Despite the ubiquitous presence of P. acnes on human tissues, concrete knowledge in relation to its pathogenic role amongst various disease states remains controversial (Portillo et al. 2013). Numerous studies have indicated the organisms’ ability to act as an opportunistic pathogen, with suggested etiological roles amongst a number of inflammatory diseases such as acne vulgaris and discitis (Achermann et al. 2014, Rollason et al. 2013).Studies have shown that this organism comprises of several distinct evolutionary clades, further discriminated based on Multilocus sequence typing into phylogenetically distinct clusters (type IA, IB, IC, II, and III) (Kilian et al. 2012). These clades possess differences in their production of virulence factors which may stimulate varying inflammatory reaction patterns in-vitro, antimicrobial resistance and disease association (Aubin et al. 2014, McDowell et al. 2008). The aim of this study was to investigate the effect of antimicrobials on P. acnes biofilm formation. Firstly, the antimicrobial susceptibility of NCTC 737 reference strain and ten (10) P. acnes strains namely acne lesion (AC (7)), acne lesion (AC (8)), IA1 (35), IA1 (56), IB (83), IB (90), II (2), II (55), III (3), and III (64), recovered from acne and disc infections to co- amoxiclav, clindamycin, rifampicin, tetracycline, and vancomycin were determined. The MIC values of all antibiotics tests against all strains was low (MICs ≤ 2 µg/ml). In table 1, co- amoxiclav, clindamycin and rifampicin demonstrated high antimicrobial activity against both skin and disc tissue isolates (MICs ≤ 0.25 µg/ml).
Several studies have been published in recent years describing the susceptibility patterns of P. acnes. Originally, P. acnes had been found to be highly susceptible to various antimicrobials such as aminoglycoside, β-lactams, lincosamide, macrolide, quinolone, and tetracycline (Goldstein et al. 2006, Gonzalez et al. 2010, Furustrand-Tafin et al. 2012), however, emergence of resistance had been reported after treatment with tetracycline, macrolides and clindamycin. In these cases, isolates usually become resistant to one single antibiotic or cross-resistant to several antibiotics. Resistance patterns comprise both isolates with moderately increased MICs and isolates with very high MICs (Oprica et al. 2006). In this study, low level resistance to vancomycin due to a slightly increased MIC (2 µg/ml) was observed in type IB (90) isolate as well as a reduced susceptibility to tetracycline due to slightly increased MIC (2 µg/ml) was observed in types IA1 (35), IA1 (56), and AC (7) test isolates. Literatures have reported the emergence of resistance of types (IA and IB) associated with moderate to severe acnes (Oprica et al. 2004, Nord and Oprica 2006). Resistance to tetracycline occurs due to a single G–C transition in the 16S rRNA of the small ribosomal subunit in P. acnes (Ross et al. 1998, Nonaka et al. 2005). Tetracycline resistance can also arise as a result of increased efflux pump activity and ribosomal protection or mutation of drug binding sites (Nakase et al. 2014). Resistance of planktonic acne isolates to vancomycin has not been reported (Oprica and Nord 2005). Tetracycline, which penetrates the bacterial cell wall by an energy dependent process binds reversibly to the 30S ribosomal subunit thus preventing the attachment of aminoacyl-tRNA to the ribosomal acceptor A-site in the RNA-ribosome complex, a bacteriostatic process (Kohanski et al. 2010). Also, clindamycin which is known to inhibit bacterial protein synthesis by binding to bacterial 50S ribosomal subunits (Smieja 1998) was bacteriostatic at concentrations tested against most strains as confirmed by the MBC assay. Interestingly, rifampicin and co-amoxiclav were bactericidal at low concentrations (≤ 4 µg/ml) suggesting their superiority in killing planktonic isolates and their use for biofilm assay. Unlike the other test antibiotics, vancomycin, a glycopeptide that inhibits cell wall synthesis (Ruzin et al. 2004) was bactericidal at (≤8× MIC) against all strains. No bactericidal concentration was determined against type II for all antibiotics, however, more interestingly, co-amoxiclav, rifampicin and vancomycin bactericidal concentrations are achievable in-vivo.
Due to reducing susceptibility being observed in P. acnes, several strategies have been proposed to prevent the development of antibiotic resistance that accompanies antimicrobial treatment. They include, restrictions in the overall use of systemic and all antibiotics and shortening of the course of antibiotic therapy itself because reports indicate long courses can be associated with the emergence of resistance, an example can be due to poor patient compliance (Nord and Oprica 2006, Lee et al. 2013).
BIOFILM FORMATION
Biofilm formation is considered to be a significant pathogenic attribute for both opportunistic and invasive pathogens, such as the P. acnes (Holmberg et al. 2009, Brandwein et al. 2016). The National Institutes of Health and Centre for Disease Control and Prevention estimates that >80% of all bacterial infections, with emphasis on chronic infections, possibly involve biofilm aetiology (Bjarnsholt 2013, Goodman et al. 2011). Biofilm infection promotes bacterial dissemination to other bodily regions stemming from its ability to serve as a reservoir (Cha et al. 2013). During infection, the processes of biofilm attachment to and detachment from host tissues or surfaces of medical devices, are a crucial part of colonisation as well as a perquisite for infection dissemination (Joo and Otto 2012).
Amongst the underlining virulence factors attributed to P. acnes, its biofilm formation ability has in recent times has been considered as a possible causative of loss of antimicrobial susceptibility as well as implications in the inflammatory process of most diseases (Feuillolay et al. 2016). The complete genome of P. acnes has been sequenced (Bruggemann et al. 2004) with detailed investigations indicating its composition of several genes that play possible significant roles in biofilm development. These genes include the LuxS genes and glucosyltransferase (GTF) responsible for EPS production as well as genes responsible adhesion proteins production (Burkhart and Burkhart 2007).
The direct visualization of P. acnes biofilms in infected and surgically removed hip arthroplasties using confocal laser immunofluorescence microscopy provides strong evidence that P. acnes can form biofilms both in-vitro, and in-vivo (Tunney et al. 2006). Recent studies carried out by Capoor et al. (2017) provide evidence of P. acnes biofilm formation within the disc spaces of patients undergoing micro-discectomy. Results obtained from several authors the likes of Rollason et al. (2013), and Capoor et al. (2017) in conjunction with the above investigative results obtained from this study (Figure 4), further strengthen the hypothesis that P. acnes produce biofilms capable of thriving within disc spaces further complicating disease progression. P. acnes biofilm formation is of major concern, as its EPS matrix provides the sessile cells with considerable resistance to host defences and antimicrobial agents (Coenye et al. 2007). Elimination of Planktonic cells via macrophage and neutrophil activity within a normal healthy host is easily attainable however, elimination of bacterial colonies contained within a self-produced polymeric sugar matrix far more resistant to oxygen radicals, phagocytosis and antibiotics than their planktonic counterparts, possess more of problem (Mah and O’Toole 2001).
ANTIBIOTIC RESISTANCE
Although MICs has been identified as the gold standard in the determination of bacterial antimicrobial sensitivity, its value is not predictive of the clinical efficacy of a particular antibiotic especially in biofilm studies (Tezel et al. 2016). The mechanisms of biofilm-associated antibiotic resistance are distinct from the well-studied mechanisms of intrinsic resistance such as, drug inactivation, drug efflux, membrane permeability and target site alterations (Fernandez and Hancock 2012). Although the basis of biofilm-associated antibiotic resistance is not fully understood, it is probable that multiple mechanisms operate simultaneously in biofilms, contributing to antibiotic resistance (Kaplan 2010). Factors such as contact time (Finch 2005), surrounding environment (Lebeaux et al. 2014), concentration of the antimicrobial agent (Tezel et al. 2016), incomplete penetration of antibiotic (Kaplan 2010), subpopulations of multidrug tolerant ‘persister cells’ (Lewis 2010), and drug indifference of slow-growing, nutrient-limited cells (Wood et al. 2013), constitute the determinants of antibiotic effectiveness in the detachment of biofilms. In this study, the antimicrobial effect of 3 clinically used antibiotics in P. acnes infections (co-amoxiclav, rifampicin and vancomycin) on biofilms formed by NCTC 737 reference strain and 5 P. acnes type strains (IA1 (35), IB (90), II (2), III (3), and acne lesion (AC (7)) recovered from disc and acne infections were determined using a modified microtiter plate assay. The results obtained revealed that all strains produced biofilms, however, the extent of biofilm formation was strain dependent as some were strong biofilm formers (types IA1 (35), IB (90), NCTC 737 and AC (7) while others formed weak biofilms (types II and III) (Figure 4). It is worthy to state however that no antibiotic was able to completely eradicate preformed biofilms (Figures 5-7). Co-amoxiclav (MIC value of 0.25 µg/ml) and vancomycin (MIC value of 2 µg/ml) caused significant reductions in only 4/6 (67%) of the test strains (T- test, P < 0.05) (Figure 5 and 7) while rifampicin at 0.125 µg/ml caused a significant reduction in 2/6 (50%) of the test strains (T- test, P < 0.05) (Figure 6). No antibiotic was able to significantly reduce biofilms formed by the type III strains (T-test, P>0.05) (Figures 5-7) while all antibiotics caused significant induction of biofilms in the NCTC 737 reference strain (Figures 5-7). The lack of antibiotic effect on type III strain could be an indication that antibiotics may only be able to target strongly adhered biofilms. To this end, it can be said that there is no fixed pattern for the effect of antibiotics on this preformed biofilm but it was noticed that the bacterial behaviour in response to the different antibiotics applied is strain dependant, which requires further investigations.
Although currently no explanation for the above observation with theses particular strain exists, this may well reflect differences in the nature and/or the biophysical properties of the biofilm produced, ultimately affecting antibiotic bioavailability and/or expression of activity. The effectiveness of co-amoxiclav corresponds to results by Albert et al (2013) who found that patients who were placed on the antibiotic for 100 days had significant improvements when compared to patients placed on placebo. Several studies have shown that infection with P. acnes involves an interaction with Toll like Receptors (TLR) 2 and (TLR) 4 on keratinocytes (Miller 2008). Activation of these pattern recognition receptors induces release of inflammatory cytokines and chemokines, including Tumour Necrosis Factor α (TNF-α) and Interleukins (IL)-8 (Arango-Duque and Descoteaux 2014), which facilitate inflammatory responses in both keratinocytes and monocytes (Arango-Duque and Descoteaux 2014). TNF-α and IL-8 are reportedly known to exacerbate skin inflammation in murine models (Kumari et al. 2013) and both lipopolysaccharide (LPS) and P. acnes are known to directly stimulate the production of TNF-α and IL-8 via TLR expression (Choi et al. 2008). Some antibiotics have been described to exhibit both antibacterial and anti-inflammatory effect, on human tissues (Hoyt and Robbins 2001). Albert et al. (2013) discovered that co-amoxiclav had inhibitory effect on pro-inflammatory mediators, interleukins IL-1 and IL-8, which is concomitant with herniated tissues. This inhibitory effect might be the probable cause for its positive result. Rifampicin (MIC value of 0.125 µg/ml) was found to be ineffective against biofilms in four of the strains that were assessed. This is in contrast with the study by Furustrand-Tafin et al. (2013) which reported a complete eradication of biofilms. Discrepancies amongst both studies could possibly result from high concentration of rifampicin and different methodology used in the biofilm assays.
Vancomycin, a glycopeptide is often the preferable systemic antibiotic of choice for the treatment of device related blood stream infections caused by invasive isolates. It was observed that vancomycin exposures at 2 µg/ml was not adequate to eradicate P. acnes sessile cells. A recent study suggests, however, that lack of activity against biofilms is due not to insufficient diffusion but, rather, to poor bioavailability, with the antibiotic possibly interacting with matrix constituents which are supposedly more abundant where bacteria are metabolically active. This gives rise to ‘persister’ cells that reduce antimicrobial activity (Bauer et al. 2013, Wood et al. 2013).
SUB MINIMUM CONCENTRATION
Currently, the sub minimum inhibitory concentrations (sub-MICs) of antibiotics are being recognized for their role in microorganisms’ persistence and in the development of antibiotic resistance (Andersson and Hughes 2011). Previously, numerous studies have reported that although sub-MICs of antibiotics are not capable of completely eradicating biofilms, they can exert anti-biofilm effects and hence reduce the density of biofilms formed (Cerca et al. 2005, Majtan et al. 2008). More recently, evidence has been provided by numerous authors stating that some antibiotics at sub-therapeutic concentrations can induce biofilm formation in microorganisms (Hoffman et al. 2005, Aka and Haji 2005). The results obtained as presented in Figures 5-7 showed that co-amoxiclav, rifampicin and vancomycin at sub inhibitory concentrations of 0.25 µg/ml, 0.125 µg/ml and 2 µg/ml respectively lost their ant biofilm effect and allowed for the significant induction of biofilms in NCTC 737 (T- test, P< 0.05). This is of clinical relevance as concentrations below the designated MICs may be encountered at the beginning and end of therapy (Aka and Haji 2015). In addition, drug – food interactions (Deppermann and Lode 1993), drug – drug interactions (Haddadin et al. 2010), and certain health condition of patients (Komori et al. 2007) can also result in reduced bioavailability of antibiotics in systemic circulation, and therefore establishing sub inhibitory concentrations of antibiotics (Komori et al. 2007).
Although, surrounding environment of biofilms may have lethal concentrations, the internal milieu may be exposed to sub-MICs of antibiotics, a situation that is credited to inactivating enzymes or physical and chemical barriers to antibiotic penetration (Haddadin et al. 2010, Fux et al. 2005). In the study reported by Matzneller et al. (2013), a poor correlation between the vascular concentrations and those within the soft tissue of patients for some antibiotics, with levels often never reaching therapeutic potentials in certain sites. Dvorchik et al. (2003) also reported that single doses therapies are always at risk of falling below the organism MIC threshold. Bacteria respond to antibiotic treatment by increasing polysaccharide synthesis and biofilm formation (Rachid et al. 2000, Nucleo et al. 2009). O’Toole and Stewart (2005) that this response is an evolutionary adaptation of bacteria to enable them to defend against various antibiotics. In a study by Hsu et al. (2011), it was found that upon vancomycin addition to Staphylococcus aureus (S. aureus) biofilms, genes involved in biofilm formation such as icaA and fnbA were upregulated. A striking finding within their study was the release of extracellular deoxyribonucleic acid (eDNA) which coincided with enhanced bacterial autolysis demonstrated to be the most important factor for vancomycin-enhanced biofilm formation. This was correlated with the study by Gomes et al. (2011) who discovered that upon addition of rifampicin to S. epidermidis biofilms, an upregulation of the icaA gene expression which possibly induced persister cells and a high ability for biofilm formation. The expression of the ica genes (Gotz 2002) and thereby the synthesis of polysaccharide intercellular adhesin (PIA) (Mack et al. 1996) are necessary for staphylococcal biofilms adhesion and formation. Another study carried out by He et al. (2017) reported that low dose of vancomycin induced biofilm formation in methicillin-resistant Staphylococcus aureus (MRSA). Mah and O’Toole (2001) observed that in Pseudomonas biofilms, Sub-MICs of antibiotics increases the intracellular second messenger cyclic dimeric guanosine monophosphate (c-di-GMP). C-di-GMP is implicated in controlling various cellular functions including virulence, motility, and adhesion, although it’s principal role is controlling the switch from motile planktonic lifestyle to the sessile biofilm forming state (Martinez and Vadyvaloo 2014). Elevated levels of c-di-GMP have been known to induce biosynthesis of adhesins, matrix polysaccharides while inhibiting motility thus enhancing biofilm formation (Jenal and Malone 2006). Although the induction of biofilms in P. acnes is not yet fully understood, it might be imperative to say that some genetic regulation might be involved. This therefore means that irrational use of antibiotics displays the potential risk of causing the induction of biofilm formation by P. acnes. Given the high level of biofilm induction, it may be advantageous to test clinical P. acnes isolates for biofilm inducibility in order to optimize antibiotic chemotherapy in clinical settings (Kaplan et al. 2012).
STRESS RESPONSE
Bactericidal antibiotics are potent instigators of the SOS response (Cirz et al. 2005, Kohanski et al. 2007, Cirz and Romesberg 2007). They induce a common cell fdeath mechanism via the stimulation of lethal amounts of oxidative radicals (Dwyer et al. 2014, Belenky et al. 2015), which activate the Recombinase A (RecA) and SOS response (Kohanski et al. 2007). Amongst most bacterial species, the (RecA) and locus for X-ray Sensitivity A (LexA) proteins govern the response, which is preserved across bacterial phyla including P. acnes., thus, RecA is crucial for increased antibiotic tolerance by enhancing DNA damage repair that occurs either directly via antibiotic-induced DNA damage or indirectly via metabolic and oxidative stress.
RecA-mediated repair also induces a hypermutable state promoting the acquisition of antibiotic resistance. If a damaged DNA is not successfully repaired, mutagenic polymerases (PolIV and PolV) are induced, resulting in mutagenesis and bacterial antibiotic resistance (Cirz et al. 2005, Kohanski et al. 2007). Bacterial antibiotic resistance can also be achieved by obtaining resistance genes from foreign DNA utilising the SOS response-mediated horizontal gene transfer pathway (Beaber et al. 2004). Mobile genetic elements such as integrating conjugative elements (ICEs) and conjugative plasmids, are key mediators for attaining antibiotic resistance genes (Fros et al. 2005). The link between bactericidal antibiotic activity, activation of the SOS response, and induction of antibiotic resistance (Cirz et al. 2005, Kohanski et al. 2007) demonstrate the potential for antimicrobial resistance reduction via targeting proteins vital for SOS response. Bactericidal antibiotic-mediated DNA damage results in the development of RecA-ssDNA filaments (Cirz et al. 2005), which are significant intermediates in DNA repair mechanisms and the SOS response. The repair of antibiotic-induced DNA damage by the SOS response has the involuntary effect of promoting the formation of a hypermutable state that stimulates mutagenesis and transmission of antibiotic resistance genes. If DNA damage repair is unsuccessful, then RecA-ssDNA filaments persist and degrade enough LexA allowing late-response SOS genes to be induced, which encode for mutagenic polymerases (PolIV and PolV). Expression of these polymerases causes mutations in genes that enable the resistance development (Cirz et al. 2005). The SOS response also promotes antibiotic resistance by enhancing horizontal gene transfer (Cirz et al. 2005, Beaber et al. 2004). RecA stimulates expression of proficient factors when bacteria are under antibiotic-induced stress, which enhances uptake and RecA-mediated chromosomal integration of exogenous DNA (Prudhomme et al. 2006). Sub MIC antibiotic concentrations have been reported to increase mutagenesis, in particular for those drugs known to interfere with DNA/RNA synthesis (Hughes and Andersson 2012). A report by Leiker and Weitao (2016) proved that deficiency of RecA in Streptococcus mutans that impedes the SOS response minimised the density and cellular viability of biofilms. Filamentation is a consequence of the SOS response. It was observed that during SOS response, premature cell division is prevented and genes for DNA damage repair are induced (Higashitani et al. 1995). It is not clearly understood yet why LexA deletion results in biofilm reduction. Although biofilm-related gene expression seems carefully regulated and programmed during biofilm development (Whiteley et al. 2001) it can be proposed that knocking out the transcription repressor LexA may repress expression of these genes, subsequently disrupting the programmatic gene expression for biofilm formation. Therefore, biofilm-related gene expression appears to be subtly controlled by the cleavable and non-cleavable LexA for biofilm development.
eDNA
Antibiotics may take a longer time duration prior to their diffusion though thicker biofilms thus allowing more time for biofilms to upregulate adaptive stress responses including excretion of antimicrobial degrading enzymes or increasing exopolysaccharide (EPS) production (Bernier and Surette 2013). Adaptive stress responses, for example antibiotic stressors or nutrient stressors, can also result to enhanced ability to survive exposures to low antibiotic concentration (Stewart 2002). Furthermore, as biofilms age, their cell density increases and extracellular DNA (eDNA) accumulates (Fux et al. 2005).
The biofilm matrix predominantly comprises of nucleic acids, polysaccharides, proteins and lipids. For most of the history of biofilm research, however, exopolysaccharides were thought to be the major and most important component. EPS was, in fact, originally an acronym for “exopolysaccharides”, rather than “exopolymeric substances”. Eventually, however, it became clear that polysaccharides are not alone in the biofilm matrix. In the last decade, the important role of nucleic acids, specifically DNA, to the biofilm matrix have been revealed. The presence of eDNA in biofilms was known, but it was not believed to play an active role in biofilm formation until the functional relevance of extracellular DNA (eDNA) to biofilms was reported for the first time in a pioneering report by Whitchurch et al. (2002) Since then, eDNA has been found to promote or modulate biofilm development of many different bacteria across several phyla. Although, the composition of P. acnes matrix is unknown (Holmberg et al. 2009), many bacteria and in particular, several Gram-positive bacteria such as Staphylococcus aureus and Streptococcus mutans biofilms have been analysed and are known to be composed of eDNA, polysaccharides and proteins (Lister and Horswill 2014, Liao et al. 2014). eDNA is involved in all stages of biofilm formation, from initial bacterial adhesion to maintenance of the biofilm’s structural integrity. eDNA influences the hydrophobicity of the bacterial cell surface. Due to its acid-base interactions and amphiphilic properties, it could theoretically influence the hydrophobicity of cells in either direction. Several studies suggest that eDNA increases the hydrophobicity of bacterial cells. Evidence has been provided by many authors that suggests that eDNA facilitates adhesion of bacteria biofilms such as those of Staphylococcus epidermidis to abiotic surfaces. (Hsu et al. 2011) reported that sub-lethal doses of vancomycin can induce more-robust biofilm formation through an enhanced autolysis- and extracellular DNA–dependent release in S. aureus. It was observed that autolysis is associated with septum formation and cell division and its separation will cause increased cell wall thickness and increased cell size thereby resulting in induction of vancomycin tolerance. Ozturk et al. (2014) also found that sub-lethal concentration of rifampicin also induced biofilms in MRSA isolates. In addition, vancomycin which binds to the terminal D-Ala-D-Ala residues of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) peptides, thereby blocking proper cell wall assembly (Abdelhady et al. 2013) has also been reported to induce biofilm formation. It was shown that sub-lethal doses induced extracellular DNA (eDNA)-dependent biofilm in several vancomycin-susceptible and vancomycin-resistant Methicillin Resistant Staphylococcus aureus MRSA strains (Singh and Ray 2012, Abdelhady et al. 2013). Therefore, it is possible that the antibiotics tested in the current study have a similar effect on expression of NCTC 737 genes that are involved in biofilm formation.
Formation of biofilms plays a crucial role in the pathogenicity of microorganisms. The formation of biofilms begins with a reversible attachment followed by the production of adhesins and extracellular polymer matrix which makes irreversibly attached to the surface (Agladze et al. 2003). The initiation of biofilm formation not only begins with quorum sensing, which may be considered a virulence regulator, after which it can activate additional virulence genes within the biofilms through quorum sensing (Rutherford and Bassler 2012, Antunes et al. 2010). Virulence genes can influence the production of virulence factors (toxins, proteases and lipases), cytotoxicity and swarming motility. Many recent studies have also suggested a link between the ability of biofilm formation and bacterial virulence, Holmberg et al. (2009), Jain and Agarwal (2009), Yamanaka et al. (2009). Experimentation by various authors have revealed that P. acnes are able to form biofilms as well as an increased production of the autoinducer AI-2 which upregulates its virulent activity. In our study, virulent strains associated with moderate to severe acnes (types IA, IB and acne lesions) had a greater ability to form more robust biofilms than a virulent strain II and III (Figure 5, 6 and 7). The genome sequence of a cutaneous type I P. acnes isolates reported that strains IA and IB produce cell surface dermatan sulphate binding adhesins which have the capacity for phase/antigenic variation, sialidase or β-haemolytic activity, essential for its adherence to surfaces and its pathogenicity (Bruggemann et al. 2004).
PHASE VARIATION
Phase variation is a gene-switching mechanism that is widely utilised in adaptation for altering environmental conditions and immune evasion (Finlay and McFadden 2006). It is a process of high-frequency, reversible, switching between phenotypes, mediated by genetic reorganization, mutation or modification and results in the continual generation of substitute phenotypes within a population. For effective colonisation and survival, pathogens must adapt to the dynamic and unpredictable changes that occur within the hosts’ environment or during transmission from one host to the other. Strategies for Bacterial adaptation involve either selection of the fittest variant form as a result of mutation or by sensing and responding to stringent and changing host conditions. Phase and antigenic variations are usually looked upon, among other things, as a strategy deployed by pathogens to evade immune defence mechanisms mounted by their hosts (Deitsch et al. 2009). Evasion of immune defence need not be the only use of phase variation; it could be a general strategy of stress adaptation. Population heterogeneity generated by phase variation could be useful in a more general way since there might be an assortment of cell types which could handle many kinds of stresses. The process could be viewed as an ‘insurance’ against catastrophe. In a sense, it is similar to bacterial persistence wherein a large population of antibiotic sensitive cells contain a small fraction of antibiotic-tolerant (not resistant) cells. Phase variation has also been shown to influence bacterial virulence and ability to initiate and maintain colonization of hosts during infection (van der Woude et al. 2004). To cite another example, turning on the expression of the fimA gene by phase variation has been shown to be essential for establishment of urinary tract infections by uropathogenic E. coli (UPEC) Synder et al. (2004). Snyder et al. (2006) constructed mutants of UPEC in which phase variation of the fim operon was ‘locked’ in the on/off states and examined their virulence in a mouse infection model. The off-locked mutants were shown to be severely impaired in their ability to colonize the kidneys and bladder of mice whereas the on locked mutants were as virulent as the wild type strain, showing the importance of the on state of fim expression in pathogenicity. Since the on state is affected by phase variation it follows that phase variation is needed for pathogenicity.
It has also been reported that types II and III have reduced lipase activity which alter their lipid metabolism and thus reduce their virulence (Bruggemann et al. 2004, Barnard et al. 2017, Tomida et al. 2013). Furthermore, due to high density and indirectly high quorum sensing, Similar to the transfer of antibiotic resistance genes, virulence genes can be acquired via a variety of mechanisms, including spontaneous mutations and deliberate exchange of genes through horizontal gene transfer. Horizontal gene transfer allows the transmission of genes both between individuals of a single species and between multiple species. Of course, such a transfer is facilitated by proximity; thus, when bacterium convert from planktonic, free floating individuals, to a sessile collective, transfer via plasmids is hastened (Madsen et al. 2012, Chia et al. 2008). The horizontal transfer of resistance determinants such as plasmids and increased mutation rates could result in the acquisition or de-novo development of reduced susceptibility to antimicrobial agents and other important microbial capabilities, such as increased virulence. The study of clustered regularly interspaced short palindromic repeats (CRISPR) in P. acnes revealed that CRISPR were present exclusively in types II and III, and differentiated type I from type II. The CRISPR mechanism may possibly inhibit these strains from acquiring virulent genes from invading foreign mobile elements (Tomida et al. 2013).
VIRULENCE
The link between virulence and antibiotic resistance is complex comprising numerous literatures that both correspond and contrast. The composition of the biofilm has been reported in some bacteria to influence susceptibility. For example, high-nutrient, high-density biofilms are less susceptible to antibiotics than low-nutrient, low-density biofilms (Condell et al. 2012). In this study, the virulent strains (IA and AC (7)) which formed stronger biofilms tended to be more resistant to antibiotics than low density forming a virulent strain (Figures 5-7). A mechanism by which cell resistance within the biofilm to antimicrobials can be enhanced, is via uptake of resistance genes by horizontal gene transfer (Schroeder et al. 2017). The high cell density, increased genetic competence and accumulation of mobile genetic elements that occur within the biofilms have been suggested to provide an ideal set of factors for efficient horizontal gene transfer, including resistance gene uptake (Aminov 2011).
Furthermore, the matrix provides a stable physical environment for cell to-cell contact, which is required for some mechanisms of gene transfer, and is a source of DNA in the form of eDNA (Kausmally et al. 2005, Kouzel et al. 2015). A common mechanism of horizontal gene transfer in biofilms is plasmid conjugation. For example, plasmids with genes that confer resistance to several antibiotics were readily transferred in Pseudomonas putida biofilms (van Meervenne et al 2014). More generally, conjugation has been shown to be up to 700 ¬fold more efficient in biofilms compared with free-living bacterial cells (Krol et al. 2013). Indeed, a study of Staphylococcus aureus showed that conjugal plasmid transfer occurred in biofilms but not in cultures of free-living bacterial cells, providing another example of a behaviour that occurs in a biofilm but that is not possible for free-living bacterial cells (Savage et al. 2013). As individual treatments can last for months to even years, it is no surprise that strains of cutaneous Propionibacterium resistant to the main antibiotics used to treat acne have emerged. Amongst the different approaches utilised by P. acnes to confer resistance to anti-acne agents, specific point mutations in the rRNA operon represent a major mechanism. Higher rates of phage transduction, which can disseminate antibiotic resistance determinants, have also been reported with treatment of some antibiotics (Zhang et al. 2000). Interestingly, environmental stress from sources other than antibiotics has similarly been shown to increase rates of horizontal gene transfer in bacterial communities Stecher et al. 2012). Biofilm formation, in addition to increasing rates of adaptive mutation, can also promote horizontal gene transfer in S. aureus (Savage et al. 2013). Thus, stress-response pathways and microbial community structures that favour the development of persistence can also potentiate horizontal gene transferAnother viable justification for the least susceptibility of types IA and AC (7) could be attributed to a robust array of multi drug resistant (MDR) transporters (Kvist et al. 2008). MDR transporters can be divided into two classes based on their source of energy: Secondary transporters, which use proton gradients to facilitate an antiporter mechanism, and adenosine triphosphate (ATP) binding cassette (ABC) transporters that couple the hydrolysis of ATP to substrate transport across the cell membrane (Wilkens 2015, Dahl 2004). Rapidly metabolizing cells in the biofilm may rely upon the conductive system to extrude a wide range of substrates, from ions and small molecules such as amino acids, sugars, xenobiotics, and vitamins up to polymers such as peptides, proteins, and polysaccharides (Eitinger et al. 2011) resulting from various biochemical activities taking place inside bacterial cells. Because of their wide substrate range, ABC transporters have been implicated in a range of cellular processes, such as nutrition uptake, xenobiotic protection, extrusion of cellular waste products, bacterial immunity and virulence, osmotic stress, lipid transport, and export of macromolecules during biogenesis, differentiation, and pathogenesis (Lubelski et al. 2007). In a study by Achermann et al. (2015), confirmed the presence of ATP transporter proteins in biofilms formed by P. acnes ATCC 11827 strain and also a clinical strain RMA 13884, which were both recovered from spinal osteomyelitis matched the IA lineage associated with acnes and could therefore be responsible for the least susceptibility observed in the study. Brzuszkiewicz et. al. (2011) also found the presence of an ABC transport system in SK137 belonging to type IA which is associated with moderate to severe acnes.
TYPE 1B
Although formation of robust biofilms has been linked to antibiotic resistance, the type IB (90) was highly susceptible to rifampicin and vancomycin (Figure 6 and 7). This occurrence may stem from the difference in EPS matrix composition. Allison et al. (2000) indicated that biofilm EPS is highly heterogeneous even amongst identical bacterial species and therefore its composition and function within the biofilms will differ. O’ Toole et al. (2000) also indicated that different biofilms produce different amounts of EPS. Some studies have identified carbohydrates as the major EPS constituents whilst others indicate proteins to be more predominant than polysaccharides (Zhang et al. 2001, Liu et al. 2002, Orgaz et al. 2007). To cite an example, it was reported that cell wall anchored proteins in Staphylococcus aureus (S. aureus) and Staphylococcus epidermis (S. epidermidis) contributed to aggregation either via homophilic interactions (Geoghegan et al. 2010, Schaeffer et al. 2015), or by interacting with matrix components originating from the host, such as collagen, fibrin and fibronectin (Buttner et al. 2015, Foster and Hook, 1998).
In Acinetobacter baumannii and S. aureus, a biofilm associated protein (Bap) was clearly required for biofilm development and maturation on both polystyrene and titanium (Goh et al. 2013, Brossard and Campagnari 2012). Bap is a high-molecular-weight protein present in many Staphylococci that is covalently anchored to the peptidoglycan through a sortase-dependent reaction recognizing the LPXTG motif (Cucarella et al 2001, Tormo et al 2005). It was hypothesized that Bap binds to other Bap molecules on neighbouring cells, thus functioning as intercellular adhesins within the biofilm and contributing to the overall structural support and integrity of the developing biofilm. S. aureus fibronectin-binding proteins (FnBPs), surface protein G (SasG) and its homologue accumulation associated protein (Aap) in S. epidermidis have also been shown to help in biofilm establishment in a polysaccharide independent background (Foster and Hook 1998). Vancomycin forms a noncovalent complex with the D-Ala-D-Ala portion of cell wall peptidoglycan, resulting in steric inhibition of the transglycosylation and/or transpeptidation steps of peptidoglycan synthesis (Kim et al 2008, Kahne et al. 2005). In addition, vancomycin alters bacteria cell membrane permeability and ribonucleic acid (RNA) synthesis, while rifampicin is thought to inhibit bacterial DNA-dependent RNA polymerase, which appears to occur as a result of drug binding in the polymerase subunit deep within the DNA/RNA channel, facilitating direct blocking of the elongating RNA (Asif 2013). This invariably means that both antibiotics targeting probably the protein content in IB (90) hence the significant reduction in biofilms formed.
Another possible cause might be attributed to the presence of degradative enzymes within its extracellular matrix that enhance the degradative activities. Mucoid strains of the human opportunistic pathogen Pseudomonas aeruginosa, for example, produce both alginate, a biofilm matrix polysaccharide composed of mannuronic and guluronic acids, and alginate lyase, an enzyme that degrades alginate. An increased expression of alginate lyase promotes cell detachment from P. aeruginosa biofilms cultured on agar surfaces (Boyd and Chakrabarty 1995), whereas, exogenously added alginate lyase increases antibiotic effectiveness against P. aeruginosa biofilms cultured in broth (Alipour et al. 2009). Polysaccharide lyases that promote biofilm detachment are also produced by P. fluorescens and P. syringae (Allison et al. 1998, Preston et al. 2000).
Another interesting example could be found in the oral bacterium Streptococcus intermedius, which produces hyaluronidase, an enzyme that degrades the glycosaminoglycan hyaluronan (HA) found in the extracellular matrix. It was recently shown by Pecharki et al. (2008) that the presence of hyaluronidase may play a role in S. intermedius biofilm dispersal. These authors found that a hyaluronidase mutant strain formed significantly more biofilm in broth supplemented with hyaluronic acid (HA) than did a wild-type strain. Also, exogenous hyaluronidase dispersed S. intermedius biofilms grown in HA-supplemented medium. This mechanism may have occurred when the type IB was treated with antibiotics and may have clinical relevance to the dispersal of P. acnes biofilms in acnes and the disc spaces.
The ability of biofilm-associated microbes to sense prevailing nutrient conditions and modulate their structural organization and species composition to aid their survival and propagation in the environment has been documented (James et al. 1995, Nielsen et al. 2000, Karthikeyan et al. 2001). Nutrient deprivation in biofilms can be one of the limiting conditions in the formation of low density biofilms as observed with the type III (Figure 5-7). Literatures have also reported that both increases and decreases in nutrient concentration have been correlated with biofilm dispersal. It has been observed that differences exist when biofilms are grown in nutrient rich and nutrient deprived environments. It was reported that nutrients boosted the biofilm cells growing in rich medium which resulted in more EPS produced. It was indicated in previous studies that biofilms growing in high nutrient medium were more abundant, densely packed and thicker (Rochex and Lebeault 2007).
TYPE III
The nutrient and oxygen depletion within biofilms may result in an altered metabolic activity, leading to slow growth of the bacteria, Likewise, growth, protein synthesis and metabolic activity are stratified in biofilms, for example, there is a high level of activity at the surface but a low level in the centre, resulting in slow or no growth. This may result in the formation of ‘persister’ cells (Werner et al. 2004) a highly multidrug-tolerant cell that constitutes a small fraction of the population. They are transiently refractory to killing, without having acquired resistance through genetic modification (Keren et al. 2004). Consequently, when the antibiotic pressure drops, the cells will give rise to a population that is as susceptible as the original one, and that again possesses a similarly small proportion of ‘persister’ cells. This discriminates ‘persister’ cells from resistant mutants, which exhibit stable, inheritable drug insensitivity.
Microscopic observation of individual bacteria grown in microfluidic devices demonstrated that ‘persisters’ have a significantly reduced growth rate. Their indifference to an antibiotic presence can therefore be explained by a global shutdown of processes essential for active growth, as the very processes that are targeted by antibiotics are no longer operational and hence not subject to inhibition any more.
Reduced growth rates have indeed been correlated with increased drug persistence both in vitro and in vivo, when bacterial growth tapers over the course of an infection due to immune pressure or lack of nutrients (Cohen et al. 2013). The reduced rates of DNA replication, translation, cell-wall synthesis, and metabolism directly targeted by antibiotics have been assumed to account for the relative drug tolerance of dormant bacteria. Although stalled biosynthesis probably promotes persistence, its effects are difficult to untangle from those of accompanying stress-response processes, and it is becoming clear that many active cellular processes occurring in parallel with growth-rate reduction are central for cellular survival in a toxic environment.it has also been reported that even antibiotics such as rifampicin that target processes essential for survival under all conditions display reduced efficacy against bacteria that are replicating only slowly or are in a low-activity metabolic state, though the relative change in drug efficacy is less profound than with the cell-wall-acting drugs. Zheng and Stewart (2002) also found that rifampicin penetrated biofilms formed by S. epidermidis but failed to effectively kill bacteria at 0.1 µg/ml. they but this protection is not due to inadequate penetration of the antibiotic through the bacterial aggregate.
Conclusion
In conclusion, the multifaceted nature of P. acnes’ interaction with antimicrobials, its biofilm formation capabilities, and the emergence of antibiotic resistance emphasizes the complexity of managing infections associated with this bacterium. Our investigation underscored the strain-dependent variability in biofilm formation and antibiotic susceptibility, revealing that certain strains, particularly types IA and IB, demonstrate robust biofilm formation and reduced susceptibility to conventional antibiotics. These findings highlight the necessity for a nuanced approach to antibiotic selection, taking into account the specific P. acnes strain and its known resistance patterns.
Critically, the study brings to light the phenomenon of biofilm-induced antibiotic resistance, advocating for the development of therapies targeting biofilm disruption as a means to enhance antibiotic efficacy. Moreover, the observed induction of biofilms at sub-MIC levels of antibiotics alerts to the potential pitfalls of sub-optimal antibiotic dosing, underscoring the importance of precise, strain-specific dosage regimens to circumvent the acceleration of resistance development.
Furthermore, our findings suggest a compelling link between antibiotic exposure and the bacterial stress response, including the SOS response and phase variation, which could further complicate treatment strategies. These adaptive mechanisms not only facilitate the persistence of P. acnes under antibiotic pressure but also enhance its capacity to evade the host immune response.
Given the significant role of P. acnes in a variety of infections, from acne vulgaris to more severe conditions like discitis, understanding the intricacies of its biofilm formation and resistance mechanisms is crucial. Future research should focus on exploring novel antimicrobial agents and treatment modalities that can effectively disrupt biofilms and mitigate the development of antibiotic resistance. Additionally, the potential for targeting specific bacterial stress response pathways presents an exciting avenue for therapeutic intervention.
In sum, this study underscores the pressing need for a comprehensive, multi-targeted approach to the treatment of P. acnes-associated infections. It highlights the importance of ongoing research into the mechanisms of biofilm formation and antibiotic resistance, with the aim of improving clinical outcomes through more effective treatment strategies and the judicious use of antibiotics. The battle against P. acnes requires not only scientific innovation but also a commitment to antibiotic stewardship to ensure the sustainability of effective treatment options for future generations.
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