An exploration of mechanisms underlying Desemzia incerta colonization resistance to methicillin-resistant Staphylococcus aureus on the skin.

Colonization of human skin and nares by methicillin-resistant Staphylococcus aureus (MRSA) leads to the community spread of MRSA. This spread is exacerbated by the transfer of MRSA between humans and livestock, particularly swine. Here, we capitalized on the shared features between human and porcine skin, including shared MRSA colonization, to study novel bacterial mediators of MRSA colonization resistance. We focused on the poorly studied bacterial species Desemzia incerta, which we found to exert antimicrobial activity through a secreted product and exhibited colonization resistance against MRSA in an in vivo murine skin model. Using parallel genomic and biochemical investigation, we discovered that D. incerta secretes an antimicrobial protein. Sequential protein purification and proteomics analysis identified 24 candidate inhibitory proteins, including a promising peptidoglycan hydrolase candidate. Aided by transcriptional analysis of D. incerta and MRSA cocultures, we found that exposure to D. incerta leads to decreased MRSA biofilm production. These results emphasize the value of exploring microbial communities across a spectrum of hosts, which can lead to novel therapeutic agents as well as an increased understanding of microbial competition.IMPORTANCEMethicillin-resistant Staphylococcus aureus (MRSA) causes a significant healthcare burden and can be spread to the human population via livestock transmission. Members of the skin microbiome can prevent MRSA colonization via a poorly understood phenomenon known as colonization resistance. Here, we studied the colonization resistance of S. aureus by bacterial inhibitors previously identified from a porcine skin model. We identify a pig skin commensal, Desemzia incerta, that reduced MRSA colonization in a murine model. We employ a combination of genomic, proteomic, and transcriptomic analyses to explore the mechanisms of inhibition between D. incerta and S. aureus. We identify 24 candidate antimicrobial proteins secreted by D. incerta that could be responsible for its antimicrobial activity. We also find that exposure to D. incerta leads to decreased S. aureus biofilm formation. These findings show that the livestock transmission of MRSA can be exploited to uncover novel mechanisms of MRSA colonization resistance.

Complete genome sequence of the bacterium Desemzia incerta isolated from the dorsal skin of a Yucatan pig.

We have shown previously that an isolate of Desemzia incerta from porcine skin has antimicrobial activity against methicillin-resistant Staphylococcus aureus. We present here the complete D. incerta genome containing one circular chromosome and five circular plasmids.

An exploration of mechanisms underlying Desemzia incerta colonization resistance to methicillin-resistant Staphylococcus aureus on the skin.

Colonization of human skin and nares by methicillin-resistant Staphylococcus aureus (MRSA) leads to community spread of MRSA. This spread is exacerbated by transfer of MRSA between humans and livestock, particularly swine. Here we capitalized on the shared features between human and porcine skin, including shared MRSA colonization, to study novel bacterial mediators of MRSA colonization resistance. We focused on the poorly studied bacterial species Desemzia incerta, which we found to exert antimicrobial activity through a secreted product and exhibited colonization resistance against MRSA in an in vivo murine skin model. Using parallel genomic and biochemical investigation, we discovered that D. incerta secretes an antimicrobial protein. Sequential protein purification and proteomics analysis identified 24 candidate inhibitory proteins, including a promising peptidoglycan hydrolase candidate. Aided by transcriptional analysis of D. incerta and MRSA cocultures, we found that exposure to D. incerta leads to decreased MRSA biofilm production. These results emphasize the value in exploring microbial communities across a spectrum of hosts, which can lead to novel therapeutic agents as well as increased understanding of microbial competition.

Harnessing diversity and antagonism within the pig skin microbiota to identify novel mediators of colonization resistance to methicillin-resistant Staphylococcus aureus.

The microbiota mediate multiple aspects of skin barrier function, including colonization resistance to pathogens such as Staphylococcus aureus. The endogenous skin microbiota limits S. aureus colonization via competition and direct inhibition. Novel mechanisms of colonization resistance are promising therapeutic targets for drug-resistant infections, such as those caused by methicillin-resistant S. aureus (MRSA). Here, we developed and characterized a swine model of topical microbiome perturbation and MRSA colonization. As in other model systems, topical antimicrobial treatment had a little discernable effect on community diversity though the overall microbial load was sensitive to multiple types of intervention, including swabbing. In parallel, we established a porcine skin culture collection and screened 7,700 isolates for MRSA inhibition. Using genomic and phenotypic criteria, we curated three isolates to investigate whether prophylactic colonization would inhibit MRSA colonization in vivo. The three-member consortium together, but not individually, provided protection against MRSA colonization, suggesting cooperation and/or synergy among the strains. Inhibitory isolates were represented across all major phyla of the pig skin microbiota and did not have a strong preference for inhibiting closely related species, suggesting that relatedness is not a condition of antagonism. These findings reveal the porcine skin as an underexplored reservoir of skin commensal species with the potential to prevent MRSA colonization and infection. IMPORTANCE The skin microbiota is protective against pathogens or opportunists such as S. aureus, the most common cause of skin and soft tissue infections. S. aureus can colonize normal skin and nasal passages, and colonization is a risk factor for infection, especially on breach of the skin barrier. Here, we established a pig model to study the competitive mechanisms of the skin microbiota and their role in preventing colonization by MRSA. This drug-resistant strain is also a livestock pathogen, and swine herds can be reservoirs of MRSA carriage. From 7,700 cultured skin isolates, we identified 37 unique species across three phyla that inhibited MRSA. A synthetic community of three inhibitory isolates provided protection together, but not individually, in vivo in a murine model of MRSA colonization. These findings suggest that antagonism is widespread in the pig skin microbiota, and these competitive interactions may be exploited to prevent MRSA colonization.

Cutibacterium acnes evolution: One pore at a time.

Cutibacterium acnes is found in the human skin microbiome. In this issue of Cell Host & Microbe, Conwill et al. investigate the coexistence of C. acnes strains on the skin and find that the skin surface harbors multiple C. acnes lineages, but individual pores are dominated by an individual lineage.

Mining for encrypted peptide antibiotics in the human proteome.

The emergence of drug-resistant bacteria calls for the discovery of new antibiotics. Yet, for decades, traditional discovery strategies have not yielded new classes of antimicrobial. Here, by mining the human proteome via an algorithm that relies on the sequence length, net charge, average hydrophobicity and other physicochemical properties of antimicrobial peptides, we report the identification of 2,603 encrypted peptide antibiotics that are encoded in proteins with biological function unrelated to the immune system. We show that the encrypted peptides kill pathogenic bacteria by targeting their membrane, modulate gut and skin commensals, do not readily select for bacterial resistance, and possess anti-infective activity in skin abscess and thigh infection mouse models. We also show, in vitro and in the two mouse models of infection, that encrypted antibiotic peptides from the same biogeographical area display synergistic antimicrobial activity. Our algorithmic strategy allows for the rapid mining of proteomic data and opens up new routes for the discovery of candidate antibiotics.

Commensal microbiota regulates skin barrier function and repair via signaling through the aryl hydrocarbon receptor.

The epidermis forms a barrier that defends the body from desiccation and entry of harmful substances, while also sensing and integrating environmental signals. The tightly orchestrated cellular changes needed for the formation and maintenance of this epidermal barrier occur in the context of the skin microbiome. Using germ-free mice, we demonstrate the microbiota is necessary for proper differentiation and repair of the epidermal barrier. These effects are mediated by microbiota signaling through the aryl hydrocarbon receptor (AHR) in keratinocytes, a xenobiotic receptor also implicated in epidermal differentiation. Mice lacking keratinocyte AHR are more susceptible to barrier damage and infection, during steady-state and epicutaneous sensitization. Colonization with a defined consortium of human skin isolates restored barrier competence in an AHR-dependent manner. We reveal a fundamental mechanism whereby the microbiota regulates skin barrier formation and repair, which has far-reaching implications for the numerous skin disorders characterized by epidermal barrier dysfunction.

Citrobacter rodentium Lysogenized with a Shiga Toxin-Producing Phage: A Murine Model for Shiga Toxin-Producing E. coli Infection.

Shiga toxin-producing E. coli (STEC) is a common foodborne pathogen in developed countries. STEC generates "attaching and effacing" (AE) lesions on colonic epithelium, characterized by effacement of microvilli and the formation of actin "pedestals" beneath intimately attached bacteria. In addition, STEC are lysogenized with a phage that, upon induction, can produce potent Shiga toxins (Stx), potentially leading to both hemorrhagic colitis and hemolytic uremic syndrome. Investigation of the pathogenesis of this disease has been challenging because STEC does not readily colonize conventional mice.Citrobacter rodentium (CR) is a related mouse pathogen that also generates AE lesions. Whereas CR does not produce Stx, a murine model for STEC utilizes CR lysogenized with an E. coli-derived Stx phage, generating CR(Φstx), which both colonizes conventional mice and readily gives rise to systemic disease. We present here key methods for the use of CR(Φstx) infection as a highly predictable murine model for infection and disease by STEC. Importantly, we detail CR(Φstx) inoculation by feeding, determination of pathogen colonization, production of phage and toxin, and assessment of intestinal and renal pathology. These methods provide a framework for studying STEC-mediated systemic disease that may aid in the development of efficacious therapeutics.

The Skin Microbiota: Balancing Risk and Reward.

The skin microbiome is an ecosystem comprised of a multitude of microbial species interacting with their surroundings, including other microbes and host epithelial and immune cells. These interactions are the basis of important roles within the skin microbiome that provide benefit to the host, boosting multiple aspects of barrier function, a critical function of this essential organ. However, with reward always comes risk; resident skin microbes function in a context-dependent manner, set on the backdrop of a dynamic host and microbial milieu. Here, we discuss the reward of hosting a microbial ecosystem on the skin, including protection from pathogens and tuning of the skin microenvironment. We also give consideration to how these skin residents, often termed "commensals" can cause disorder, damage, and promote skin disease.

Research Techniques Made Simple: Profiling the Skin Microbiota.

Skin is colonized by microbial communities (microbiota) that participate in immune homeostasis, development and maintenance of barrier function, and protection from pathogens. The past decade has been marked by an increased interest in the skin microbiota and its role in cutaneous health and disease, in part due to advances in next-generation sequencing platforms that enable high-throughput, culture-independent detection of bacteria, fungi, and viruses. Various approaches, including bacterial 16S ribosomal RNA gene sequencing and metagenomic shotgun sequencing, have been applied to profile microbial communities colonizing healthy skin and diseased skin including atopic dermatitis, psoriasis, and acne, among others. Here, we provide an overview of culture-dependent and -independent approaches to profiling the skin microbiota and the types of questions that may be answered by each approach. We additionally highlight important study design considerations, selection of controls, interpretation of results, and limitations and challenges.

Synchronous Disease Kinetics in a Murine Model for Enterohemorrhagic E. coli Infection Using Food-Borne Inoculation.

Upon colonization of the intestinal epithelium, the attaching and effacing (AE) pathogen Enterohemorrhagic Escherichia coli (EHEC) effaces microvilli and forms pedestal-like structures beneath the adherent bacterium. The production of one of its virulence factors, the phage-encoded Shiga toxin (Stx) results in systemic disease, including the development of renal failure. Although EHEC does not productively infect conventional mice, EHEC infection can be modeled in mice utilizing a derivative of the natural murine AE pathogen Citrobacter rodentium (CR). Gavage of mice with CR(ΦStx2dact), a C. rodentium lysogenized by a phage encoding an Stx variant with high potency in mice, features AE lesion formation on intestinal epithelium and Stx-mediated systemic disease, including renal damage. This model is somewhat limited by mouse-to-mouse variation in the course of disease, with the time to severe morbidity (and required euthanasia) varying by as many as 5 days, a feature that limits pathological analysis at defined stages of disease. In the current study, we altered and optimized the preparation, dose, and mode of delivery of CR(ΦStx2dact), using food-borne route of infection to generate highly synchronous disease model. We found that food-borne inoculation of as few as 3 × 104 CR(ΦStx2dact) resulted in productive colonization and severe systemic disease. Upon inoculation of 1 × 108 bacteria, the majority of infected animals suffered weight loss beginning 5 days post-infection and all required euthanasia on day 6 or 7. This enhanced murine model for EHEC infection should facilitate characterization of the pathology associated with specific phases of Stx-mediated disease.