Pore Hopping
by Christoph
To study bacterial ecology, Roberto said in his recent post, "you'd want a setup whose scale corresponds to the size of the bacteria in question." He was pleased to learn of a study addressing bacterial chemical ecology within a very small ecosystem, namely the hair follicles of human skin, where Cutibacterium acnes contend with Staphylococci. Here now, it's again about C. acnes, but this time about several strains competing for growth in human hair follicles, colloquial pores. An ecological family affair, so to speak.
Figure 1. C. acnes biogeography at high spatial resolution and high genetic resolution. (A) We collected C. acnes colonies from sebaceous skin regions (forehead, nose, right cheek, left cheek, chin, shoulder, back) from 16 healthy adult subjects. Samples were acquired at coarse spatial resolution with a toothpick (scrapes; N=155) and fine spatial resolution (pore extracts and pore strips; N=145). This approach enabled us to examine C. acnes biogeography with spatial resolution down to a single pore (sebaceous follicle). (B) To understand how these samples were related to each other, we performed whole genome sequencing on 947 colonies (1–15 per sample), each of which represents the genetic content of a single cell that originated on the skin of one of our subjects. This approach enabled us to examine C. acnes biogeography with genetic resolution down to a single nucleotide variant (SNV). Source. Frontispiece: Propionibacterium acnes. SEM image Matthias Mörgelin, Lund University, SE. Source
A whiff of taxonomy. Cutibacterium acnes, a slow-growing, aerotolerant anaerobic, non-spore forming Gram‑positive rod-shaped bacterium, had been known among microbiologists as Bacillus acnes since 1900, and was re‑named Propionibacterium acnes in 1946. Scholz & Kilian (2016), through a comprehensive genome analysis of 162 whole‑genome sequences of strains representing species of the family Propionibacteriaceae (phylum Actinomycetota), found that the commonly used 16S RNA phylogeny had too low a resolution and that re‑classification to a new genus, Cutibacterium, was warranted (see frontispiece).
The species kept its "acnes" name, but it remains unclear to what extent it is involved in the etiology of acne (med. acne vulgaris). "Cutis" is Latin for skin, and indeed C. acnes is a highly abundant bacterium in the skin microbiota of almost all humans. It predominates in invaginations of oily skin sites such the face and back, the follicles, which are anoxic and enriched in sebum. "Highly abundant" meaning that Claesen et al. (2020) estimated that individual pores contain up to 108 colony-forming units (CFU) of C. acnes (average 105 CFU). Individuals are typically colonized by several C. acnes strains that coexist over time and at different body sites. However, it is unclear whether coexisting strains occupy distinct ecological niches on the skin surface and within skin substructures, such as pores, and why unique sets of C. acnes strains in individuals are so robust to change. That is, it was unclear until now.
Figure 2. C. acnes dominates within-pore bacterial populations. A subset of samples from two subjects was analyzed for community composition via 16S rRNA amplicon sequencing, in addition to colony-based C. acnes whole-genome sequencing. Each species is colored in if its mean relative abundance across samples is >5% and each genus is colored if its max relative abundance across samples is >33%; everything else is categorized as "Other". Actinomycetota: C. acnes, Lawsonella sp., Corynebacterium sp.; Bacillota: Staphylococcus sp., Anaerococcus sp., Peptoniphilus sp.; Pseudomonadota: Delftia sp.. Samples are categorized by collection method and subject. Source
A bit of background on why microbiologists find the last point so intriguing: with prolonged passages of a bacterial species in shake flasks under constant growth conditions − such as in Richard Lenski's LTEE − mutants always prevail in the population that have increased fitness compared to the parent strain. So, why are unique sets of C. acnes strains in individuals so robust to change?
Enter Conwill et al. (2022) from Tami Lieberman's lab at MIT-CEE, Cambridge MA, USA. They collected C. acnes from sebaceous skin regions from 16 healthy adults (=subjects). Sampling was done with toothpicks and, for fine spatial resolution, as pore extracts and with pore strips (Figure 1A). To understand how these samples were related to each other, they performed whole genome sequencing on 947 colonies (1−15 per sample), each of which represents the genetic content of a single cell that originated on the skin of one of their subjects. This approach led them to examine C. acnes biogeography with spatial resolution down to a single pore (sebaceous follicle) (Figure 1B). And they could thus capture interstrain and intrastrain diversity at a single-nucleotide variant (SNV) level. They assigned each isolate a strain type, and, by using genetic distance-based clustering, they grouped the isolates into lineages defined by <100 mutation distance.
Figure 3. C. acnes lineages from distinct colonization sources coexist on individuals. © The phylogenetic relationship of the 53 C. acnes lineages detected across all subjects is shown and colored by C. acnes strain-type. All six distinct coexisting lineages found on Subject 1 are highlighted. (D) A zoom-in of strain-type C illustrates that lineages within a strain-type are separated by large genetic distances relative to intralineage diversity. The heights of triangles are proportional to the number of colonies in each lineage and their widths represent the extent of intralineage genetic divergence. Lineages are named by subject number and then indexed by size within each subject using lowercase letters. Source
Across all samples, C. acnes largely dominated within-pore bacterial populations (Figure 2). They disentangled the phylogenetic relationship of the 53 C. acnes lineages detected across all subjects and I show here, as example, the results for the six distinct coexisting lineages found on Subject 1 (Figure 3). In Figure 3D, a zoom-in of strain-type C illustrates that lineages within a strain-type are separated by large genetic distances relative to intralineage diversity.
This diversification of the C. acnes lineages found by Conwill et al. (2022), that is, their ongoing evolution in the sense of adaptation, is not exceptional but well described, for example, for the within‑host adaptation of Pseudomonas aeruginosa in patients with cystic fibrosis (see here). An aside, and a point not addressed by the researchers: this type of analysis may allow a more realistic estimate of mutation rates than the most commonly used method based on differences in 16S RNA sequences.
Interestingly, this coexistence was not explained by niche specificity, because the authors did not observe strain-type exclusivity to the skin surface or skin pores, nor did they observe strain-type preferences for body site (for example, forehead versus back). These findings implicate neutral, rather than selective, effects to explain C. acnes strain type localization on skin. They found that although individual subjects harbored multiple lineages, implying multiple colonization events, individual pores within subjects were each dominated by an individual lineage (Figure 4). Even within lineages, the authors found a lack of C. acnes intrapore diversity at an SNV level.
Figure 4. C. acnes lineages are spatially segregated into different pores despite coexistence on skin regions. A pore strip from Subject 3 (left cheek section) illustrates how pores housing different lineages can be in close vicinity to each other. Each black circle represents a single pore sample, and each interior symbol represents a colony from that follicle. Symbol colors indicate strain-type and symbol shapes indicate lineage membership. Source
Conwill et al. (2022) conclude from the results of their spatially-resolved sequencing that most likely C. acnes populations within each pore typically descend from a single ancestral genotype introduced stochastically. The special anatomy and physiology of the pores then leads to lineage coexistence on individuals (theoretical terminology: neutral bottlenecking). Sebum flow out of the pore, the environmental gradient along the length of the pore, and physical crowding may make it more difficult for a cell to colonize a pore before a resident bacterium proliferates (Figure 5A). Thus, bottlenecking at the level of individual pores reduces competition between lineages by spatially segregating populations with different genotypes. The skin surface allows for little growth of the aerotolerant C. acnes and thus limits, in addition, opportunities for competition even among strains/lineages that have different growth rates in lab experiments (Figure 5B).
Figure 5. Pore physiology may contribute to neutral bottlenecking and to lineage coexistence. (A) Sebum flow out of the pore, the environmental gradient along the length of the pore, and physical crowding may make it more difficult for a cell to colonize a pore before a resident bacterium proliferates. (B) Bottlenecking at the level of individual pores reduces competition between lineages by spatially segregating populations with different genotypes. The skin surface allows for little growth and thus limits opportunities for competition. Source
Learning from the study by Conwill et al. (2022) that various strains of one bacterial species can well coexist under identical ecological conditions if their spatial separation is possible at the micro-scale, I remembered "One Grain of Sand," a piece I wrote for STC years ago. Probandt et al. (2018) went from studying bacterial diversity in marine sediment to their study on individual sand grains. Bacteria gather as clusters in indents and cavities of sub-millimeter-sized sand grains, while avoiding exposed convex areas. They found that "the 10 most sequence-abundant family-level clades were the same on all sand grains, and not grossly different from those in bulk sediment samples," and that "each sand grain investigated in this study was the habitat for around 105 cells representing several thousand species." They did not go so far as to identify individual species and strains in their study, but it is reasonable to suspect that the amazing diversity of closely related organisms and their coexistence under identical ecological conditions is due to, well, "grain hopping."
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