Understanding the role of Propionibacterium acnes in acne vulgaris: the
critical importance of skin sampling methodologies

Hélène Omer PhD, Andrew McDowell PhD, Oleg A. Alexeyev MD,

PhD

PII: S0738-081X(16)30264-4
DOI: doi: 10.1016/j.clindermatol.2016.10.003
Reference: CID 7101

To appear in: Clinics in Dermatology

Please cite this article as: Omer Hélène, McDowell Andrew, Alexeyev Oleg A.,
Understanding the role of Propionibacterium acnes in acne vulgaris: the critical
importance of skin sampling methodologies, Clinics in Dermatology (2016), doi:
10.1016/j.clindermatol.2016.10.003

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Title:

Understanding the role of Propionibacterium acnes in acne vulgaris: the critical importance of

skin sampling methodologies

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Authors:

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Hélène Omer PhD1, Andrew McDowell PhD2, Oleg A. Alexeyev MD, PhD1

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Affiliations:
1
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Department of Medical Biosciences/Pathology, Umeå University, Umeå, Sweden.
2
Northern Ireland Centre for Stratified Medicine, School of Biomedical Sciences, University of
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Ulster, Londonderry, UK
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Correspondence
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Oleg A. Alexeyev Department of Pathology, Umeå University Hospital, S-90185 Umeå, Sweden,

Tel.: +46-90-7852841, Fax: +46-90-139853, e-mail: oleg.alexeyev@umu.se

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Conflicts of interest

None

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Abstract

Acne vulgaris is a chronic inflammatory skin condition, classified by the Global Burden of

Disease Study as the eighth most prevalent disease worldwide. The pathophysiology of the

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condition has been extensively studied, with an increase in sebum production, abnormal

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keratinization of the pilosebaceous follicle, and an inflammatory immune response all implicated

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in its etiology. One of the most disputed points, however, is the role of the Gram-positive

anaerobic bacterium Propionibacterium acnes in the development of acne, particularly when this

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organism is also found in normal sebaceous follicles of healthy skin. Against this background, we
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now describe the different sampling strategies that have been adopted for qualitative and

quantitative study of P. acnes within intact hair follicles of the skin, and discuss the strengths and
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weaknesses of such methodologies for investigating the role of P. acnes in the development of

acne.
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Key words: Skin, acne, Propionibacterium acnes, inflammation, gel biopsy, skin biopsy,

phylogroups
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Introduction

Acne vulgaris is a chronic, inflammatory skin disorder of the pilosebaceous follicles 1. It is

estimated that virtually all adolescents will be affected by acne at some point in their life, with

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15-20% suffering moderate-to-severe forms of the condition 2. The disease can also persist into

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adulthood, affecting 20-40% of all individuals 3. Strikingly, The Global Burden of Disease Study

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has estimated acne to affect approximately 10% of the global population, ranking it as the eighth

most prevalent disease worldwide, and the third most prevalent dermatological condition 4.

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The earliest subclinical ‘lesion’ in acne, known as the microcomedone, can evolve into open and

closed comedones and into different inflammatory lesions, such as papules, pustules, nodules,
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and cysts 5. Historically, comedogenesis has been linked to a combination of increased sebum

production and hyperproliferation of keratinocyte epithelium prior to the development of

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inflammation; however, more recent studies are now providing evidence that inflammation may
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actually play a role in all stages of acne lesion progression, including subclinically before the

formation of the comedo 6.

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The Gram-positive, anaerobic skin commensal Propionibacterium acnes has long been suspected

to play a key role in the pathophysiology of acne because antimicrobials that reduce skin surface

P. acnes result in clinical improvement, and acne, which is recalcitrant to antibiotic treatment, is

associated with the presence of antibiotic resistant strains 7. Despite these observations, the exact

role played by P. acnes in the development of acne has been consistently questioned based on a

number of important findings. These include the anti-inflammatory properties of antimicrobials,

which may explain, at least in part, their effectiveness in acne treatment, combined with the
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observations that counts of P. acnes on acneic and healthy skin are very similar, levels of P.

acnes within lesions do not correlate with the degree of inflammation, and some inflamed lesions

show no evidence of bacterial colonization 8.

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Kligman 5 estimated the number of pilosebaceous follicles on the human face to total

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approximately 5,000. This number may actually be significantly under-estimated, because the

number of hair follicles on the face could total at least 292 follicles/cm2 7, 9, which for a skin

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surface estimated to be 675 cm2 in area 10 would result in a total of 200,000 follicles. In general,
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acne is considered to be severe when the patient has more than 50 comedones and 30

inflammatory lesions on the face 11. This would mean that < 0.04 % of the follicles on the face
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are actually involved in the disease. If hyperkeratinization and increased sebum production were

indeed factors triggering acne then one would expect many more follicles to be affected.
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In this review, we critically appraise the different methodologies which have been used to study

the association of P. acnes with acne vulgaris, and describe the current ‘state of play’ based on
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the results obtained with some of these various sampling approaches. We also consider the

importance and relevance of biofilm formation and inflammatory processes in the context of

acne.

P. acnes population structure and key genomic and biochemical properties potentially

related to acne vulgaris development

Before we describe the current methodologies used for sampling and detection of P. acnes in

healthy and acneic skin, we will first provide a brief overview of the phylogenetics and
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population structure of the bacterium, along with relevant genomic and biochemical properties

that will be important for our discussion of later sections.

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P. acnes is an opportunistic pathogen and a member of the ‘cutaneous’ group of human

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propionibacteria along with P. avidum, P. granulosum and P. humerusii, although it can also be

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isolated from the oral cavity and the genitourinary and gastrointestinal tracts. The original

seminal work on the population structure of P. acnes by Johnson and Cummins 12 revealed two

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distinct serotypes of P. acnes, designated types I and II, which differed in the sugar composition
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of their cell walls, as well as bacteriophage susceptibility 13. More recently, our understanding of

the genetic population structure of this bacterium has increased tremendously based on single,
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multilocus and whole genome sequence (WGS) analyses 14-19. These studies have demonstrated

that types I and II represent phylogenetically distinct divisions or clades, and have further
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identified a new and distinct genetic group, known as type III, which has the capacity to adopt a
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long filamentous cell morphology which is not observed with types I and II 14, 15.
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Strains within this type III clade have recently been proposed as Propionibacterium acnes

subspecies elongatum subsp. nov., with strains of type I and II classified as Propionibacterium

acnes subspecies acnes subsp. nov 20. Further subdivisions within the type I clade, designated

types IA1, IA2, IB, IC, have now also been described based on the higher phylogenetic resolution

obtained with multiple gene analysis 17, 21-23. These latter groups also display differences in

biochemical phenotype, their association with disease, production of putative virulence

determinants and resistance to anti-acne antibiotics 21, 22, 24.

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Overall, P. acnes is in linkage disequilibrium and has a clonal, epidemic population structure

characterized by a number of highly successful and globally disseminated lineages 16, 17, 22.

Multilocus sequence typing (MLST) and ribotyping schemes developed for the bacterium have

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shown that within the large type I clade, strains from the type IA1 phylogroup cluster into three

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major clonal complexes (CC) (CC1, CC3, CC4; MLST8 scheme), while type IA2, IB and IC

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phylogroups are tighter phylogenetic clusters currently comprised of only one CC each 17, 22. The

large type II clade also clusters into three distinct CCs (CC6, CC71, CC72), while strains from

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the type III division comprise a single CC (CC77) 17, 22 (Figure 1). In addition to these CCs, a
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number of singleton sequence types (STs) have also been described (Figure 1). The availability of

over 100 complete and draft whole genome sequences for P. acnes has also facilitated detailed
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comparative genomic studies, which have been well described in the literature 18, 25-27; these

analyses have found an average genome size and gene content of 2.5 Mb and 2,500, respectively,
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for the bacterium.

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While overall genome plasticity is low, differences between the phylogroups due to the presence
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of island-like genomic regions with aberrant G+C content and flanking insertion sequences have

been discovered 18, 25, 26, 28. This ‘flexible gene pool’ encodes putative virulence factors and traits

which may be associated with fitness and niche adaptation, including antimicrobial defense,

adhesion and metabolic functions 28. One of the key observations from these studies has been the

discovery in type II strains of clustered regularly interspaced short palindromic repeats

(CRISPR)/Cas loci that confer immunity to bacteriophage and foreign mobile genetic elements 18,
26, 29
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In contrast, type I and also type III strains contain CRISPR/cas gene remnants indicating deletion

of the locus during their evolutionary history. As a consequence, these latter clades may be much

more susceptible to horizontal gene transfer and the acquisition of fitness or virulence traits that

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are important in the context of human disease, especially acne. For example, strains from type

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IA1 CC3 (MLST8), contain a novel plasmid with a tight adhesion (Tad) locus and two unique

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genomic islands, designated loci 1 and 2, that contain genes which are proposed to enhance

virulence via increased bacterial adhesion and host immune response 18, 26, 30. This may be

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important in the association of these strains with acne as discussed later.
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Transcriptomic, proteomic, and biochemical studies have also revealed important host-interacting
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properties common to different strains of P. acnes which may be important in the context of

human disease, including the production of proteins with degrading activities, such as proteases,
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glycoside hydrolases and esterases 15, 16, 25, 31, 32. Differences between phylogroups in
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inflammatory potential and the expression of various putative virulence factors, some of which

may be important in the emerging association of type IA1 strains with acne, have, however, also
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been described. These include haemolysins, neuraminidase, lipase, polyunsaturated fatty acid

isomerase, the iron acquisition protein HtaA, Christie-Atkins-Munch-Peterson (CAMP) factors,

and immunogenic phase and antigenically variable dermatan sulphate-binding proteins (DsA1,

PPA2127; DsA2, PPA2210) 15-17, 21, 23, 25, 31-33. These latter proteins have a variable number of C

residues in a CnTCn motif which is upstream of a putative signal peptide, along with variable

numbers of repeats towards the mid-region and the carboxy-terminus that alter molecular mass

and protein expression 23, 33. This variation is most likely generated by slipped strand mispairing,

leading to phenotypic variation and possible evasion of the immune system as well as
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dissemination via alteration of adhesin specificity. Recently, type IA1 strains isolated from

patients with acne (CC3; MLST8) were also found to produce significantly higher levels of

porphyrins, proinflammatory metabolites important in acne development, which were further

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enhanced by vitamin B12 supplementation 24. In contrast, strains believed to be associated with

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skin health produced low levels and did not respond to vitamin B12.

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Establishing the role of P. acnes in acne vulgaris

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Despite over 100 years of history, the role of P. acnes in acne vulgaris is still controversial.
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Numerous reviews have been published discussing arguments for and against the role of the

bacterium in the condition 6, 34-39. In order to critically evaluate a plethora of studies using
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different sampling and detection techniques, we need to define the extent to which Koch

postulates can be applied to support or to refute an etiological link between P. acnes and acne
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vulgaris. The very first postulate stating that the offending organism must be found in diseased
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but not in healthy individuals would formally reject a role for P. acnes in the condition since it is

present in persons without acne and in unaffected hair follicles from acne patients 40-42. As the
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organism is an opportunistic pathogen that forms part of the indigenous skin microbiota, its

presence in non-diseased skin would, however, be expected; a causative microbial component

that completely satisfies Koch’s postulates has rarely been identified in skin diseases 43. As a

result, other quantitative or qualitative differences must be demonstrated between acne-affected

and intact hair follicles if we are to establish a clear link to the disease. We therefore suggest a

modification of Koch’s postulates, and propose that a key finding should be significantly higher

bacterial counts of viable P. acnes within individual acne lesions compared to unaffected hair

follicles. To meet this requirement, the content of an entire hair follicle must be sampled and
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contamination from nearby anatomical structures (skin surface, stratum corneum) avoided. The

choice of a control hair follicle is vital as age, gender, diet, usage of cosmetics, localization of

skin biopsy, hair follicle functional status and a magnitude of other known and unknown factors

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can influence the abundance of P. acnes in the skin at any given time. An anatomically intact hair

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follicle derived from the same patient and the same area as the acne lesion is required in order to

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avoid sampling bias. Furthermore, both the acne lesion and control skin must be sampled at the

same point in time. In addition, our improved understanding of P. acnes phylogeny and the

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identification of distinct genetic divisions and lineages with apparent heightened capacity to
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cause infection also raises the possibility that differences in the nature of the phylogroups and

STs isolated from individual acneic lesions versus normal follicles may also be an important,
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although not conclusive, observation in the association of P. acnes with the etiology of acne

development 16, 17, 22.

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Contamination from other non-follicular skin structures is also a major concern in studies

investigating the role of P. acnes in acne, especially when applying sensitive nucleic acid assays.
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A recent study showed that P. acnes DNA can be detected in all next generation sequencing

datasets generated from multiple samples of different origin, including non-template controls 44.

This highlights the importance of careful interpretation of data generated by molecular methods,

and further experiments to validate any association identified.

Overview of methodologies to sample P. acnes in the skin

The skin is a diverse environment, containing different habitats such as invaginations,

appendages, glands and follicles 43. As bacteria may inhabit many different skin appendices,
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different methodologies have been developed to sample these various areas. Within a clinical

setting, swab, scrub ,and gel biopsy are used to sample the upper skin levels, while a skin biopsy

is required in order to sample hair follicles and subcutaneous fat tissue (Table 1) 45. Because acne

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vulgaris is a disease of the hair follicle, it is imperative that sampling strategies target the whole

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follicular P. acnes community. Some studies have utilized multiple sampling methodologies

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with their cohorts, making meaningful comparison of the data across healthy skin and lesion

types very difficult 46. In the following section, we will discuss the advantages and disadvantages

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of current sampling methods in their capacity to target the follicular population.
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Swab, scrub and tape stripping techniques
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The swab technique refers to surface sampling using a sterile tissue to brush the skin. The method

is non-invasive, cheap and can be applied to the sampling of many patients in a short period of
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time. Its limitation, however, is the difficulty of standardization since variation in the user-
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dependent force applied during sampling may affect microbe retrieval 47. As a result, it is difficult

to compare quantitative data between and within studies, which is further compounded by
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variation in the structure, chemical composition and toxicity of swab tissues which may also

affect bacterial retrieval 48. The method only samples skin surface bacteria, which is appropriate

when screening for antibiotic resistant strains, but tells us little regarding the microbiology of

individual follicles.

Williamson and Kligman 40 developed a technique known as the “surface scrub technique” or

“detergent scrub technique” that also allows isolation of skin surface microbiota. This technique

uses a blade to gently scrape the skin surface but, similar to swab sampling, considerable
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variations in quantification are observed. To minimize this variation, triplicate samples at a given

site on a given day are recommended 40. While comparing the swab and scrub method, Keyworth
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concluded that they gave similar concentrations of the bacterial populations sampled.

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Another way to collect skin surface bacteria is the direct application of a contact plate, velvet pad

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or stripping tape to the skin. The plate and pad only target surface microbiota and, therefore, are

generally used for aerobic bacteria. The tape application was developed to study the different

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layers of human epidermis 50 and was later adapted by Updegraff 51 to quantify the bacterial
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population of up to 14 stratum corneum layers of the skin. The bacteriostatic activity of different

tapes against some bacteria is, however, an issue 51. Tape stripping can also remove different
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quantities of material again making it difficult for the method to be standardized.

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Overall, these techniques should be considered unsuitable for quantitative studies examining the
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relationship between P. acnes and acne vulgaris, because they target surface and stratum corneum

bacterial populations, provide very little material from inside the hair follicle, and are difficult to
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standardize.

Gel biopsy

In order to provide insight into follicular microbial populations, Holland and Roberts in 1974 52

developed a sampling technique based on cyanoacrylate gel (Permabond). In this approach, a

drop of cyanoacrylate gel is deposited on the skin, enabling varying amounts of stratum corneum

and follicular infundibulum content to be sampled 53. Cyanoacrylate gel polymerizes quickly, and

will immediately form a very strong adhesive whenever exposed to water and amino acid content
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present in the skin 54; the kinetics of gel polymerization upon contact with sebum/lipids is not

known. The extent of penetration into the hair follicle will likely be confined to the superficial

region, and again this method is highly variable due to skin humidity, pH and other unaccounted

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environmental factors.

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Gel sampling will yield numerous follicular casts, which makes it extremely difficult to link an

individual cast to an acne-affected or intact but dilated hair follicle; acneic follicles are

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intermixed with healthy follicles which are numerically predominant, even in severe cases of
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disease. Another limitation is that varying amounts of intrafollicular content will be extracted

from individual hair follicles, while unknown and unpredictable amounts of material will be left
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in the skin. While the gel biopsy method remains an attractive approach to make an approximate

quantitative analysis of hair follicle infundibulum, it is unsuitable for analysis of the entire hair
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follicle.
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Microcomedone / Comedone extraction methods

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The contents of an individual inflamed follicle (or lesion) can be extracted by using a special

device such as stylet, lancet or comedone extractor. In an elegant experiment, Plewig 55 showed

that by taking a biopsy directly after extraction, the hair follicles still retained abundant horny

materials. While the technique can be attractive for larger skin lesions it may be problematic for

smaller ones and is not suitable for retrieving the contents of healthy hair follicles needed as

controls.

The same applies for the vacuum extractor technique largely advertised by dermatologic

companies. For example, the presence of P. acnes in different parts of the skin with and without
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acne has been investigated using a constant negative pressure to aspirate the contents of

sebaceous follicles, followed by DNA extraction and quantitative PCR (qPCR) for numeration of

P. acnes populations.56 Another major limitation of the vacuum extraction method is that the

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number of sebaceous follicles sampled is unknown, as well as the quantity of material extracted.

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A more recent approach to sample the contents within pilosebaceous units, including the

anaerobic portion, is the utilization of commercially available pore strips (eg., Bioré®) 18.

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Individual follicular contents can then be plucked of the resulting strip using microforceps,
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pooled and processed for further analyses. With this approach, minimal sampling of the skin

surface or human cells within the follicular lining appears to occur as judged by metagenomic
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shotgun sequencing and determination of human sequence reads 57. While the method can be

used for small (microcomedones) and healthy follicles, it is limited in its ability to separate
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lesional from non-lesional comedones, but it has the advantage of reaching deeper into the
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sebaceous follicle.
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Biopsy

Skin biopsy represents the gold standard approach, enabling the whole hair follicle to be

retrieved. This approach is, however, invasive and therefore requires approval from ethical

regulatory bodies. Isolation of an intact hair follicle from the surrounding tissue is also

problematic. A 1 M CaCl2 solution has been used to isolate pilosebaceous units from skin

biopsies, a technique first described by Kellum 58. 59. Briefly, a punch biopsy is excised from

the skin and soaked in cold calcium chloride to facilitate separation of the epidermis and follicles

from the dermis; each follicle is then excised using surgical microscissors. The cumbersome

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nature of the procedure, exposure of biological material to chemicals, and unavoidable risks of

cross-contamination of skin structures during manipulation makes it a poor method for

quantitative analysis.

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As an alternative, the biopsy can be subjected to direct visualization by various staining assays

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such as immuno-fluorescence microscopy (IFM) 42, 60 and fluorescence in-situ hybridization

(FISH) 61-63. With this approach, however, a conventional tissue section which is between 4-10

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µm thick provides only a limited insight or glimpse into the hair follicle content. As we have
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shown that P. acnes biofilms can be as large as 2 mm, as many as 400 skin sections may need to

be evaluated in order to visualize the whole hair follicle 64.

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Overview of methodologies to detect P. acnes sampled from the skin

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Three main methods used to detect microorganisms in skin samples are culture, analysis of
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nucleic acid content, and direct visualization by IFM / FISH. These methods detect the bacteria in

different physiologic states and this information needs to be considered since such bacteria may
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have different pathogenic potentials (Table 2) 65. The physiologic states of bacteria in natural

environments include (i) live bacteria that are viable and metabolically active, (ii) dormant

bacteria that are alive but metabolically inactive and (iii) dead bacteria that are non-viable,

usually due to membrane defects.

The direct culture of a sample will only reveal viable bacteria at a snapshot in time, and will be

biased towards those organisms that flourish under the nutritional milieu presented to them 43.

Restriction of typing schemes to only a small number of colonies may also underrepresent
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specific lineages that may be present in a mixed type sample in low numbers; this may be

especially relevant to P. acnes 19. Nucleic acid detection is usually done on samples by PCR or

qPCR using highly conserved regions of the 16S rDNA gene ubiquitously present in bacteria.

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The availability of low cost methods for WGS analysis also makes this an attractive alternative,

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although both approaches will amplify viable and non-viable bacterial DNA, as well as

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extracellular DNA which is abundant in many natural environments 66.

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Microscopic visualization also enables the direct quantitative analysis of bacteria in the skin, and
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is an extremely attractive approach for the analysis of P. acnes and visualization of probable

biofilm formation. Furthermore, as bacteria in the skin can appear in biofilms, pretreatment of
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samples by sonication may also be important, especially to maximize cell numbers for

quantitative analyses 67.

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P. acnes analysis based on swab and scrub sampling

The swab and scrub techniques have been used to define the pattern of Propionibacterium
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colonization on normal human skin and acneic skin with similar results. Propionibacteria are

ubiquitous on the human skin, and P. acnes is dominant in oily regions with high sebum

concentration, including the face and the upper trunk. Definitive quantitative differences linking

P. acnes levels on the skin to acne have not, however, been found based on the culture or

sequencing of organisms sampled using these techniques 68-70, although age-dependent

differences in P. acnes levels have been described 71. P. acnes appears more prevalent in acne

patients during the time when the disease begins (age 11-20), whereas after the age of 20 years,

the levels of P. acnes in healthy subjects is comparable to patients with acne.

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A limited number of early studies utilizing low resolution biotyping of P. acnes isolated from

healthy and acneic skin via the swab sampling technique have been described; 46, 72, 73.however,

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much more recent high resolution phylogenetic and population genetic analysis of P. acnes

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isolated from healthy and acneic skin by swab and scrub sampling have now been reported,

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although in some cases this was also combined with gel biopsy methods (which we discuss later)

making interpretation of the results slightly more complicated 16, 17, 19, 20, 22, 23, 74. These latter

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independent studies, which have been based on subjects in geographically widespread regions,
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have consistently demonstrated that acneic skin of patients with moderate-to-severe acne appears

associated with, or enriched for, particular strains from the type IA1 phylogroup, while strains
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from the type IA2, IB, IC and II divisions are infrequently recovered, and those from the type III

phylogroup never recovered. Other strains have also been identified that appear associated with
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skin health 16.

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In particular, patients with acne appear to be enriched for epidemic clones and other STs from the
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type IA1 clonal complexes CC1, CC3 and CC4 (MLST8 scheme). Nevertheless, while these

recent studies clearly indicate significant differences in the phylogroup composition of the skin

from acne sufferers and healthy individuals, the swab and scrub sampling methods do not directly

sample follicular contents, but measure the content or output of all follicles, both healthy and

acneic, on the skin surface. It therefore remains to be determined exactly what role P. acnes

strains from these different CCs may play in the pathophysiology of acne.

P. acnes analysis based on gel biopsy

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P. acnes distribution has also been studied using the gel biopsy method 52, 75 followed by bacterial

culture. Holland and Roberts 52 described their Permabond-based method as more effective than

the scrub technique of Williamson and Kligman, 40 because the procedure can be applied several

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times to the same region of the skin, enabling part of the hair follicle microbiota and varying

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amounts of the stratum corneum populations to be sampled. In order to investigate the early

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follicular changes in acne, the structural organization and colonization of follicular casts of

prepuberal children has been studied 76. This work demonstrated by microscopy that even though

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the follicular casts were present in all samples and had the same anatomy as those present in acne
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patients, they were not as abundant and were also sterile as assessed by microscopy and culture.

This led to the conclusion that there was no evidence for any bacterial role in the initiation of
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acne lesions. Additional studies 77 focused on the collection of cutaneous microbiota from various

areas of facial skin using cyanoacrylate gel, followed by the measurement of bacterial numbers
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after culture. There were significantly higher numbers of P. acnes in follicular casts from patients
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compared with their control volunteers for female facial skin and male back skin, and that

approximately 90% of normal follicles had no detectable viable microorganisms versus only 10%
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in acne follicles. No differences in the skin microbiology of adolescent acne patients, persistent

acne patients or late-onset acne patients were, however, observed.

A later study 78 used a molecular biology-based approach to investigate the microbial diversity of

follicles from acne patients and healthy individuals sampled by cyanoacrylate gel biopsies. In this

study, pooled follicular casts were microdissected before DNA sequence analysis of cloned 16s

rRNA genes. Interestingly, and in contrast to the data previously generated 77, the researchers

observed that follicles from healthy skin were exclusively colonized by P. acnes, and that the
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follicular microbiota of acne patients included, in addition to P. acnes, Staphylococcus

epidermidis and minor proportions of other species. As this was a molecular based project, no

information on the physiological state of the bacteria was available, which is a major limitation of

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the study.

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A more recent proteomic investigation examined the protein content of follicular casts extracted

from healthy and acne-affected individuals using cyanoacrylate-gel biopsies processed for mass

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spectrometry analysis 79. Bacterial proteins were identified in both normal and acne affected skin,
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and were exclusively from P. acnes; the most prevalent P. acnes proteins identified were surface-

exposed dermatan sulphate adhesins (DsA1; DsA2), CAMP factors, and an uncharacterized
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lipase. Since DsA1 and DsA2 were abundantly found in the follicular cast proteome, this

provides clear evidence that healthy as well as diseased individuals are colonized with P. acnes
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strains from the type IA phylogroup, although other types may also be present. In addition to
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bacterial proteins, normal follicular casts were enriched in proteins (prohibitins and

peroxiredoxins) which are involved in protection from various stresses, including reactive oxygen
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species. By contrast, follicular casts extracted from acne-affected skin contained proteins

involved in inflammation, wound healing and tissue remodeling, including myeloperoxidase,

lactotransferrin, and neutrophil elastase inhibitor. This indicates that the biological processes

taking place within acneic lesions reflect a ‘host response to a bacterium’. In addition to the

problems previously described utilizing a gel-based sampling methodology, this study did

highlight other potential limitations including variation due to differences in sampling sites,

which was the nose for healthy controls and facial and back skin areas for patients with acne.

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While some of these studies provide tantalizing hints of a role for P. acnes in the pathological

processes leading to acne, the variability in results, especially in relation to P. acnes colonization

of follicles in healthy subjects, restrict our ability to make solid conclusions; such differences

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may reflect the nature of cohorts studied along with other inherent study variabilities arising from

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gel biopsy sampling.

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P. acnes analysis based on microcomedone/ comedone extraction methods

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To determine the microbiota specific to acne, methods based on follicle extraction have been
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used as described earlier. Some of these techniques, however, make it difficult to extract control

follicles, but they do give insight into the follicular microbiota of acneic lesions. Using such
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approaches, a number of studies have found that the organisms present in inflamed and non-

inflamed acne lesions are similar to that of normal microbiota 58, 70, 80-84. In most of these studies,
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a varying number of sterile lesions have been found supporting the hypothesis that the presence
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of P. acnes in a follicle is not necessary to trigger comedogenesis 8, 85-87, but the lack of a

matched control population makes these comparisons between “lesional” and “normal” bacterial
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populations difficult to interpret.

The contents of sebaceous follicles have also been sampled by aspiration using a constant

negative pressure, with subsequent quantification of the P. acnes population by qPCR56. This

sampling approach demonstrated that numbers of P. acnes genome-equivalents were extremely

low in control subjects less than 10 years of age, but higher in older subjects. This investigation

also observed that P. acnes counts on the forehead and nose were higher in acne patients aged 10-

14 years than in the age-matched controls in both sexes, and that the proportion of P. acnes
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present on one subject is highly individual and variable between subjects, even in the same age

range. Overall, their results suggested that hair follicles containing many P. acnes cells are not

particularly prone to acne, except in younger teenagers. While this method is sensitive it doesn’t

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discriminate between acne and non-acne follicles and takes into account all DNA present,

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including non-viable bacteria.

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A more recent study.examined the P. acnes strain populations on the skin of patients with acne

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and healthy subjects18. The contents of pilosebaceous follicles on the nose of both cohorts,
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irrespective of the presence or absence of acneic lesions, were extracted using commercially

available pore strips, followed by metagenomic analysis. While the relative abundances of P.
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acnes were found to be similar between the two cohorts, the strain population structures were

significantly different. In particular, using a novel ribotyping scheme, a sub-population of type

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IA1 strains representing ribotypes (RT) 4 and 5 appeared highly enriched on the skin of patients
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with acne, while other lineages (RT7, RT8, RT9, RT10) were also associated with the disease and

a subpopulation of type II strains (RT6) was also strongly associated with the skin of healthy
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subjects.

Interestingly, although this study utilised a metagenomics approach, thereby identifying all strain

populations, the results were in part agreement with data from previous culture-based MLST

studies, which similarly found that strains corresponding to RT4/5 (CC3; MLST8) and RT8

(CC4; MLST8) dominated isolates from acneic skin 16, 17, 22. However, strains from CC1

(MLST8), also found to be strongly associated with moderate-to-severe acne based on previous

MLST studies 16, 17, 22, were not found to be enriched within the skin of acne patients versus
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controls using this metagenomic approach 18. This study not only differed from previous

investigations in the nature of the detection methodology utilised, which would measure non-

viable DNA, but also in the fact that it attempted to look for differences in the nature of the

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underlying follicular microbiome of pre-lesional samples at the same anatomical site of subjects,

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rather than focus on regions where diseased, lesional skin was present.

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P. acnes analysis based on biopsies

As the sebaceous follicle population is heterogeneous, methods isolating each single follicle by
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microdissection of skin biopsies are a particularly potent means to investigate the correlation

between the presence of bacteria and inflammation. Such studies have emphasized the high
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variability of the follicular content between and within different individuals, as well as the large
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proportion of sterile lesions, which has led to the conclusion that the presence of microorganisms

is not essential for the initiation of comedogenesis and inflammation in acne 58, 85, 88. Using an
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IFM-based detection method on skin biopsies, Imamura et al. 60 found P. acnes in the sebaceous
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follicles of the face in normal subjects and in the uninvolved skin of acne subjects, but not the

normal skin on the back of healthy subjects and the uninvolved skin on the back of acne patients.

Evidence for P. acnes in 7/11 comedones and in all 30 inflammatory papules, pustules, and

nodules investigated was also found. Most of the fluorescence in the inflammatory lesion was

present within the confines of the follicle, with small amounts of bacterial antigen present in the

dermal infiltrate and within macrophages.

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Using a similar approach, a more recent IFM study by Jahns et al. 42 found a statistically

significant higher prevalence of P. acnes in follicles from acne patients compared to controls

(47% vs 21%). Furthermore, these studies suggest that P. acnes may be present in the follicles in

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the form of macrocolonies / biofilms (Figure 2).

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P. acnes biofilm in the follicle

Most bacteria exist as biofilms in their natural habitat. A biofilm is defined as a bacterial

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community embedded in an extracellular matrix attached to a surface; both mono- and poly-

species biofilms have been described

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89, 90
. Biofilms are usually more resistant to antibiotics than
91-93 94
planktonic cells , at least in part due to the presence of persister cells . They are associated
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with a variety of infections and diseases, including chronic wounds, miliaria, atopic dermatitis,

onychomycosis, impetigo and furuncles, and are often challenging to treat 95-97.
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98
Biofilm formation by P. acnes was first described in relation to prosthetic hip infections and
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various orthopedic biomaterials 99. The sequence of the P. acnes genome 100 has revealed clusters
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of genes involved in the polysaccharide biosynthesis of a glycocalyx polymer, which can be used
101 102-
for biofilm matrix formation . Several studies have investigated P. acnes biofilms in vitro
104
and, as expected, P. acnes growing within a biofilm is more resistant to antibiotics than its

planktonic counterparts; the biofilms can even develop in the presence of some antibiotics 105 and

biofilm dispersal and removal by antibiotics does not necessarily correlate with the death of the
106
bacteria . Furthermore an increase in the lipase activity and auto-inducer 2 (AI-2) production

was detected in the cells grown as a biofilm 106.

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P. acnes biofilms have been directly observed in acne lesions more frequently than in control

sebaceous follicles (37% vs 13%) 42. The biofilms are present in the stratum corneum as well as

matrix-encased macro-colonies in the hair follicles (Figure 2). Most interestingly, these large

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macro-colonies appear to consist of different P. acnes phylogroups existing within the same

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follicle 42, which is intriguing given the association of type IA1 strains with acne. To further

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characterize P. acnes biofilms in the skin, a 3-D model has been reconstructed by sequential

cutting of the entire skin biopsy 63, 64. The biofilms are found attached to the follicle wall, the hair

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shaft, spreading over the lumen, and/or in the centre of the hair follicles without any evident
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attachment. They may start from the stratum corneum and go as deep as 2 mm under the skin

surface. Such observations therefore highlight the need for correct sampling methods when
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studying the presence or absence of P. acnes in skin samples.

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P. acnes biofilms have also been observed in other skin diseases such as folliculitis 107, folliculitis
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decalvans 108 and hidradenitis suppurativa (acne inversa) 109. Interestingly, in the above

conditions the biofilms were seen in terminal hair follicles.

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Acne vulgaris and inflammation

P. acnes can activate a variety of immune pathways and induce the production of interleukin

(IL)-1α, tumor necrosis factor (TNF)-α and granulocyte-macrophage colony-stimulating factor

(GM-CSF) 110 in keratinocytes 111, 112. Pro-inflammatory cytokines have also been directly

detected in acne lesions including IL-1α in open comedones and IL-1 expression in early
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inflamed lesions 111, 113. Other inflammatory components, such as CD4+ T-cells, macrophages,

cytokines and integrins, have also been observed in the perifollicular area of uninvolved skin

from acne patients. Recent in vitro studies have revealed that P. acnes can induce IL-17

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production by PBMC 114 and T-cells (Th1/Th17) 115, and that IL-17-expressing cells are present

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in acne biopsies but not detectable in healthy controls 114. P. acnes can activate monocytes via

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TLR2 or TLR4, and these receptors are co-localized with CD14 in acne lesions but not in

controls 116.

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The activation of TLR2 induces the production of pro-inflammatory cytokines including IL-1,

TNF-α, prostaglandins, leukotrienes and chemokines (IL-8). An increased expression of TLR2

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and TLR4 was detected in the epidermis of inflammatory acne lesions, and this was more intense

with TLR2 than with TLR4, as observed in normal skin; the expression level increased with the
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degree of differentiation of the keratinocytes 117. P. acnes can also trigger the activation of the
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NLRP3-inflammasome pathway in antigen-presenting cells 118 and myeloid cells via IL-1β 119.

Expression of the G-coupled protein receptor PAR-2 was also shown to be increased in
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keratinocytes from acne 120, and this was correlated with a production of IL-1α, β-defensin 2,

antimicrobial peptide LL-37 and different matrix metalloproteinases.

Studies of the evolution of acne lesions in vivo have shown that the initial infiltrate consisted of

mononuclear cells, predominantly CD4+ T cells 121. Neutrophils could be seen appearing in some

of the later lesions (72h), and were associated with disruption of the duct. The study of gene

expression by array profiling on skin biopsies from unaffected control, normal skin of acne

patients, and from inflammatory papules of the same patients showed 211 genes upregulated in
122
acne lesions when compared to controls . Most of these genes were involved in inflammation
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and matrix remodeling, including matrix metalloproteinases, IL-8, β-defensin 4, and granulysin.
113
Jeremy et al. showed the presence of elevated numbers of macrophages and T-cells in

perifollicular and papillary dermis of uninvolved acne skin compared to control skin in the

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absence of keratinocytes hyperproliferation.

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All these findings suggest that inflammation and not keratinocyte hyperproliferation is the prime

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event in acne pathogenesis, placing the role of P. acnes in the later stages of inflammation 6. The

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major limitation of these studies is, however, a lack of information on the presence or absence of

P. acnes in acne lesions. We previously demonstrated clusters of CD3+ cells in the vicinity of the
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P. acnes-positive comedones, but those cells were virtually absent from the surroundings of P.
63
acnes-positive inflamed lesions . On this basis, the co-localization of P. acnes and CD3+
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lymphocytes in early acne stage may actually suggest a role of P. acnes in the initiation of the
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inflammation.
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Conclusio

Over the years, a large number of studies have examined the relationship between P. acnes and
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acne vulgaris. Despite this, the role played by the bacterium in the condition still remains unclear,

especially as various investigations have found no difference in the counts of P. acnes on the skin

of patients with acne versus healthy subjects, alongside the detection of inflammation in lesions

apparently devoid of bacteria. While such studies have appeared to question the importance of P.

acnes in acne development, we would argue that in many cases they may not have been based on

the most appropriate sampling methods we described earlier, making the interpretation and

relevance of the results unclear. In the future, consideration of such methods when designing

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experiments aimed at understanding the role of P. acnes in acne development should be a critical

component of any study.

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Figure legends

Figure 1.

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eBURST population snapshot of P. acnes based on data from the P. acnes MLST8 database

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(http://pubmlst.org/pacnes/). A total of nine clonal complexes, where the isolates share 7/8 loci

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with at least one other ST in the group, and 21 singletons are illustrated. The frequency of each

ST within the isolate database is indicated by the size of each circle. Founding genotypes are

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highlighted in blue and sub-founders in yellow. Note, the spacing between the singletons and
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clonal complexes is not related to the genetic distance between them. Adapted from McDowell et

al. 22.
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Figure 2.
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P. acnes biofilm within a hair follicle (stained by IFM using specific monoclonal anti-P. acnes
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antibodies).
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Table 1.

Overview of various P. acnes skin populations targeted by different sampling methods

(adapted from Alexeyev et al. 45).

Table 2.

Physiological states of bacteria found on human skin (adapted from Alexeyev et al. 65).

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126:1071-1079.
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Figure 1.

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Figure 2.

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Table 1.

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P. acnes population Sampling method

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Swab Scrape Cyanoacrylate gel Biopsy

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Superficial stratum + + + -a
corneum

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Within stratum corneum - + + +
Infudibulum - -
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Lower hair follicle - - - +
a
Likely removed during pre-treatment with antiseptics.
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Table 2.

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Physiological state Phenotype

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Viable (high metabolic Can proliferate and form colonies on culture media
activity) (if available)

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Dormant (low metabolic Temporarily lost or markedly diminished capability
activity) to proliferate and form colonies on culture media

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Non-viable (dead) Permanently lost capability to proliferate and form
colonies on culture media
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