Propionibacterium acnes biofilm – A sanctuary for Staphylococcus aureus?

doi:10.1016/j.anaerobe.2016.05.014

Anaerobe

Volume 40, August 2016, Pages 63-67

https://doi.org/10.1016/j.anaerobe.2016.05.014 Get rights and content

Highlights

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Propionibacterium acnes enhances virulence factors of Staphylococcus aureus under anaerobic conditions.
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S. aureus grows well under anaerobic conditions.
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The inside of a human body is more consistent with an anaerobic environment.
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Aerobic organisms may behave differently when grown anaerobically.

Abstract

The purpose of this study was to measure the effect of combined culture of Propionibacterium acnes and Staphylococcus aureus on biofilm formation under different oxygen concentrations. We measured planktonic growth and biofilm formation of P. acnes and S. aureus alone and together under aerobic and anaerobic conditions. Both P. acnes and S. aureus grew under anaerobic conditions. When grown under anaerobic conditions, P. acnes with or without S. aureus formed a denser biomass biofilm than did S. aureus alone. Viable S. aureus was recovered from a16-day old combined P. acnes and S. aureus biofilm, but not a monomicrobial S. aureus biofilm.

Introduction

Propionibacterium acnes is an aerotolerant Gram-positive bacillus found commensally and asymptomatically in and on the skin of humans, especially in the sebaceous glands of oily skin on the face, chest, and back; P. acnes may contribute to the inflammatory component of comedonal acne [1], [2], [3]. P. acnes is frequently isolated from clinical specimens, and, although it is most commonly considered a contaminant, it is increasingly recognized as a primary pathogen [4], [5], especially in shoulder arthroplasty infections [6]. The pathogenesis of P. acnes infections is poorly understood, and such infections do not always manifest clinical characteristics of other bacterial infections, such as elevated white blood count, eythrocyte sedimentation rate or C-reactive protein.

An interesting clinical observation is that, when recovered from clinical specimens, P. acnes may be co-isolated with Staphylococcus species, including Staphylococcus aureus and Staphylococcus epidermidis [7], [8], [9], [10], [11], [12]. While this may be a simple matter of probability since these organisms are often found together as commensal flora, it may alternatively be that the combination of organisms is found together due to mutualism. When cultured with P. acnes, for example, S. aureus manifests a greater degree of β-hemolysis than when grown alone [13]. And, P. acnes produces extracellular coproporphyrin III, which induces aggregation and biofilm formation of S. aureus isolated from skin sources [14]. These observations suggest that there may be a relationship between P. acnes and S. aureus; given their co-isolation from prosthetic joints and cerebrospinal fluid shunts, this interaction may be particularly germane to biofilm-associated infections. Further, if P. acnes enhances the virulence of other pathogens, its presence in clinical specimens may have greater significance than previously appreciated.

A challenge in the culture-based diagnosis of P. acnes infection is that P. acnes grows slowly and preferentially under anaerobic conditions [15]. That anaerobic organisms, including P. acnes, can cause bone and joint infections, suggests that the environment at these infection sites may be anaerobic, as high oxygen tensions would not permit robust growth of anaerobes. Room air at sea level has an average FiO₂ of 21%. Oxygen levels in human cartilage have been estimated to range from 1 to 6% [16], and, oxygen tension has been noted to be lower in diseased compared to healthy joints [17], [18].

Culture-based diagnosis and study of aerobic pathogens such as S. aureus typically involves growth under ideal, usually aerobic, conditions. Although many of these organisms are able to grow anaerobically, their growth characteristics, including their ability to form biofilms, under anaerobic conditions, and how they interact with other bacteria under conditions of anaerobiosis, have not been well-defined. Even though it has been broadly observed that organisms interact with symbiotic relationships and complex signaling networks, this is best described for gut and environmental flora. Herein, we evaluated planktonic growth and biofilm formation of P. acnes and S. aureus alone and in combination under aerobic and anaerobic conditions.

Section snippets

Methods

P. acnes IDRL-7676 and S. aureus IDRL-4284, both of which are clinical isolates, were grown on sheep blood agar. S. aureus required 24 h of growth aerobically; P. acnes required 48 h of growth anaerobically. A 1 McFarland solution of each organism was prepared and diluted 1:50 with brain heart infusion (BHI) broth supplemented with 1% glucose. Each organism, or combination of organisms (detailed below), was prepared in quadruplicate in 96-well flat-bottom tissue culture plates. Experimental

Results

When grown planktonically under aerobic conditions, S. aureus demonstrated an expected growth pattern, and as expected, P. acnes showed little growth (Fig. 1). The mixture of the two followed the growth curve of S. aureus. When S. aureus was added to P. acnes, the growth curve mirrored that of S. aureus. Under aerobic conditions, biofilm formation recapitulated planktonic findings except that the OD₄₉₂ tended to fall after rising (Fig. 2).

When cultured under anaerobic conditions, both organisms

Discussion

Biofilm formation plays a role in device-associated infections, such as prosthetic joint infection, making these infections challenging to cure. P. acnes is a biofilm-forming organism frequently found in culture, sometimes in the setting of other organisms, and often dismissed as a contaminant. The environment of the prosthetic joint space likely has a low oxygen content [16], [17], [18]. Aerobic bacteria and interactions of aerobic and anaerobic bacteria are infrequently studied under

Acknowledgements

We thank James M. Steckelberg, MD, for providing content review. R.P. is supported by the National Institutes of Health [grant numbers R01 AR056647 and R01 AI91594].

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There are more references available in the full text version of this article.

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Cutibacterium acnes (previously known as Propionibacterium acnes) is frequently found on lipid-rich parts of the human skin. While C. acnes is most known for its role in the development and progression of the skin disease acne, it is also involved in many other types of infections, often involving implanted medical devices. C. acnes readily forms biofilms in vitro and there is growing evidence that biofilm formation by this Gram-positive, facultative anaerobic micro-organism plays an important role in vivo and is also involved in treatment failure. In this brief review we present an overview on what is known about C. acnes biofilms (including their role in pathogenesis and reduced susceptibility to antibiotics), discuss model systems that can be used to study these biofilms in vitro and in vivo and give an overview of interspecies interactions occurring in polymicrobial communities containing C. acnes.
Cutibacterium acnes biofilm forming clinical isolates modify the formation and structure of Staphylococcus aureus biofilms, increasing their susceptibility to antibiotics
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Within infection sites C. acnes is frequently co-isolated with Staphylococcus aureus and Staphylococcus epidermidis [13]. A recent study demonstrated that combining C. acnes and S. aureus cultures led to increased biofilm biomass and better survival of C. acnes in aerobic conditions [14]. In contrast, others have shown that S. epidermidis is able to inhibit growth of C. acnes clinical isolates, and vice versa [15].
Cutibacterium acnes (formally Propionibacterium acnes) is frequently identified within surgical device related infections. It is often co-isolated from infection sites with other opportunistic pathogens. Recent studies have demonstrated that C. acnes is able to form biofilms and when co-cultured with Staphylococcus spp. both inhibitory and stimulatory effects have been reported across several studies. Here, we investigated the biofilm-forming ability of 100 clinical C. acnes isolates from various infection sites in human patients, both deep tissue and superficial, followed by an investigation of how the supernatants of C. acnes cultures influenced the attachment and maturation of Staphylococcus aureus NCTC 6571 biofilms. All of the C. acnes isolates were able to form biofilms in vitro, although biofilm biomass varied between isolates. Nineteen isolates were weakly adherent, 33 were moderately adherent and the majority (48) showed strong adherence. The presence of C. acnes sterile supernatants reduced the biomass of S. aureus cultures, with a > 90% reduction observed in the presence of several of the C. acnes isolates. We observed that this decrease was not due to C. acnes affecting S. aureus viability, nor due to the presence of propionic acid. Biofilm maturation was however delayed over a 24-h period as was biofilm surface structure, although initial (up to 8 h) surface attachment was not affected. We hypothesis that this defective biofilm maturation is the cause of the observed biomass decrease. In turn, these altered biofilms showed a greater susceptibility to antibiotic treatments. In contrast the presence of C. acnes supernatant in planktonic (defined as a free moving, non-surface attached population within the liquid column) S. aureus cultures increased antibiotic tolerance, via a currently undefined mechanism. This study suggests that complex interactions between C. acnes and other opportunistic pathogens are likely to exist during colonisation and infection events. Further investigation of these interactions may lead to increased treatment options and a better prognosis for patients.
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Indeed, the microenvironment within the biofilm protects bacteria from the antibiotic action, thus making any treatment difficult. Polymicrobial biofilm formation is a possibility which makes therapy even harder [22]. The lack of an accepted definition of PJI may delay diagnosis and beginning of therapy, thus prolonging the time of healing and pushing costs higher.
Prosthetic joint infection (PJI) is a common complication of the knee and hip arthroplasty and represents a huge challenge for physicians. PJI raises serious social, economic and clinical concerns in the public health that need a comprehensive approach to better focus on proven strategies for disease prevention and treatment. History and clinical signs on joint site are useful means for suspecting PJI that need to be confirmed through major and minor diagnostic criteria. The pathogen isolation and the resulting antibiogram are crucial to guide the correct antibiotic strategy and together with surgical treatment (prosthesis revision and spacer implantation) represent the cornerstones to eradicate the infection before attempting a new arthroplasty. External fixator with removal of the spacer may be an option before performing a new arthroplasty when the infection does not heal. Arthrodesis may also be considered if the arthroplasty is contraindicated. Limb amputation is the last chance when pathogen eradication failed and might lead to life-threatening situations.
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The aim was to determine the characteristics of patients who developed Cutibacterium acnes spinal implant-associated infection (SIAI) and the associated risk factors.
We conducted two parallel case–control studies comparing 59 patients with SIAI caused by C. acnes (cases 1) and 93 patients with SIAI caused by other microorganisms (cases 2) diagnosed during 2010–2015 with 302 controls who underwent spinal instrumentation without subsequent infection.
Late-onset infections (median time to diagnosis, 843 days versus 23 days; p < 0.001) were more common in cases 1 than in cases 2. However, 20/59 (34%) of cases 1 occurred within the first 3 months after the index surgery. In addition, cases 1 were less likely to have fever (27%, 16/59 versus 58%, 54/93; p 0.001) or wound inflammation (39%, 23/59 versus 72%, 67/93; p < 0.001). Moreover, 24/59 (40%) of cases 1 presented with polymicrobial infections, and staphylococcal pathogens accounted for 22/24 (92%) of the co-infections. By comparing and contrasting the two multivariate risk models (cases 1 versus controls and cases 2 versus controls), the following factors associated with C. acnes SIAI development were identified: age <54 years (adjusted odds ratio (aOR) 2.43, 95% confidence interval (CI) 1.09–5.58, p 0.03), a body mass index <22 kg/m² (aOR 2.47, 95% CI 1.17–5.29, p 0.02), and thoracic instrumentation (aOR 16.1, 95% CI 7.57–37.0, p < 0.001).
Future therapeutic and prophylactic studies on C. acnes SIAI should focus on young, thin patients who undergo spinal instrumentation procedures involving the thoracic spine.
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Certain types of microorganisms may be more likely to grow together than separately, and there may be benefits in doing so (Tyner and Patel, 2016). For example, we showed that dual-species S. aureus and C. acnes biofilms enhanced survival of S. aureus (Tyner and Patel, 2016). This led us to hypothesize that some species may be more likely than others to form polymicrobial biofilms on prosthetic joints.
Prosthetic joint infection (PJI) is a rare but challenging complication of arthroplasty. Herein, we describe the epidemiology and microbiology of PJI, with a focus on analyzing differences between the microbiology of polymicrobial versus monomicrobial infection of hip, knee, and shoulder prostheses. In addition, we report the most frequent co-pathogens in polymicrobial infections, as detected by culture. A total of 373 patients diagnosed with PJI at Mayo Clinic were studied. For hip and knee arthroplasties, a higher proportion of fractures (P = 0.02) and a shorter time between the implantation and symptom onset (P < 0.0001) were noted in polymicrobial versus monomicrobial PJI. The most common microorganism detected, Staphylococcus epidermidis, was more frequently detected in polymicrobial (60%) versus monomicrobial (35%) PJI (P = 0.0067). Among polymicrobial infections, no co-pathogens were more frequently found than others, except S. epidermidis and Enterococcus faecalis which were found together in 5 cases. In addition to coagulase-negative staphylococci and enterococci, Corynebacterium species and Finegoldia magna were common in polymicrobial infections. Conversely, there was no difference between the prevalence of Staphylococcus aureus, Gram-negative bacilli, or Cutibacterium acnes between the polymicrobial and monomicrobial groups. The microbiology of polymicrobial PJI is different from that of monomicrobial PJI.
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Citation Excerpt :
Recently, the ability of C. acnes to generate bacterial persister cells in order to remain in the tissue in a stationary “sleeping phase” was discovered.22 The pathogenicity of C. acnes also becomes clear under the conditions of coinfection with S. aureus.23 Tyner et al23 report that coinfection with both bacteria leads to a more stable and denser biofilms, which makes it more difficult for antibiotics to penetrate the bacteria.24
Genome studies of heart valve tissue (HVT) in patients with structural valvular heart disease (sVHD) and acute infective endocarditis (aIE) showed polymicrobial infections. Subject of this study is the quantification of bacterial DNA in HVT of sVHD in comparison to aIE. It will be examined whether the bacterial DNA concentration can be used as surrogate marker to differentiate chronic and acute infections. DNA was isolated from HVT of 100 patients with sVHD and 23 microbiologically positively tested patients with aIE. Selected pathogens (Cutibacterium acnes, Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Streptococcus pyogenes, Streptococcus agalactiae, Clostridium difficile, and Klebsiella pneumoniae) were quantified using TaqMan-qPCR. Polymicrobial infiltration of HVT was investigated by immunohistologic methods. Of 100 sVHD patients, 94 tested positive for bacteria by 16S-rDNA and 72 by TaqMan-qPCR. In 29% of the sVHD cohort and in 70% of the aIE cohort, a coinfection with more than 2 bacteria was observed as indication of a polymicrobial infection. The most common pathogens in the sVHD patients were C. acnes (59%; 5–4074 pg/mL), E. faecalis (16%, 174–2781 pg/mL), and S. aureus (15%, 8–105 pg/mL). The DNA concentration of E. faecalis (P = 0.0285) and S. aureus (P = 0.0149) is significantly lower in the sVHD cohort than in the aIE cohort. sVHD is associated with bacterial infection and infiltration of the HVT in a majority of cases. TaqMan-qPCR is a valid instrument for the specific detection of bacteria in HVT and allows discrimination between sVHD and aIE for E. faecalis and S. aureus.

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Anaerobe

Highlights

Abstract

Introduction

Section snippets

Methods

Results

Discussion

Acknowledgements

References (18)

Clinical meaning of unexpected positive cultures (UPC) in revision shoulder arthroplasty

J. Shoulder Elb. Surg.

Synovial fluid leukocyte count and differential for the diagnosis of prosthetic knee infection

Am. J. Med.

Staphylococcus aureus hijacks a skin commensal to intensify its virulence: immunization targeting beta-hemolysin and CAMP factor

J. Investig. Dermatol.

An increased incidence of Propionibacterium acnes biofilms in acne vulgaris: a case-control study

Br. J. Dermatol.

The production of inflammatory compounds by Propionibacterium acnes and other skin organisms

Br. J. Dermatol.

Induction of proinflammatory cytokines by a soluble factor of Propionibacterium acnes: implications for chronic inflammatory acne

Infect. Immun.

Infections caused by Propionibacterium species

Rev. Infect. Dis.

Propionibacterium acnes: an under-appreciated cause of post-neurosurgical infection

J. Antimicrob. Chemother.

Rapid molecular microbiologic diagnosis of prosthetic joint infection

J. Clin. Microbiol.

Cited by (32)

The role of biofilm formation in the pathogenesis and antimicrobial susceptibility of Cutibacterium acnes

Cutibacterium acnes biofilm forming clinical isolates modify the formation and structure of Staphylococcus aureus biofilms, increasing their susceptibility to antibiotics

Prosthetic joint infection. A relevant public health issue

Risk factors for Cutibacterium acnes spinal implant-associated infection: a case–case–control study

Microbiology of polymicrobial prosthetic joint infection

Quantification of Multiple Bacteria in Calcified Structural Valvular Heart Disease

Pathogenesis and toxinsPropionibacterium acnes biofilm – A sanctuary for Staphylococcus aureus?

Highlights

Abstract

Introduction

Section snippets

Methods

Results

Discussion

Acknowledgements

J. Shoulder Elb. Surg.

Am. J. Med.

J. Investig. Dermatol.

An increased incidence of Propionibacterium acnes biofilms in acne vulgaris: a case-control study

Br. J. Dermatol.

The production of inflammatory compounds by Propionibacterium acnes and other skin organisms

Br. J. Dermatol.

Induction of proinflammatory cytokines by a soluble factor of Propionibacterium acnes: implications for chronic inflammatory acne

Infect. Immun.

Infections caused by Propionibacterium species

Rev. Infect. Dis.

Propionibacterium acnes: an under-appreciated cause of post-neurosurgical infection

J. Antimicrob. Chemother.

Rapid molecular microbiologic diagnosis of prosthetic joint infection

J. Clin. Microbiol.

Pathogenesis and toxins
Propionibacterium acnes biofilm – A sanctuary for Staphylococcus aureus?