageome in normal-weight and obese children with metabolic syndrome. It is also suitable to conduct high-throughput studies for other diseases. For complete details on the use and execution of this profile, please refer to Bikel et al. (2021).

Gut dsDNA virome shows diversity and richness alterations associated with childhood obesity and metabolic syndrome.

Changes in the human gut microbiome are associated with obesity and metabolic syndrome, but the role of the gut virome in both diseases remains largely unknown. We characterized the gut dsDNA virome of 28 school-aged children with healthy normal-weight (NW, n = 10), obesity (O, n = 10), and obesity with metabolic syndrome (OMS, n = 8), using metagenomic sequencing of virus-like particles (VLPs) from fecal samples. The virome classification confirmed the bacteriophages' dominance, mainly composed of Caudovirales. Notably, phage richness and diversity of individuals with O and OMS tended to increase, while the VLP abundance remained the same among all groups. Of the 4,611 phage contigs composing the phageome, 48 contigs were highly prevalent in ≥80% of individuals, suggesting high inter-individual phage diversity. The abundance of several contigs correlated with gut bacterial taxa; and with anthropometric and biochemical parameters altered in O and OMS. To our knowledge, this gut phageome represents one of the largest datasets and suggests disease-specific phage alterations.

Metatranscriptomic analysis to define the Secrebiome, and 16S rRNA profiling of the gut microbiome in obesity and metabolic syndrome of Mexican children.

BACKGROUND: In the last decade, increasing evidence has shown that changes in human gut microbiota are associated with diseases, such as obesity. The excreted/secreted proteins (secretome) of the gut microbiota affect the microbial composition, altering its colonization and persistence. Furthermore, it influences microbiota-host interactions by triggering inflammatory reactions and modulating the host's immune response. The metatranscriptome is essential to elucidate which genes are expressed under diseases. In this regard, little is known about the expressed secretome in the microbiome. Here, we use a metatranscriptomic approach to delineate the secretome of the gut microbiome of Mexican children with normal weight (NW) obesity (O) and obesity with metabolic syndrome (OMS). Additionally, we performed the 16S rRNA profiling of the gut microbiota. RESULTS: Out of the 115,712 metatranscriptome genes that codified for proteins, 30,024 (26%) were predicted to be secreted, constituting the Secrebiome of the gut microbiome. The 16S profiling confirmed an increased abundance in Firmicutes and decreased in Bacteroidetes in the obesity groups, and a significantly higher richness and diversity than the normal weight group. We found novel biomarkers for obesity with metabolic syndrome such as increased Coriobacteraceae, Collinsela, and Collinsella aerofaciens; Erysipelotrichaceae, Catenibacterium and Catenibacterium sp., and decreased Parabacteroides distasonis, which correlated with clinical and anthropometric parameters associated to obesity and metabolic syndrome. Related to the Secrebiome, 16 genes, homologous to F. prausniitzi, were overexpressed for the obese and 15 genes homologous to Bacteroides, were overexpressed in the obesity with metabolic syndrome. Furthermore, a significant enrichment of CAZy enzymes was found in the Secrebiome. Additionally, significant differences in the antigenic density of the Secrebiome were found between normal weight and obesity groups. CONCLUSIONS: These findings show, for the first time, the role of the Secrebiome in the functional human-microbiota interaction. Our results highlight the importance of metatranscriptomics to provide novel information about the gut microbiome's functions that could help us understand the impact of the Secrebiome on the homeostasis of its human host. Furthermore, the metatranscriptome and 16S profiling confirmed the importance of treating obesity and obesity with metabolic syndrome as separate conditions to better understand the interplay between microbiome and disease.

A meta-analysis reveals the environmental and host factors shaping the structure and function of the shrimp microbiota.

The shrimp or prawn is the most valuable traded marine product in the world market today and its microbiota plays an essential role in its development, physiology, and health. The technological advances and dropping costs of high-throughput sequencing have increased the number of studies characterizing the shrimp microbiota. However, the application of different experimental and bioinformatics protocols makes it difficult to compare different studies to reach general conclusions about shrimp microbiota. To meet this necessity, we report the first meta-analysis of the microbiota from freshwater and marine shrimps using all publically available sequences of the 16S ribosomal gene (16S rRNA gene). We obtained data for 199 samples, in which 63.3% were from marine (Alvinocaris longirostris, Litopenaeus vannamei and Penaeus monodon), and 36.7% were from freshwater (Macrobrachium asperulum, Macrobrachium nipponense, Macrobranchium rosenbergii, Neocaridina denticulata) shrimps. Technical variations among studies, such as selected primers, hypervariable region, and sequencing platform showed a significant impact on the microbiota structure. Additionally, the ANOSIM and PERMANOVA analyses revealed that the most important biological factor in structuring the shrimp microbiota was the marine and freshwater environment (ANOSIM R = 0.54, P = 0.001; PERMANOVA pseudo-F = 21.8, P = 0.001), where freshwater showed higher bacterial diversity than marine shrimps. Then, for marine shrimps, the most relevant biological factors impacting the microbiota composition were lifestyle (ANOSIM R = 0.341, P = 0.001; PERMANOVA pseudo-F = 8.50, P = 0.0001), organ (ANOSIM R = 0.279, P = 0.001; PERMANOVA pseudo-F = 6.68, P = 0.001) and developmental stage (ANOSIM R = 0.240, P = 0.001; PERMANOVA pseudo-F = 5.05, P = 0.001). According to the lifestyle, organ, developmental stage, diet, and health status, the highest diversity were for wild-type, intestine, adult, wild-type diet, and healthy samples, respectively. Additionally, we used PICRUSt to predict the potential functions of the microbiota, and we found that the organ had more differentially enriched functions (93), followed by developmental stage (12) and lifestyle (9). Our analysis demonstrated that despite the impact of technical and bioinformatics factors, the biological factors were also statistically significant in shaping the microbiota. These results show that cross-study comparisons are a valuable resource for the improvement of the shrimp microbiota and microbiome fields. Thus, it is important that future studies make public their sequencing data, allowing other researchers to reach more powerful conclusions about the microbiota in this non-model organism. To our knowledge, this is the first meta-analysis that aims to define the shrimp microbiota.

Microbiome of Pacific Whiteleg shrimp reveals differential bacterial community composition between Wild, Aquacultured and AHPND/EMS outbreak conditions.

Crustaceans form the second largest subphylum on Earth, which includes Litopeneaus vannamei (Pacific whiteleg shrimp), one of the most cultured shrimp worldwide. Despite efforts to study the shrimp microbiota, little is known about it from shrimp obtained from the open sea and the role that aquaculture plays in microbiota remodeling. Here, the microbiota from the hepatopancreas and intestine of wild type (wt) and aquacultured whiteleg shrimp and pond sediment from hatcheries were characterized using sequencing of seven hypervariable regions of the 16S rRNA gene. Cultured shrimp with AHPND/EMS disease symptoms were also included. We found that (i) microbiota and their predicted metagenomic functions were different between wt and cultured shrimp; (ii) independent of the shrimp source, the microbiota of the hepatopancreas and intestine was different; (iii) the microbial diversity between the sediment and intestines of cultured shrimp was similar; and (iv) associated to an early development of AHPND/EMS disease, we found changes in the microbiome and the appearance of disease-specific bacteria. Notably, under cultured conditions, we identified bacterial taxa enriched in healthy shrimp, such as Faecalibacterium prausnitzii and Pantoea agglomerans, and communities enriched in diseased shrimp, such as Aeromonas taiwanensis, Simiduia agarivorans and Photobacterium angustum.