Development of early life gut resistome and mobilome across gestational ages and microbiota-modifying treatments.

BACKGROUND: Gestational age (GA) and associated level of gastrointestinal tract maturation are major factors driving the initial gut microbiota composition in preterm infants. Besides, compared to term infants, premature infants often receive antibiotics to treat infections and probiotics to restore optimal gut microbiota. How GA, antibiotics, and probiotics modulate the microbiota's core characteristics, gut resistome and mobilome, remains nascent. METHODS: We analysed metagenomic data from a longitudinal observational study in six Norwegian neonatal intensive care units to describe the bacterial microbiota of infants of varying GA and receiving different treatments. The cohort consisted of probiotic-supplemented and antibiotic-exposed extremely preterm infants (n = 29), antibiotic-exposed very preterm (n = 25), antibiotic-unexposed very preterm (n = 8), and antibiotic-unexposed full-term (n = 10) infants. The stool samples were collected on days of life 7, 28, 120, and 365, and DNA extraction was followed by shotgun metagenome sequencing and bioinformatical analysis. FINDINGS: The top predictors of microbiota maturation were hospitalisation length and GA. Probiotic administration rendered the gut microbiota and resistome of extremely preterm infants more alike to term infants on day 7 and ameliorated GA-driven loss of microbiota interconnectivity and stability. GA, hospitalisation, and both microbiota-modifying treatments (antibiotics and probiotics) contributed to an elevated carriage of mobile genetic elements in preterm infants compared to term controls. Finally, Escherichia coli was associated with the highest number of antibiotic-resistance genes, followed by Klebsiella pneumoniae and Klebsiella aerogenes. INTERPRETATION: Prolonged hospitalisation, antibiotics, and probiotic intervention contribute to dynamic alterations in resistome and mobilome, gut microbiota characteristics relevant to infection risk. FUNDING: Odd-Berg Group, Northern Norway Regional Health Authority.

Supplementation with a probiotic mixture accelerates gut microbiome maturation and reduces intestinal inflammation in extremely preterm infants.

Probiotics are increasingly administered to premature infants to prevent necrotizing enterocolitis and neonatal sepsis. However, their effects on gut microbiome assembly and immunity are poorly understood. Using a randomized intervention trial in extremely premature infants, we tested the effects of a probiotic product containing four strains of Bifidobacterium species autochthonous to the infant gut and one Lacticaseibacillus strain on the compositional and functional trajectory of microbiome. Daily administration of the mixture accelerated the transition into a mature, term-like microbiome with higher stability and species interconnectivity. Besides infant age, Bifidobacterium strains and stool metabolites were the best predictors of microbiome maturation, and structural equation modeling confirmed probiotics as a major determinant for the trajectory of microbiome assembly. Bifidobacterium-driven microbiome maturation was also linked to an anti-inflammatory intestinal immune milieu. This demonstrates that Bifidobacterium strains are ecosystem engineers that lead to an acceleration of microbiome maturation and immunological consequences in extremely premature infants.

Metaproteomic profiling of fungal gut colonization in gnotobiotic mice.

BACKGROUND: Eukaryotic microbes can modulate mammalian host health and disease states, yet the molecular contribution of gut fungi remains nascent. We previously showed that mice exclusively colonised with fungi displayed increased sensitivity to allergic airway inflammation and had fecal metabolite profiles similar to germ-free mice. This marginal effect on the host metabolome suggested that fungi do not primarily use metabolites to modulate the host immune system. METHODS: To describe functional changes attributed to fungal colonisation, we performed mass spectrometry-based analyses of feces (Label-Free Quantitative; LFQ) and the small intestine (labeling with Tandem Mass Tag; TMT) of gnotobiotic mice colonised with defined consortia of twelve bacterial species, five fungal species, or both. We also evaluated the effect of microbiome perturbances on the metaproteome by analysing feces from mouse pups treated with an antibiotic or antifungal. RESULTS: We detected 6675 proteins in the mice feces, of which 3845 had determined LFQ levels. Analysis of variance showed changes in the different gnotobiotic mouse groups; specifically, 46% of 2860 bacterial, 15% of 580 fungal, and 76% of 405 mouse quantified proteins displayed differential levels. The antimicrobial treatments resulted in lasting changes in the bacterial and fungal proteomes, suggesting that the antimicrobials impacted the entire community. Fungal colonisation resulted in changes in host proteins functional in innate immunity as well as metabolism, predicting specific roles of gut fungi on host systems during early developmental stages. Several of the detected fungal proteins (3% of 1492) have been previously reported as part of extracellular vesicles and having immunomodulating properties. Using an isobaric labelling TMT approach for profiling low abundant proteins of the jejunal tissue, we confirmed that the five fungal species differentially impacted the host intestinal proteome compared to the bacterial consortium. The detected changes in mouse jejunal proteins (4% of 1514) were mainly driven by metabolic proteins. CONCLUSIONS: We used quantitative proteomic profiling of gnotobiotic conditions to show how colonisation with selected fungal species impacts the host gut proteome. Our results suggest that an increased abundance of certain gut fungal species in early life may affect the developing intracellular attributes of epithelial and immune cells.

Inferring early-life host and microbiome functions by mass spectrometry-based metaproteomics and metabolomics.

Humans have a long-standing coexistence with microorganisms. In particular, the microbial community that populates the human gastrointestinal tract has emerged as a critical player in governing human health and disease. DNA and RNA sequencing techniques that map taxonomical composition and genomic potential of the gut community have become invaluable for microbiome research. However, deriving a biochemical understanding of how activities of the gut microbiome shape host development and physiology requires an expanded experimental design that goes beyond these approaches. In this review, we explore advances in high-throughput techniques based on liquid chromatography-mass spectrometry. These omics methods for the identification of proteins and metabolites have enabled direct characterisation of gut microbiome functions and the crosstalk with the host. We discuss current metaproteomics and metabolomics workflows for producing functional profiles, the existing methodological challenges and limitations, and recent studies utilising these techniques with a special focus on early life gut microbiome.

Label-free quantitative proteomic analysis of the oral bacteria Fusobacterium nucleatum and Porphyromonas gingivalis to identify protein features relevant in biofilm formation.

BACKGROUND: The opportunistic pathogens Fusobacterium nucleatum and Porphyromonas gingivalis are Gram-negative bacteria associated with oral biofilm and periodontal disease. This study investigated interactions between F. nucleatum and P. gingivalis proteomes with the objective to identify proteins relevant in biofilm formation. METHODS: We applied liquid chromatography-tandem mass spectrometry to determine the expressed proteome of F. nucleatum and P. gingivalis cells grown in biofilm or planktonic culture, and as mono- and dual-species models. The detected proteins were classified into functional categories and their label-free quantitative (LFQ) intensities statistically compared. RESULTS: The proteomic analyses detected 1,322 F. nucleatum and 966 P. gingivalis proteins, including abundant virulence factors. Using univariate statistics, we identified significant changes between biofilm and planktonic culture (p-value ≤0.05) in 0,4% F. nucleatum, 7% P. gingivalis, and 14% of all proteins in the dual-species model. For both species, proteins involved in vitamin B2 (riboflavin) metabolism had significantly increased levels in biofilm. In both mono- and dual-species biofilms, P. gingivalis increased the production of proteins for translation, oxidation-reduction, and amino acid metabolism compared to planktonic cultures. However, when we compared LFQ intensities between mono- and dual-species, over 90% of the significantly changed P. gingivalis proteins had their levels reduced in biofilm and planktonic settings of the dual-species model. CONCLUSIONS: The findings suggest that P. gingivalis reduces the production of multiple proteins because of the F. nucleatum presence. The results highlight the complex interactions of bacteria contributing to oral biofilms, which need to be considered in the design of prevention strategies.

Distinct Metabolic Features of Pathogenic Escherichia coli and Shigella spp. Determined by Label-Free Quantitative Proteomics.

Escherichia coli and Shigella spp. causing illnesses in humans represent a genotypically and phenotypically diverse group of pathogens. Although E. coli diversity has been studied by comparative genomics, the intra-species variation at the proteome level is currently unknown. The proteomes of 16 pathogenic E. coli, 2 non-pathogenic E. coli, and 5 Shigella strains originating from 18 phylogenetic lineages are investigated. By applying label-free quantitative proteomics on trypsin-digested cell extracts from bacteria grown on blood agar, 4018 proteins are detected, 3285 of which arequantified, and 261 represented virulence factors. Of 753 proteins quantified in all strains, the levels of 153 vary substantially between strains and are functionally associated mostly with stress response and peripheral metabolism. The levels of proteins associated with the central metabolism vary considerably less than the levels of proteins from other metabolic pathways. Hierarchical clustering analysis based on the protein levels results in strains grouping that differ from that obtained by gene-based phylogenetic analysis. Finally, strains of some E. coli pathotypes have more similar protein profiles even when the strains are not genetically closely related. The results suggest that the degree of genetic relatedness may not necessarily be a good predictor of E. coli phenotypic characteristics.

Intestinal fungi are causally implicated in microbiome assembly and immune development in mice.

The gut microbiome consists of a multi-kingdom microbial community. Whilst the role of bacteria as causal contributors governing host physiological development is well established, the role of fungi remains to be determined. Here, we use germ-free mice colonized with defined species of bacteria, fungi, or both to differentiate the causal role of fungi on microbiome assembly, immune development, susceptibility to colitis, and airway inflammation. Fungal colonization promotes major shifts in bacterial microbiome ecology, and has an independent effect on innate and adaptive immune development in young mice. While exclusive fungal colonization is insufficient to elicit overt dextran sulfate sodium-induced colitis, bacterial and fungal co-colonization increase colonic inflammation. Ovalbumin-induced airway inflammation reveals that bacterial, but not fungal colonization is necessary to decrease airway inflammation, yet fungi selectively promotes macrophage infiltration in the airway. Together, our findings demonstrate a causal role for fungi in microbial ecology and host immune functionality, and therefore prompt the inclusion of fungi in therapeutic approaches aimed at modulating early life microbiomes.

Host-microbiome intestinal interactions during early life: considerations for atopy and asthma development.

PURPOSE OF REVIEW: The body's largest microbial community, the gut microbiome, is in contact with mucosal surfaces populated with epithelial, immune, endocrine and nerve cells, all of which sense and respond to microbial signals. These mutual interactions have led to a functional coevolution between the microbes and human physiology. Examples of coadaptation are anaerobes Bifidobacteria and Bacteroides, which have adjusted their metabolism to dietary components of human milk, and infant immune development, which has evolved to become reliant on the presence of beneficial microbes. Current research suggests that specific composition of the early-life gut microbiome aligns with the maturation of host immunity. Disruptions of natural microbial succession patterns during gut colonization are a consistent feature of immune-mediated diseases, including atopy and asthma. RECENT FINDINGS: Here, we catalog recent birth cohorts documenting associations between immune dysregulation and microbial alterations, and summarize the evidence supporting the role of the gut microbiome as an etiological determinant of immune-mediated allergic diseases. SUMMARY: Ecological concepts that describe microbial dynamics in the context of the host environment, and a portray of immune and neuroendocrine signaling induced by host-microbiome interactions, have become indispensable in describing the molecular role of early-life microbiome in atopy and asthma susceptibility.

Comparative Proteomics of Enterotoxigenic Escherichia coli Reveals Differences in Surface Protein Production and Similarities in Metabolism.

Enterotoxigenic Escherichia coli (ETEC) infections are an important cause of diarrhea among young children living in low- and middle-income countries and visiting travelers. The development of effective vaccines is complicated by substantial genomic diversity that exists among ETEC isolates. To investigate how ETEC genomic variation is reflected at expressed proteome level, we applied label-free quantitative proteomics to seven human ETEC strains representing five epidemiologically important lineages. We further determined the proteome profile of the nonpathogenic E. coli B strain BL21(DE3) to discriminate features specific for ETEC. The analysis yielded a data set of 2893 proteins, of which 1729 were present in all strains. Each ETEC strain produced on average 27 plasmid- or chromosomally-encoded proteins with known or putative connections to virulence, and a number of strain-specific proteins associated with the biosynthesis of surface antigens. Statistical comparison of protein levels between the ETEC strains and BL21(DE3) revealed several proteins with considerably increased levels only in BL21(DE3) including enzymes of arginine biosynthesis and metabolism of melibiose, galactitol, and gluconate. ETEC strains displayed consistently increased levels of proteins that were functional in iron acquisition, maltose metabolism, and acid resistance. The latter results suggest that specific metabolic functions might be shared among ETEC isolates.

Quantitative proteomic analysis of extracellular matrix extracted from mono- and dual-species biofilms of Fusobacterium nucleatum and Porphyromonas gingivalis.

The Gram-negative bacteria Fusobacterium nucleatum and Porphyromonas gingivalis are members of a complex dental biofilm associated with periodontal disease. In this study, we cultured F. nucleatum and P. gingivalis as mono- and dual-species biofilms, and analyzed the protein composition of the biofilms extracellular polymeric matrix (EPM) by high-resolution liquid chromatography-tandem mass spectrometry. Label-free quantitative proteomic analysis was used for identification of proteins and sequence-based functional characterization for their classification and prediction of possible roles in EPM. We identified 542, 93 and 280 proteins in the matrix of F. nucleatum, P. gingivalis, and the dual-species biofilm, respectively. Nearly 70% of all EPM proteins in the dual-species biofilm originated from F. nucleatum, and a majority of these were cytoplasmic proteins, suggesting an enhanced lysis of F. nucleatum cells. The proteomic analysis also indicated an interaction between the two species: 22 F. nucleatum proteins showed differential levels between the mono and dual-species EPMs, and 11 proteins (8 and 3 from F. nucleatum and P. gingivalis, respectively) were exclusively detected in the dual-species EPM. Oxidoreductases and chaperones were among the most abundant proteins identified in all three EPMs. The biofilm matrices in addition contained several known and hypothetical virulence proteins, which can mediate adhesion to the host cells and disintegration of the periodontal tissues. This study demonstrated that the biofilm matrix of two important periodontal pathogens consists of a multitude of proteins whose amounts and functionalities vary largely. Relatively high levels of several of the detected proteins might facilitate their potential use as targets for the inhibition of biofilm development.

Zoonotic necrotizing myositis caused by Streptococcus equi subsp. zooepidemicus in a farmer.

BACKGROUND: Streptococcus equi subsp. zooepidemicus is a beta-hemolytic group C streptococcus mainly causing infections in domesticated animals. Here we describe the first case of zoonotic necrotizing myositis caused by this bacterium. CASE PRESENTATION: The patient was a 73-year-old, previously healthy farmer with two asymptomatic Shetland ponies in his stable. After close contact with the ponies while feeding them, he rapidly developed erythema of his left thigh and sepsis with multiple organ failure. The clinical course was severe and complicated, requiring repetitive surgical excision of necrotic muscle, treatment with vasopressors, mechanical ventilation and continuous venovenous hemofiltration, along with adjunctive hyperbaric oxygen therapy. The patient was discharged from hospital at day 30, without obvious sequelae. The streptococcal isolate was identified as Streptococcus equi by MALDI-ToF MS, and was later assigned subspecies identification as S. equi subsp. zooepidemicus. Multilocus sequence typing identified the strain as a novel sequence type (ST 364), closely related to types previously identified in horses and cattle. A focused proteomic analysis revealed that the ST 364 expressed putative virulence factors similar to that of Streptococcus pyogenes, including homologues of the M protein, streptodornases, interleukin 8-protease and proteins involved in the biosynthesis of streptolysin S. CONCLUSION: This case illustrates the zoonotic potential of S. equi subsp. zooepidemicus and the importance of early clinical recognition, rapid and radical surgical therapy, appropriate antibiotics and adequate supportive measures when necrotizing soft tissue infection is suspected. The expression of Streptococcus pyogenes-like putative virulence determinants in ST 364 might partially explain the fulminant clinical picture.

Coordination of Metabolism and Virulence Factors Expression of Extraintestinal Pathogenic Escherichia coli Purified from Blood Cultures of Patients with Sepsis.

One of the trademarks of extraintestinal pathogenic Escherichia coli is adaptation of metabolism and basic physiology to diverse host sites. However, little is known how this common human pathogen adapts to permit survival and growth in blood. We used label-free quantitative proteomics to characterize five E. coli strains purified from clinical blood cultures associated with sepsis and urinary tract infections. Further comparison of proteome profiles of the clinical strains and a reference uropathogenic E. coli strain 536 cultivated in blood culture and on two different solid media distinguished cellular features altered in response to the pathogenically relevant condition. The analysis covered nearly 60% of the strains predicted proteomes, and included quantitative description based on label-free intensity scores for 90% of the detected proteins. Statistical comparison of anaerobic and aerobic blood cultures revealed 32 differentially expressed proteins (1.5% of the shared proteins), mostly associated with acquisition and utilization of metal ions critical for anaerobic or aerobic respiration. Analysis of variance identified significantly altered amounts of 47 proteins shared by the strains (2.7%), including proteins involved in vitamin B6 metabolism and virulence. Although the proteomes derived from blood cultures were fairly similar for the investigated strains, quantitative proteomic comparison to the growth on solid media identified 200 proteins with substantially changed levels (11% of the shared proteins). Blood culture was characterized by up-regulation of anaerobic fermentative metabolism and multiple virulence traits, including cell motility and iron acquisition. In a response to the growth on solid media there were increased levels of proteins functional in aerobic respiration, catabolism of medium-specific carbon sources and protection against oxidative and osmotic stresses. These results demonstrate on the expressed proteome level that expression of extraintestinal virulence factors and overall cellular metabolism closely reflects specific growth conditions. Data are available via ProteomeXchange with identifier PXD002912.

Improving genome annotation of enterotoxigenic Escherichia coli TW10598 by a label-free quantitative MS/MS approach.

The most commonly used genome annotation processes are to a great extent based on computational methods. However, those can only predict genes that have been described earlier or that have sequence signatures indicative of a gene function. Here, we report a synonymous proteogenomic approach for experimentally improving microbial genome annotation based on label-free quantitative MS/MS. The approach is exemplified by analysis of cell extracts from in vitro cultured enterotoxigenic Escherichia coli (ETEC) strain TW10598, as part of an effort to create a new reference ETEC genome sequence. The proteomic analysis yielded identification of 2060 proteins, out of which 312 proteins were originally described as hypothetical. For 84% of the identified proteins we have provided description of their relative quantitative levels, among others, for 20 abundantly expressed ETEC virulence factors. Proteogenomic mapping supported the existence of four protein-coding genes that had not been annotated, and led to correction of translation start positions of another nine. The addition of the proteomic analysis into TW10598 genome re-annotation project improved quality of the annotation, and provided experimental evidence for a significant portion of ETEC expressed proteome. Data are available via ProteomeXchange with identifier PXD002473 (http://proteomecentral.proteomexchange.org/dataset/PXD002473).