Biology subjects

Kalan, L.

Publications and source records attributed to Kalan, L..

17 recordsLinked to original sources

bioRxiv2026

Bioactive Natural Products Produced by Streptomyces from the Microbiome of Cadaveric Fly Larvae

Streptomyces are prolific producers of bioactive compounds and increasingly recognized as members of insect microbiomes, yet the microbiome of cadaveric fly larvae remain an overlooked system for discovering metabolically versatile Streptomyces species. Here, we conduct targeted bacterial isolations from the microbiome of fly larvae collected from pig cadavers, generating 42 Streptomyces isolates of interest, and systematically evaluated their metabolic potential through genomic analysis, antimicrobial screening, biosynthetic gene cluster assessment, untargeted LC-MS/MS metabolomics, and compound purification. The Streptomyces isolates spanned nine species, including underrepresented lineages for which we added genomic representatives. Streptomyces from carrion fly larvae exhibited broad-spectrum antimicrobial activity and substantial BGC diversity, supported by metabolomic detection of antimycins, surugamides, and macrotetrolides. From a deep phylogenetic lineage, we purified JBIR-68 and Simamycin and demonstrated their potent anthelmintic activity against Brugia malayi microfilariae. GNPS molecular networking revealed three additional JBIR-68 analogs, establishing the first taxonomically resolved Streptomyces lineage capable of producing these rare metabolites. Our findings position cadaveric fly larvae as a rich ecological reservoir for discovering Streptomyces with the potential to produce chemically diverse natural products with biomedical applications.

biochemistry↗

bioRxiv2025

Establishing an ex vivo porcine skin model to investigate the effects of broad spectrum antiseptic on viable skin microbial communities

Incomplete antiseptic efficacy against potentially pathogenic microbial taxa places some patients at disproportionate risk for developing a surgical site infection. Laboratory models capable of interrogating the effects of antiseptics on the skin and its complex microbial communities are desperately needed to improve and better tailor antiseptic formulations. This work aims to establish an ex vivo porcine skin model to explore the impact of topical antiseptics on complex skin microbial communities and superficial skin lipids. Microbiome samples were treated with propidium-monoazide to selectively evaluate DNA from viable microorganisms. Bacterial abundances were assessed via viability-qPCR and quantitative culture. Viable community populations were evaluated with 16S rRNA gene sequencing. Epidermal biopsies were collected at multiple timepoints for lipidomic assessment via LC/MS. The ex vivo environment promoted shifts in porcine skin lipid composition and microbial communities over the experiments duration. Compared to water treated control skin, skin treated with the antiseptic chlorhexidine gluconate had significantly lower culturable and bioburden determined by viability-qPCR. Compared to water treated skin, viable microbial communities on CHG treated skin displayed greater relative abundance of several gut associated and Gram-negative bacterial taxa, including SMB53, Turicibacter, Pseudomonas and Proteus. Collectively these findings highlight the utility of an ex vivo porcine skin system for interrogating the impacts of antimicrobial disruption to complex microbial ecosystems, and ultimately for the future testing and development of improved antiseptic formulations.

microbiology↗

bioRxiv2024

Large-scale investigation for antimicrobial activity reveals novel defensive species across the healthy skin microbiome

The human skin microbiome constitutes a dynamic barrier that can impede pathogen invasion by producing antimicrobial natural products. Gene clusters encoding for production of secondary metabolites, biosynthetic gene clusters (BGCs), that are enriched in the human skin microbiome relative to other ecological settings, position this niche as a promising source for new natural product mining. Here, we introduce a new human microbiome isolate collection, the EPithelial Isolate Collection (EPIC). It includes a large phylogenetically diverse set of human skin-derived bacterial strains from eight body sites. This skin collection, consisting of 980 strains is larger and more diverse than existing resources, includes hundreds of rare and low-abundance strains, and hundreds of unique BGCs. Using a large-scale co-culture screen to assess 8,756 pairwise interactions between skin-associated bacteria and potential pathogens, we reveal broad antifungal activity by skin microbiome members. Integrating 287 whole isolate genomes and 268 metagenomes from sampling sites demonstrates that while the distribution of BGC types is stable across body sites, specific gene cluster families (GCFs), each predicted to encode for a distinct secondary metabolite, can substantially vary. Sites that are dry or rarely moist harbor the greatest potential for discovery of novel bioactive metabolites. Among our discoveries are four novel bacterial species, three of which exert significant and broad-spectrum antifungal activity. This comprehensive isolate collection advances our understanding of the skin microbiomes biosynthetic capabilities and pathogen-fighting mechanisms, opening new avenues towards antimicrobial drug discovery and microbiome engineering.

microbiology↗

bioRxiv2024

Farm exposure is associated with human breast milk immune profile and microbiome

Prenatal and early life farm exposure, and breastfeeding, are associated with protection from allergic diseases. We hypothesize that farm exposure influences the human breast milk microbiome and immune proteins. The immune protein profiles and microbial communities of 152 human breast milk samples were compared among three maternal farm exposure groups (traditional agrarian, farm, and non-farm) in rural Wisconsin to identify signatures associated with farm status and atopic disease. We found significant differences between farm groups for 23 immune proteins (p-adj<0.05), microbiome diversity (p=2.2E-05), and microbiome richness (p=8.0e-06). Traditional agrarian human breast milk had the highest immune protein levels and microbiome diversity and richness, followed by farm and non-farm human breast milk. Furthermore, Gram-positive bacterial species correlated with IL-23 mediated signaling events (p-adj<1.0E-05). These data suggest that increased farm exposures promotes human breast milk that is more microbially-diverse and rich in immune-associated proteins, ultimately influencing immune development in the infant.

microbiology↗

bioRxiv2024

Altered oral microbiota of drug-resistant organism carriers exhibit impaired gram-negative pathogen inhibition

The oral microbiome has been understudied as a reservoir for clinical pathogens, including drug-resistant strains. Understanding how alterations in microbiome functioning render this site vulnerable to colonization is essential, as multidrug-resistant organisms (MDRO) carriage is a major risk factor for developing serious infections. To advance our knowledge of oral MDRO carriage and protection against pathogen colonization conferred by native microbiota, we examined microbiomes from individuals colonized by MDROs (n=33) and non-colonized age-matched controls (n=30). Shotgun metagenomic analyses of oral swabs from study participants revealed significant differences in microbial communities with depletion of Streptococcus spp. among those colonized by multidrug-resistant gram-negative bacilli (RGNB), compared to non-carriers. We utilized metagenomic sequencing to characterize the oral resistome and find antimicrobial resistance genes are present in higher abundance among RNGB carriers versus non-carriers. High-throughput co-culture screening revealed oral bacteria isolated from MDRO non-carriers demonstrate greater inhibition of gram-negative pathogens, compared to isolates from carriers. Moreover, biosynthetic gene clusters from streptococci are found in higher abundance from non-carrier microbiomes, compared to RGNB carrier microbiomes. Bioactivity-guided fractionation of extracts from Streptococcus isolate SID2657 demonstrated evidence of strong E. coli and A. baumannii inhibition in a murine model of infection. Together, this provides evidence that oral microbiota shape this dynamic microbial community and may serve as an untapped source for much-needed antimicrobial small-molecules.

microbiology↗

bioRxiv2024

Still Not Sterile: Chlorhexidine gluconate treatment does not completely reduce skin microbial bioburden and promotes pathogen overabundance in patients undergoing elective surgeries.

Surgical site infections (SSI) continue to occur despite widespread adoption of surgical antiseptics. The effects of chlorhexidine gluconate (CHG)-based antiseptics on the skin microbiome also remains undefined due to confounding effects of CHG persistence on skin. Patients undergoing elective surgery were enrolled to characterize the immediate and long-term impact of pre-surgical preparation with CHG antiseptic on skin microbial communities. Due to the broad-spectrum antimicrobial activity of CHG and its propensity to bind extracellular DNA, methods to selectively identify live microorganisms are critical to this process and to fully elucidate the effectiveness of pre-surgical protocols and potential disruptions to the healthy skin microbiome. Swabs of the surgical site skin microbiome were collected at multiple timepoints before and after surgery. Microbial bioburden and community compositions were evaluated with viability qPCR and 16S ribosomal RNA gene profiling. Pre-operative CHG induced a measurable reduction in the viable microbial bioburden at the surgical site. On the day of surgery, surgical sites displayed a significant increase in the relative abundance of several SSI associated bacterial genera, including, Acinetobacter, Bacillus, Escherichia-Shigella, and Pseudomonas, compared to baseline. Bacillus species isolated from subjects at baseline showed resistance to CHG with MICs exceeding 1000 {micro}g/ml. Despite major shifts in the skin microbiome upon exposure to CHG, they were transient in the majority of individuals. Skin microbial community structure recovered by the post-surgical follow-up. In short, this study shows that pre-surgical application of CHG can significantly reduce viable skin microbial bioburden, however, complete sterility is not achieved. While CHG induces temporary shifts in the skin microbiome, including enrichment for potentially pathogenic taxa, the skin microbiome recovers back to near baseline. Collectively, these findings identify tangible avenues for improving antiseptic formulations and offer further support that the skin microbiome is viable, stable, and resilient to chemical perturbation.

microbiology↗

bioRxiv2024

Skin-associated Corynebacterium amycolatum shares cobamides

The underlying interactions that occur to maintain skin microbiome composition, function, and overall skin health are largely unknown. Often, these types of interactions are mediated by microbial metabolites. Cobamides, the vitamin B12 family of cofactors, are essential for metabolism in many bacteria, but are only synthesized by a small fraction of prokaryotes, including certain skin-associated species. Therefore, we hypothesize that cobamide sharing mediates skin community dynamics. Preliminary work predicts that several skin-associated Corynebacterium species encode de novo cobamide biosynthesis and that their abundance is associated with skin microbiome diversity. Here, we show that commensal Corynebacterium amycolatum produces cobamides and that this synthesis can be tuned by cobalt limitation. To demonstrate cobamide sharing by C. amycolatum, we employed a co-culture assay using an E. coli cobamide auxotroph and show that C. amycolatum produces sufficient cobamides to support E. coli growth, both in liquid co-culture and when separated spatially on solid medium. We also generated a C. amycolatum non-cobamide-producing strain (cob-) using UV mutagenesis that contains mutated cobamide biosynthesis genes cobK and cobO and confirm that disruption of cobamide biosynthesis abolishes support of E. coli growth through cobamide sharing. Our study provides a unique model to study metabolite sharing by microorganisms, which will be critical for understanding the fundamental interactions that occur within complex microbiomes and for developing approaches to target the human microbiota for health advances.

microbiology↗

bioRxiv2023

What is Slough?A pilot study to define the proteomic and microbial composition of wound slough and its implications for wound healing.

Slough is a well-known feature of non-healing wounds. This study aims to determine the proteomic and microbiologic components of slough as well as interrogate the associations between wound slough components and wound healing. Twenty-three subjects with slow-to-heal wounds and visible slough were enrolled. Etiologies included venous stasis ulcers, post-surgical site infections, and pressure ulcers. Patient co-morbidities and wound healing outcome at 3-months post-sample collection was recorded. Debrided slough was analyzed microscopically, through untargeted proteomics, and high-throughput bacterial 16S-ribosomal gene sequencing. Microscopic imaging revealed wound slough to be amorphous in structure and highly variable. 16S-profiling found slough microbial communities to associate with wound etiology and location on the body. Across all subjects, slough largely consisted of proteins involved in skin structure and formation, blood-clot formation, and immune processes. To predict variables associated with wound healing, protein, microbial, and clinical datasets were integrated into a supervised discriminant analysis. This analysis revealed that healing wounds were enriched for proteins involved in skin barrier development and negative regulation of immune responses. While wounds that deteriorated over time started off with a higher baseline Bates-Jensen Wound Assessment Score and were enriched for anerobic bacterial taxa and chronic inflammatory proteins. To our knowledge, this is the first study to integrate clinical, microbiome, and proteomic data to systematically characterize wound slough and integrate it into a single assessment to predict wound healing outcome. Collectively, our findings underscore how slough components can help identify wounds at risk of continued impaired healing and serves as an underutilized biomarker.

microbiology↗

bioRxiv2023

zol & fai: large-scale targeted detection and evolutionary investigation of gene clusters

Many universally and conditionally important genes are genomically aggregated within clusters. Here, we introduce fai and zol, which together enable large-scale comparative analysis of different types of gene clusters and mobile-genetic elements (MGEs), such as biosynthetic gene clusters (BGCs) or viruses. Fundamentally, they overcome a current bottleneck to reliably perform comprehensive orthology inference at large scale across broad taxonomic contexts and thousands of genomes. First, fai allows the identification of orthologous instances of a query gene cluster of interest amongst a database of target genomes. Subsequently, zol enables reliable, context-specific inference of ortholog groups for individual protein-encoding genes across gene cluster instances. In addition, zol performs functional annotation and computes a variety of evolutionary statistics for each inferred ortholog group. Importantly, in comparison to tools for visual exploration of homologous relationships between gene clusters, zol can scale to thousands of gene cluster instances and produce detailed reports that are easy to digest. To showcase fai and zol, we apply them for: (i) longitudinal tracking of a virus in metagenomes, (ii) discovering novel population-level genetic insights of two common BGCs in the fungal species Aspergillus flavus, and (iii) uncovering large-scale evolutionary trends of a virulence-associated gene cluster across thousands of genomes from a diverse bacterial genus.

bioinformatics↗

bioRxiv2022

Comparative genomic and metagenomic investigations of the Corynebacterium tuberculostearicum species complex reveals potential mechanisms underlying associations to skin health and disease

Corynebacterium are a diverse genus and dominant member of the human skin microbiome. Recently, we reported that the most prevalent Corynebacterium species found on skin - including Corynebacterium tuberculostearicum and Corynebacterium kefirresidentii - comprise a narrow species complex despite the diversity of the genus. Here, we apply high-resolution phylogenomics and comparative genomics to describe the structure of the C. tuberculostearicum species complex. We find this species complex is missing a fatty acid biosynthesis gene family which is often found in multi-copy in approximately 99% of other Corynebacterium species. Conversely, this species complex is enriched for multiple genetic traits, including a gene encoding for a collagen-like peptide. Further, through metagenomic investigations, we find that one species within the complex, C. kefirresidentii, increases in relative abundance during atopic dermatitis flares and show that most members of this species possess a colocalized set of putative virulence genes.

microbiology↗

bioRxiv2022

Sweat and sebum preferences of the human skin microbiota

The microorganisms that inhabit human skin, collectively termed the skin microbiome, must overcome numerous challenges that typically impede microbial growth, including low pH, osmotic pressure, and low nutrient availability. Yet, the skin microbiota thrive on the skin and have adapted to these stressful conditions. Limited skin nutrients are available for microbial use in this unique niche, including those from host-derived sweat, sebum, and corneocytes. Here, we have developed physiologically-relevant, skin-like growth media that is composed of compounds present in human sweat and sebum. We find that skin-associated bacterial species exhibit unique growth profiles in different concentrations of sweat and sebum. The majority of strains evaluated demonstrate a preference for high sweat concentrations, while sebum preference is highly variable, suggesting that the capacity for sebum utilization may be an important driver of skin microbial community structure. Furthermore, these findings provide experimental rationale for why different skin microenvironments harbor distinct microbiome communities. In all, our study further emphasizes the importance of studying microorganisms in an ecologically-relevant context, which is critical for our understanding of their physiology, ecology, and function on the skin.

microbiology↗

bioRxiv2022

lsaBGC provides a comprehensive framework for evolutionary analysis of biosynthetic gene clusters within focal taxa

We developed lsaBGC, a bioinformatics suite that introduces several new methods to expand on the available infrastructure for genomic and metagenomic-based comparative and evolutionary investigation of biosynthetic gene clusters (BGCs). Through application of the suite to four genera commonly found in skin microbiomes, we uncover multiple novel findings on the evolution and diversity of their BGCs. We show that the virulence associated carotenoid staphyloxanthin in Staphylococcus aureus is ubiquitous across the Staphylococcus genus but has largely been lost in the skin-commensal species Staphylococcus epidermidis. We further identify thousands of novel single nucleotide variants (SNVs) within BGCs from the Corynebacterium tuberculostearicum sp. complex, which we describe here to be a narrow, multi-species clade that features the most prevalent Corynebacterium in healthy skin microbiomes. Although novel SNVs were approximately ten times as likely to correspond to synonymous changes when located in the top five percentile of conserved sites, lsaBGC identified SNVs which defied this trend and are predicted to underlie amino acid changes within functionally key enzymatic domains. Ultimately, beyond supporting evolutionary investigations, lsaBGC provides important functionalities to aid efforts for the discovery or synthesis of natural products.

microbiology↗

bioRxiv2021

vRhyme enables binning of viral genomes from metagenomes

Genome binning has been essential for characterization of bacteria, archaea, and even eukaryotes from metagenomes. Yet, no approach exists for viruses. We developed vRhyme, a fast and precise software for construction of viral metagenome-assembled genomes (vMAGs). vRhyme utilizes single- or multi-sample coverage effect size comparisons between scaffolds and employs supervised machine learning to identity nucleotide feature similarities, which are compiled into iterations of weighted networks and refined bins. Using simulated viromes, we displayed superior performance of vRhyme compared to available binning tools in constructing more complete and uncontaminated vMAGs. When applied to 10,601 viral scaffolds from human skin, vRhyme advanced our understanding of resident viruses, highlighted by identification of a Herelleviridae vMAG comprised of 22 scaffolds, and another vMAG encoding a nitrate reductase metabolic gene, representing near-complete genomes post-binning. vRhyme will enable a convention of binning uncultivated viral genomes and has the potential to transform metagenome-based viral ecology.

bioinformatics↗

bioRxiv2021

Trade-offs between antiseptic cytotoxicity and efficacy in a human ex vivo wound contamination model

Wound cleansing agents are routine in wound care, even in the absence of signs of infection. Antiseptic activity prevents contaminating microbes from establishing an infection while also raising concerns of cytotoxicity and delayed wound healing. Here, we used an ex vivo human skin excisional wound model to evaluate the cytotoxicity of five clinically-used wound cleaning agents (saline, povidone iodine, Dove(R) soap, Dial(R) soap, and chlorhexidine gluconate). We established a wound contamination model using [~]100 cells of Pseudomonas aeruginosa per wound to evaluate antiseptic efficacy and microbial biofilm spatial organization. We found that Dial(R) soap and chlorhexidine gluconate significantly reduced metabolic activity of the biopsies, while all treatments except saline affected local cellular viability. Within the contamination model, only chlorhexidine gluconate treatment resulted in significantly lower P. aeruginosa counts at 24 hours post-treatment, driven by sub-limit-of-detection counts immediately post-treatment. Later applications of chlorhexidine gluconate had no effects on microbial growth, with microscopy showing extensive surface colonization of the wound bed. We present a clinically-relevant model for evaluating antiseptic cytotoxicity and efficacy, with the ability to resolve spatial localization and temporal dynamics of tissue viability and microbial growth.

microbiology↗

bioRxiv2020

Corynebacterium Comparative Genomics Reveals a Role for Cobamide Sharing in the Skin Microbiome

The human skin microbiome is a key player in human health, with diverse functions ranging from defense against pathogens to education of the immune system. While recent studies have begun to shed light on the valuable role that skin microorganisms have in maintaining a healthy skin barrier, a detailed understanding of the complex interactions that shape healthy skin microbial communities is limited. Cobamides, the vitamin B12 class of cofactor, are essential for organisms across the tree of life. Because this vitamin is only produced by a limited fraction of prokaryotes, cobamide sharing has been shown to mediate community dynamics within microbial communities. Here, we provide the first large-scale unbiased metagenomic assessment of cobamide biosynthesis and utilization in the skin microbiome. We show that while numerous and diverse taxa across the major bacterial phyla on the skin are cobamide dependent, relatively few species encode for de novo cobamide biosynthesis. We find that cobamide sharing shapes the network structure in microbial communities across the different microenvironments of the skin and that changes in community structure and microbiome diversity are driven by the abundance of cobamide producers in the Corynebacterium genus, in both healthy and disease skin states. Lastly, we find that de novo cobamide biosynthesis is enriched only in host-associated Corynebacterium species, including those prevalent on human skin. We confirm that the cofactor is produced in excess through quantification of cobamide production by skin-associated species isolated in the laboratory. Taken together, our results support a role for cobamide sharing within skin microbial communities, which we predict stabilizes the microbiome and mediates host interactions.

microbiology↗

bioRxiv2019

Effects of Topical Antimicrobial Formulations on Pseudomonas aeruginosa Biofilm in an In vivo Porcine Burn Wound Model

Silver has been incorporated into a variety of wound dressings and topical agents to prevent and combat wound infections. Pseudomonas aeruginosa is a common cause of burn wound infections and well-known biofilm producer. The objective of this study was to evaluate the effects of a panel of wound dressings containing different silver formulations on P. aeruginosa biofilms using an in vivo porcine burn wound model. Second-degree burns were created on the skin of specific pathogen-free pigs (n = 3) and inoculated with 2.14 x 105 cfu P. aeruginosa per wound. Biofilms were allowed to develop for 24 h, and then each wound was treated with one of 6 treatments: silver oxynitrate dressing (OXY), silver oxynitrate powder (POWD), nanocrystalline silver dressing (NANO), silver chloride dressing (AGCL), silver sulfadiazine (SSD), or a negative control polyurethane film with no silver-based formulation (NEG). Wounds were cultured at D3 post-infection (n = 3 per pig per treatment) and at D6 post-infection (n = 3 per pig per treatment) for quantification of bacteria. On D6, biopsies (n = 3 per treatment) were taken from POWD, SSD, and NEG wounds and wound healing progress was evaluated histologically. At the time of treatment initiation, 24 h post-infection, 8.71 log cfu P. aeruginosa were present in burn wounds. On D3 and D6, all treatments significantly reduced bacterial counts in wounds as compared to NEG, but POWD caused an approximately 7-log reduction in bacterial counts on both days and was the only treatment to reduce the bacterial counts to below the threshold for detecting bacteria. The OXY, NANO, and SSD treatments had similar reductions in bacterial recovery on D3 and D6 of approximately 2.5-4 log. The histological healing metrics of reepithelialization percentage, epithelial thickness, white cell infiltration, angiogenesis, and granulation tissue formation were similar among wounds from POWD, SSD, and NEG groups at 6 days post-infection. Silver oxynitrate powder reduced P. aeruginosa growth in burn wounds more effectively than other silver-based dressings but did not impact wound healing.

microbiology↗

bioRxiv2018

The microbial basis of impaired wound healing: differential roles for pathogens, \"bystanders\", and strain-level diversification in clinical outcomes

Chronic, non-healing wounds are a major complication of diabetes associated with high morbidity and health care expenditures estimated at $9-13 billion annually in the US. Though microbial infection and critical colonization is hypothesized to impair healing and contribute to severe outcomes such as amputation, antimicrobial therapy is inefficacious and the role of microbes in tissue repair, regeneration, and healing remains unclear. Here, in a longitudinal prospective cohort study of 100 subjects with non-infected neuropathic diabetic foot ulcer (DFU), we performed metagenomic shotgun sequencing to elucidate microbial temporal dynamics at strain-level resolution, to investigate pathogenicity and virulence of the DFU microbiome with respect to outcomes, and to determine the influence of therapeutic intervention on the DFU microbiota. Slow healing DFUs were associated with signatures of biofilm formation, host invasion, and virulence. Though antibiotic resistance was widespread at the genetic level, debridement, rather than antibiotic treatment, significantly shifted the DFU microbiome in patients with more favorable outcomes. Primary clinical isolates of S. aureus, C. striatum, and A. faecalis induced differential biological responses in keratinocytes and in a murine model of diabetic wound healing, with the S. aureus strain associated with non-healing wounds eliciting the most severe phenotype. Together these findings implicate strain-level diversification of the wound pathogen S. aureus in chronic wound outcomes, while revealing potential contributions from skin commensals and other previously underappreciated constituents of the wound microbiota.

genomics↗