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Cited by 131Microbial Community Ecology and PhysiologyBacterial biofilms and quorum sensingAntimicrobial Resistance in Staphylococcus

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Biotic Interactions Shape the Ecological Distributions of Staphylococcus Species

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Kastman, Erik K., Kamelamela, Noelani, Norville, Josh W., Cosetta, Casey M., Dutton, Rachel J., Wolfe, Benjamin E.·2016·mBio
DOI 10.1128/mbio.01157-16PMC PMC5082897
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Abstract

Many metagenomic sequencing studies have observed the presence of closely related bacterial species or genotypes in the same microbiome. Previous attempts to explain these patterns of microdiversity have focused on the abiotic environment, but few have considered how biotic interactions could drive patterns of microbiome diversity. We dissected the patterns, processes, and mechanisms shaping the ecological distributions of three closely related Staphylococcus species in cheese rind biofilms. Paradoxically, the most abundant species (S. equorum) is the slowest colonizer and weakest competitor based on growth and competition assays in the laboratory. Through in vitro community reconstructions, we determined that biotic interactions with neighboring fungi help resolve this paradox. Species-specific stimulation of the poor competitor by fungi of the genus Scopulariopsis allows S. equorum to dominate communities in vitro as it does in situ Results of comparative genomic and transcriptomic experiments indicate that iron utilization pathways, including a homolog of the S. aureus staphyloferrin B siderophore operon pathway, are potential molecular mechanisms underlying Staphylococcus-Scopulariopsis interactions. Our integrated approach demonstrates that fungi can structure the ecological distributions of closely related bacterial species, and the data highlight the importance of bacterium-fungus interactions in attempts to design and manipulate microbiomes. IMPORTANCE: Decades of culture-based studies and more recent metagenomic studies have demonstrated that bacterial species in agriculture, medicine, industry, and nature are unevenly distributed across time and space. The ecological processes and molecular mechanisms that shape these distributions are not well understood because it is challenging to connect in situ patterns of diversity with mechanistic in vitro studies in the laboratory. Using tractable cheese rind biofilms and a focus on coagulase-negative staphylococcus (CNS) species, we demonstrate that fungi can mediate the ecological distributions of closely related bacterial species. One of the Staphylococcus species studied, S. saprophyticus, is a common cause of urinary tract infections. By identifying processes that control the abundance of undesirable CNS species, cheese producers will have more precise control on the safety and quality of their products. More generally, Staphylococcus species frequently co-occur with fungi in mammalian microbiomes, and similar bacterium-fungus interactions may structure bacterial diversity in these systems.

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FIG 1 Ecological distributions of Staphylococcus species in cheese rind microbiomes. (A) A natural rind cheese showing the surface biofilm and curd. (B) Colonies of three of the most abundant CNS species found in cheese rinds were grown on plate count agar and photographed in natural light. Most strains of S. saprophyticus and S. xylosus are yellow or orange, while most strains of S. equorum are white. (C) Relative abundances of four CNS species in 25 cheese rind communities determined via metagenomic mapping of reads to reference genomes. Data are presented as relative abundances of reads that mapped to a Staphylococcus genome. Numbers at the bottom of the figure panel represent cheese sample identification numbers. (D) Relative abundances of four CNS species across a temporal sampling of 3 wheels of cheese aged over 56 days. Data for fungal and bacterial communities are from amplicon data presented in reference 26. Data for Staphylococcus communities were determined and are presented as described for panel C. (E) A model of Staphylococcus interactions with the biotic and abiotic environment during cheese rind development.
FIG 1 Ecological distributions of Staphylococcus species in cheese rind microbiomes. (A) A natural rind cheese showing the surface biofilm and curd. (B) Colonies of three of the most abundant CNS species found in cheese rinds were grown on plate count agar and photographed in natural light. Most strains of S. saprophyticus and S. xylosus are yellow or orange, while most strains of S. equorum are white. (C) Relative abundances of four CNS species in 25 cheese rind communities determined via metagenomic mapping of reads to reference genomes. Data are presented as relative abundances of reads that mapped to a Staphylococcus genome. Numbers at the bottom of the figure panel represent cheese sample identification numbers. (D) Relative abundances of four CNS species across a temporal sampling of 3 wheels of cheese aged over 56 days. Data for fungal and bacterial communities are from amplicon data presented in reference 26. Data for Staphylococcus communities were determined and are presented as described for panel C. (E) A model of Staphylococcus interactions with the biotic and abiotic environment during cheese rind development.CC BY · PubMed Central
FIG 2 Ecological processes impacting the distributions of Staphylococcus (Staph) species in cheese rinds. (A) Colonization of different strains of S. equorum, S. saprophyticus, and S. xylosus on cheese curd agar. Points represent means (n = 3) ± 1 standard error. Strain (F11,24 = 115.19, P < 0.0001) and time of harvest (F3,72 = 151.09, P < 0.001) and their interaction (F33,72 = 16.187, P < 0.001) were significant based on a repeated-measures analysis of variance (ANOVA), with all S. equorum isolates having significantly lower growth than the S. saprophyticus and S. xylosus isolates based on Tukey’s honestly significant difference (HSD) test. Red asterisks next to strain names indicate the strains of each species used for the remaining experiments. (B) Pairwise competition of the three species of Staphylococcus (S. equorum [S.eq] strain BC9, S. saprophyticus [S.sap] strain BC4, and S. xylosus [S.xyl] strain BC10), with colors as defined for panel A. Bars represent means (n = 3) ± 1 standard error. Asterisks indicate significant differences in growth rates (P < 0.01) in the presence of an interspecific competitor compared to growth in the absence of an interspecific competitor (Dunnett’s test). (C) Growth of same species/strains as described for panel B over a range of pH and salt concentrations in BHI medium as measured by OD600 after 48 h. Points represent means (n = 3) ± 1 standard error. S. xylosus has a greater niche breadth (OD600 sum = 21.47 ± 0.50) than S. saprophyticus (OD600 sum = 19.187 ± 0.28) or S. equorum (OD600 sum = 19.08 ± 0.33) based on ANOVA with Tukey’s HSD test (F2,5 = 13.336, P < 0.01). (D) Relative abundances of the three species of Staphylococcus in experimental in vitro communities over 21 days. The three species were inoculated at initially identical densities and grown alone or with 5 single-species neighbors or an entire community containing all 5 neighboring species. Both biotic treatment (F6,20 = 48.268, P < 0.0001) and time of harvest (F2,40 = 29.310, P < 0.001) and their interaction (F12,40 = 12.528, P < 0.001) had significant effects on the abundance of S. equorum. Asterisks indicate significant stimulation of S. equorum compared to Staph-alone treatment based on Tukey’s HSD tests of absolute numbers of CFU. (E) Absolute abundances of the three species of Staphylococcus in the same experimental in vitro communities as described for panel D but only for molds Penicillium and Scopulariopsis. Solid lines represent the species growth in treatments without neighbors, while dashed lines represent the species growth in treatments with the fungal partner. Points represent means (n = 4) ± 1 standard error.
FIG 2 Ecological processes impacting the distributions of Staphylococcus (Staph) species in cheese rinds. (A) Colonization of different strains of S. equorum, S. saprophyticus, and S. xylosus on cheese curd agar. Points represent means (n = 3) ± 1 standard error. Strain (F11,24 = 115.19, P < 0.0001) and time of harvest (F3,72 = 151.09, P < 0.001) and their interaction (F33,72 = 16.187, P < 0.001) were significant based on a repeated-measures analysis of variance (ANOVA), with all S. equorum isolates having significantly lower growth than the S. saprophyticus and S. xylosus isolates based on Tukey’s honestly significant difference (HSD) test. Red asterisks next to strain names indicate the strains of each species used for the remaining experiments. (B) Pairwise competition of the three species of Staphylococcus (S. equorum [S.eq] strain BC9, S. saprophyticus [S.sap] strain BC4, and S. xylosus [S.xyl] strain BC10), with colors as defined for panel A. Bars represent means (n = 3) ± 1 standard error. Asterisks indicate significant differences in growth rates (P < 0.01) in the presence of an interspecific competitor compared to growth in the absence of an interspecific competitor (Dunnett’s test). (C) Growth of same species/strains as described for panel B over a range of pH and salt concentrations in BHI medium as measured by OD600 after 48 h. Points represent means (n = 3) ± 1 standard error. S. xylosus has a greater niche breadth (OD600 sum = 21.47 ± 0.50) than S. saprophyticus (OD600 sum = 19.187 ± 0.28) or S. equorum (OD600 sum = 19.08 ± 0.33) based on ANOVA with Tukey’s HSD test (F2,5 = 13.336, P < 0.01). (D) Relative abundances of the three species of Staphylococcus in experimental in vitro communities over 21 days. The three species were inoculated at initially identical densities and grown alone or with 5 single-species neighbors or an entire community containing all 5 neighboring species. Both biotic treatment (F6,20 = 48.268, P < 0.0001) and time of harvest (F2,40 = 29.310, P < 0.001) and their interaction (F12,40 = 12.528, P < 0.001) had significant effects on the abundance of S. equorum. Asterisks indicate significant stimulation of S. equorum compared to Staph-alone treatment based on Tukey’s HSD tests of absolute numbers of CFU. (E) Absolute abundances of the three species of Staphylococcus in the same experimental in vitro communities as described for panel D but only for molds Penicillium and Scopulariopsis. Solid lines represent the species growth in treatments without neighbors, while dashed lines represent the species growth in treatments with the fungal partner. Points represent means (n = 4) ± 1 standard error.CC BY · PubMed Central
FIG 3 Putative molecular mechanisms underlying interactions between Staphylococcus equorum and fungi. (A) Comparative genomics of Staphylococcus species reveals species-specific genes, which are unevenly distributed across functional categories. Data are from 12 S. equorum (gray), 5 S. saprophyticus (yellow), and 5 S. xylosus (orange) strains. The Venn diagram shows overlap of clusters of protein-coding genes across the three species. The bar graph shows the distribution of species-specific genes (found in all strains of one species but not other species) across SEED subsystems. (B) Genome size estimates based on genome assemblies and percentages of prophage with intact genomes, as determined through PHAST annotations. (C) Changes in genome expression of S. equorum grown with the fungus Penicillium (left panel) or Scopulariopsis (right panel). Panels show scatter plots of global gene expression for S. equorum alone (x axis) versus with a fungus (y axis). Each dot represents a transcript from across the genome. Orange dots represent those transcripts that were differentially expressed when grown with the fungus (>5-fold change in expression; Rockhopper q value < 0.05). (D) Distribution of differentially expressed genes represented in panel C across SEED subsystems. (E) Representative read mapping at the staphyloferrin B operon from treatments with S. equorum alone (top), S. equorum plus Penicillium (middle), and S. equorum plus Scopulariopsis (bottom). Blue-shaded regions represent average coverage from read mappings. (F) Mean (± standard error) free iron concentration in cheese curd agar (CCA), CCA plus Scopulariopsis, and CCA plus Penicillium. All three conditions had significantly different iron concentrations based on ANOVA with Tukey’s HSD test (F2,10 = 10.30, P < 0.01). (G) Lyophilized and concentrated supernatants of CCA incubated without a fungus, with Penicillium, and with Scopulariopsis were spotted onto cellulose disks on chrome azurol S (CAS) medium. Shown is a representative plate after 1 day of incubation. (H) The supernatants used in the experiment whose results are shown in panel G were spotted onto cheese curd agar plates seeded with the three Staphylococcus species. The radius of zones of stimulation was measured around the cellulose disk after 3 days. (I) Mean methionine concentrations (± standard error) determined in cheese curd alone and cheese curd plus Scopulariopsis using capillary electrophoresis-time of flight mass spectrometry (CE-TOF MS). See Table S5 in the supplemental material for more details on metabolomics.
FIG 3 Putative molecular mechanisms underlying interactions between Staphylococcus equorum and fungi. (A) Comparative genomics of Staphylococcus species reveals species-specific genes, which are unevenly distributed across functional categories. Data are from 12 S. equorum (gray), 5 S. saprophyticus (yellow), and 5 S. xylosus (orange) strains. The Venn diagram shows overlap of clusters of protein-coding genes across the three species. The bar graph shows the distribution of species-specific genes (found in all strains of one species but not other species) across SEED subsystems. (B) Genome size estimates based on genome assemblies and percentages of prophage with intact genomes, as determined through PHAST annotations. (C) Changes in genome expression of S. equorum grown with the fungus Penicillium (left panel) or Scopulariopsis (right panel). Panels show scatter plots of global gene expression for S. equorum alone (x axis) versus with a fungus (y axis). Each dot represents a transcript from across the genome. Orange dots represent those transcripts that were differentially expressed when grown with the fungus (>5-fold change in expression; Rockhopper q value < 0.05). (D) Distribution of differentially expressed genes represented in panel C across SEED subsystems. (E) Representative read mapping at the staphyloferrin B operon from treatments with S. equorum alone (top), S. equorum plus Penicillium (middle), and S. equorum plus Scopulariopsis (bottom). Blue-shaded regions represent average coverage from read mappings. (F) Mean (± standard error) free iron concentration in cheese curd agar (CCA), CCA plus Scopulariopsis, and CCA plus Penicillium. All three conditions had significantly different iron concentrations based on ANOVA with Tukey’s HSD test (F2,10 = 10.30, P < 0.01). (G) Lyophilized and concentrated supernatants of CCA incubated without a fungus, with Penicillium, and with Scopulariopsis were spotted onto cellulose disks on chrome azurol S (CAS) medium. Shown is a representative plate after 1 day of incubation. (H) The supernatants used in the experiment whose results are shown in panel G were spotted onto cheese curd agar plates seeded with the three Staphylococcus species. The radius of zones of stimulation was measured around the cellulose disk after 3 days. (I) Mean methionine concentrations (± standard error) determined in cheese curd alone and cheese curd plus Scopulariopsis using capillary electrophoresis-time of flight mass spectrometry (CE-TOF MS). See Table S5 in the supplemental material for more details on metabolomics.CC BY · PubMed Central
FIG 4 An overview of traits of cheese rind Staphylococcus species and potential mechanisms driving their interactions with fungi. Solid lines indicate interactions/mechanisms established in this work or in previous studies. Dashed lines indicate potential or unknown interactions/mechanisms.
FIG 4 An overview of traits of cheese rind Staphylococcus species and potential mechanisms driving their interactions with fungi. Solid lines indicate interactions/mechanisms established in this work or in previous studies. Dashed lines indicate potential or unknown interactions/mechanisms.CC BY · PubMed Central

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Metadata

Authors
Kastman, E. K., Kamelamela, N., Norville, J. W., Cosetta, C. M., Dutton, R. J., & Wolfe, B. E.
Affiliations
  • Tufts University (US)
  • University of California San Diego (US)
Corresponding author
Wolfe, Benjamin E.
ORCID
Venue
mBio
Publisher
American Society for Microbiology
Published
2016-11-02
Volume
7
Issue
5
ISSN
  • 2150-7511 (ISSN-L)
  • 2161-2129
DOI
10.1128/mbio.01157-16
PMC
PMC5082897
OpenAlex
W2538439703
Cited by
127
References
88
Citations by year
2026: 7 · 2025: 14 · 2024: 14 · 2023: 18 · 2022: 4 · 2021: 15 · 2020: 13 · 2019: 14 · 2018: 18 · 2017: 9 · 2016: 1
FWCI
12.24
Type
journal article
Language
en
Update notices
correction (2017-03-21)
Open access
Licence
Locations
  • mBio (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • eScholarship (California Digital Library) (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • eScholarship (California Digital Library) (submittedVersion)
Funders
  • HHS | National Institutes of Health (P50 GM068763)
  • Tufts University
  • UC | University of California, San Diego
Topics
  • Physical Sciences › Environmental Science › Ecology › Microbial Community Ecology and Physiology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Bacterial biofilms and quorum sensing
  • Health Sciences › Medicine › Infectious Diseases › Antimicrobial Resistance in Staphylococcus
Keywords
Staphylococcus, Staphylococcus equorum, Staphylococcus saprophyticus, Scopulariopsis, coagulase-negative staphylococci, ecological distribution, bacterial competition, colonization, iron utilization, siderophores, microbiota composition, urinary tract infection
MeSH terms
Food Microbiology, Iron, Staphylococcus, Gene Expression Profiling, Genomics, Microbial Interactions, Biota, Scopulariopsis

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