Tert-butyl benzoquinone: mechanism of biofilm eradication and potential for use as a topical antibiofilm agent.

OBJECTIVES: Tert-butyl benzoquinone (TBBQ) is the oxidation product of tert-butyl hydroquinone (TBHQ), an antimicrobial food additive with >40 years of safe use. TBBQ displays potent activity against Staphylococcus aureus biofilms in vitro. Here, we report on studies to further explore the action of TBBQ on staphylococcal biofilms, and provide a preliminary preclinical assessment of its potential for use as a topical treatment for staphylococcal infections involving a biofilm component. METHODS: The antibacterial properties of TBBQ were assessed against staphylococci growing in planktonic culture and as biofilms in the Calgary Biofilm Device. Established assays were employed to measure the effects of TBBQ on biofilm structure and bacterial membranes, and to assess resistance potential. A living-skin equivalent was used to evaluate the effects of TBBQ on human skin. RESULTS: TBBQ eradicated biofilms of S. aureus and other staphylococcal species at concentrations ≤64 mg/L. In contrast to other redox-active agents exhibiting activity against biofilms, TBBQ did not cause substantial destructuring of the biofilm matrix; instead, the antibiofilm activity of the compound was attributed to its ability to kill slow- and non-growing cells via membrane perturbation. TBBQ acted synergistically with gentamicin, did not damage a living-skin equivalent following topical application and exhibited low resistance potential. CONCLUSIONS: The ability of TBBQ to eradicate biofilms appears to result from its ability to kill bacteria regardless of growth state. Preliminary evaluation suggests that TBBQ represents a promising candidate for development as a topical antibiofilm agent.

Redox-active compounds with a history of human use: antistaphylococcal action and potential for repurposing as topical antibiofilm agents.

OBJECTIVES: To investigate the antistaphylococcal/antibiofilm activity and mode of action (MOA) of a panel of redox-active (RA) compounds with a history of human use and to provide a preliminary preclinical assessment of their potential for topical treatment of staphylococcal infections, including those involving a biofilm component. METHODS: Antistaphylococcal activity was evaluated by broth microdilution and by time-kill studies with growing and slow- or non-growing cells. The antibiofilm activity of RA compounds, alone and in combination with established antibacterial agents, was assessed using the Calgary Biofilm Device. Established assays were used to examine the membrane-perturbing effects of RA compounds, to measure penetration into biofilms and physical disruption of biofilms and to assess resistance potential. A living skin equivalent model was used to assess the effects of RA compounds on human skin. RESULTS: All 15 RA compounds tested displayed antistaphylococcal activity against planktonic cultures (MIC 0.25-128 mg/L) and 7 eradicated staphylococcal biofilms (minimum biofilm eradication concentration 4-256 mg/L). The MOA of all compounds involved perturbation of the bacterial membrane, whilst selected compounds with antibiofilm activity caused destructuring of the biofilm matrix. The two most promising agents [celastrol and nordihydroguaiaretic acid (NDGA)] in respect of antibacterial potency and selective toxicity against bacterial membranes acted synergistically with gentamicin against biofilms, did not damage artificial skin following topical application and exhibited low resistance potential. CONCLUSIONS: In contrast to established antibacterial drugs, some RA compounds are capable of eradicating staphylococcal biofilms. Of these, celastrol and NDGA represent particularly attractive candidates for development as topical antistaphylococcal biofilm treatments.

Interleukin-10 secretion from CD14+ peripheral blood mononuclear cells is downregulated in patients with acne vulgaris.

BACKGROUND: Acne is a common chronic inflammatory dermatosis of the pilosebaceous unit. It is characterized by seborrhoea, comedone formation and an inflammatory response consistent with defective cellular immunity to Propionibacterium acnes. OBJECTIVES: The objective of this study was to investigate the immune reactivity of patients with acne compared with healthy controls by examining the response of peripheral blood mononuclear cells (PBMCs) to stimulation with P. acnes. Particular focus was placed upon measuring the production of interleukin (IL)-10, which has an established immunoregulatory role. PATIENTS AND METHODS: Venous blood was collected from 47 patients with acne and 40 age- and sex-matched healthy controls with no prior history of acne. PBMCs were cultured and their cytokine response to P. acnes investigated. RESULTS: Proinflammatory IL-8 and tumour necrosis factor (TNF)-alpha secretion from PBMCs was higher in patients with acne when stimulated with P. acnes. In contrast, a statistically significant reduction in PBMC secretion of anti-inflammatory IL-10 in patients with acne was identified. The impaired production of IL-10 by PBMCs from patients with acne was confined to CD14+ cells presumed to be monocytes. The ability of CD14 cells from patients with acne to phagocytose P. acnes bacteria was also observed to be defective but the addition of exogenous IL-10 to PBMC cultures restored phagocytic activity. CONCLUSIONS: These data suggest that patients with acne have a proinflammatory cytokine milieu and crucially are unable to contain early inflammatory changes due to a specific defect in immunosurveillance, namely low monocyte IL-10 production. Our observations raise the possibility that acne therapeutics might profitably target IL-10 both as a regulator of proinflammatory cytokines and in augmenting the CD14+ cell phagocytic response.

Modulation of comedonal levels of interleukin-1 in acne patients treated with tetracyclines.

To understand the basis for the anti-inflammatory activity of tetracyclines in acne, we compared the cytokine profiles [interleukin 1 (IL-1) alpha and beta, tumor necrosis factor (TNF) alpha, and IL-6] and bacterial flora of 66 open comedones removed from eleven patients before and after at least 8 weeks treatment with either tetracycline or minocycline. Pre-treatment, the only cytokine regularly recovered from comedones was bioactive IL-1 alpha-like material. The mean concentration of IL-1 alpha-like bioactivity/mg comedonal material rose from 272.0 +/- 88.6 pg to 844.3 +/- 196.7 pg following treatment (p < 0.05, Wilcoxon matched pairs). All six minocycline-treated patients showed an increase in bioactive IL-1 alpha-like material compared with three of five tetracycline-treated patients. The incidence (p < 0.001, chi 2) and concentration (p < 0.05, Wilcoxon) of immunochemical IL-beta were also raised post-treatment, although significantly more patients assigned to minocycline therapy had detectable levels of this cytokine before therapy was initiated. However, the mean concentration of IL-1 beta/mg comedonal material post-treatment was similar in both groups (72.5 +/- 23.3 pg for tetracycline-treated compared with 78.6 +/- 41.9 pg for minocycline-treated patients). The other cytokines were either absent (IL-6) or present in < 10% of comedones (TNF alpha) before and after therapy. Following treatment, only three of 11 patients showed a decrease of > or = 1 log10 in propionibacterial numbers/mg comedonal material, whereas six patients showed an increase of > 0.5 log10 in numbers of staphylococci. In eight patients, the increase or decrease in staphylococcal numbers correlated with the change in concentration of IL-1 alpha-like bioactivity. This is the first study to show an effect of antibiotic therapy on cytokine levels in vivo. Increased levels of IL-1 in comedones destined to become inflamed may enhance resolution and promote repair of the damaged follicular epithelium. Hence, these results provide further evidence of the augmentation of immune responses by tetracyclines and support the hypothesis that epidermal IL-1 plays a physiologic role in wound healing.

Pro-inflammatory levels of interleukin-1 alpha-like bioactivity are present in the majority of open comedones in acne vulgaris.

The factors that initiate the inflammatory response in acne are not known. The presence of pro-inflammatory cytokines in acne comedones was therefore investigated. One hundred eight open comedones were collected from 18 untreated acne patients (10 male, 8 female). Each comedone was homogenized and centrifuged, and the supernatant was analyzed for bioactive and immunochemically detectable IL-1 alpha, IL-1 beta, and TNF alpha. Viable counts of propionibacteria, staphylococci, and Malassezia spp. were determined in the comedone pellet. Bioactive IL-1 alpha-like material was demonstrated in 76% of open comedones (range of 23-4765 pg IL-1 alpha-like bioactivity/mg of comedone material). In 58% of comedones, levels exceeded 100 pg/mg. There was no correlation between IL-1 alpha-like bioactivity and IL-1 alpha determined immunochemically. Bioactive IL-1 beta was not detected in any comedones. Twenty-four percent contained low levels of immunochemical IL-1 beta (range 12-103 pg IL-1 beta/mg comedone material). Bioactive TNF alpha was detected in three comedones with a further five comedones containing immunochemical TNF alpha (range of 61-820 pg TNF alpha/mg comedone material). The majority of open comedones (97%) contained microorganisms. There was, however, no significant correlation (Spearman's rank) between levels of any cytokine, in particular IL-1 alpha-like bioactivity, and numbers of microorganisms. Thus, bioactive IL-1 alpha-like material in the majority of open comedones may be concerned in the initiation of inflammation in acne following spongiosis or rupture of the pilosebaceous follicle wall.

Identification of a chromosomally encoded ABC-transport system with which the staphylococcal erythromycin exporter MsrA may interact.

The energy-dependent efflux of erythromycin (Er) in staphylococci is due to the presence of msr A, which encodes an ATP-binding protein. MsrA is related to the multi-component ATP-binding cassette (ABC) transporters which characteristically also contain membrane-spanning domains. Since MsrA functions in a heterologous host in the absence of other plasmid-encoded products, the requirement for a transmembrane (TM) complex might be fulfilled by hijacking a chromosomally encoded protein. Two genes, stpA and smpA, were identified upstream from msrA on the original Staphylococcus epidermidis plasmid, encoding an ATP-binding protein and a hydrophobic TM protein, respectively. Sequences highly similar to stpA and smpA (stpB and smpB) were also found adjacent to a chromosomal copy of msrA in S. hominis. In Southern blots, internal fragments of stpA or smpA hybridized to the chromosome of the Ers S. aureus RN4220. Cloning and sequence analysis of the region identified revealed the presence of two genes, stpC and smpC, related to stpA and smpA. The deduced amino-acid sequences of the gene products showed that StpA and StpC were 85% identical, whereas SmpA and SmpC were 65% identical. A gene similar to msrA was not present in the S. aureus chromosome. There was no further sequence similarity outside these conserved regions. These results indicate that the chromosomes of S. hominis and S. aureus contain sequences encoding a potential TM protein with which MsrA might interact.

Minimal functional system required for expression of erythromycin resistance by msrA in Staphylococcus aureus RN4220.

Previous studies have suggested that inducible erythromycin (Er) resistance in staphylococci mediated by the plasmid-borne ABC-transporter msrA is dependent on additional unidentified chromosomally encoded transmembrane (TM) domains. The requirement for two S. aureus candidate sequences, stpC and smpC, highly similar to sequences adjacent to msrA on the original S. epidermidis plasmid was investigated. Deletion of the sequences by allelic replacement was accomplished by electroporation of S. aureus RN4220 with a nonreplicating suicide vector. S. aureus strains carrying a delta(stpC-smpC) mutation showed an identical ErR phenotype to those arising from single crossover events and unmutated RN4220 containing msrA. This proves that neither stpC nor smpC is required for ErR. To further define the minimal functional unit required for MSR, the control region within the leader sequence of msrA was deleted. This resulted in constitutive resistance to Er and type B streptogramins (Sg), proving that SgR does not require the presence of Er. Deletion constructs containing the N- or C-terminal ABC regions of MsrA did not confer ErR in RN4220 singly or in combination.

Physiological factors affecting streptomycin production by Streptomyces griseus ATCC 12475 in batch and continuous culture.

Conditions of growth are described for the production of streptomycin by Streptomyces griseus ATCC 12475 using chemically defined minimal medium and complex medium. It was found using batch cultures that early synthesis of the antibiotic occurred during growth in minimal medium but was delayed until the onset of stationary phase in complex medium. This effect was independent of whether spores or vegetative cells were used as inoculum. Stability of streptomycin biosynthesis in continuous culture was dependent on dilution rate and medium employed. Cultures were highly unstable when grown on complex medium but could be maintained in steady states in continuous culture using minimal medium when the dilution rate was increased in a stepwise manner, starting at a dilution rate of 0.02 h-1 (15% of mumax). The effect of changing dilution rate on growth, streptomycin production and the level of streptomycin phosphotransferase was examined using this technique.

The use of PCR to isolate a putative ABC transporter from Saccharopolyspora erythraea.

A gene (ertX) encoding a putative ABC transporter was cloned from the erythromycin producer Saccharopolyspora erythraea, using PCR. The primers were based on regions of homology from ABC transporters which confer resistance to macrolide antibiotics. While ertX encodes a protein with a strong degree of similarity to other macrolide ABC transporters from streptomycetes and staphylococci, it did not confer resistance to erythromycin, tylosin, spiramycin, oleandomycin, josamcin, chalcomycin or midecamycin when subcloned into sensitive streptomycete hosts. Southern blot analysis suggested that ertX did not constitute part of the erythromycin gene cluster as identified to date.

Is acne an infection of blocked pilosebaceous follicles? Implications for antimicrobial treatment.

A model is proposed which is based on the assumption that acne is due to infection of functionally blocked pilosebaceous follicles by propionibacteria. Noninflamed lesions, which are first visible during the adrenarche in acne-prone individuals, do not contain propionibacteria. Comedogenesis appears to be independent of bacterial infection and may be driven by high levels of bioactive interleukin-1 alpha derived from ductal hyperkeratinocytes. The stimulus which triggers interleukin-1 alpha production is unknown. Formalin killed Propionibacterium acnes failed to stimulate production of the cytokine by cultured human keratinocytes in vitro. Inflamed lesions are thought to arise from microcomedones, but the initiating events are unknown. Evidence that propionibacteria are involved in the generation of inflammatory lesions is inconclusive. The cellular infiltrate is consistent with a type IV hypersensitivity response to one or more persistent lesional antigens, not necessarily bacterial. The potent adjuvant activity of P. acnes would up-regulate the immune response to any antigen which came into contact with the mononuclear cell infiltrate. Antibiotics are widely used in the treatment of acne, and their effects in selecting a predominantly resistant commensal population are well recognized. Although they reduce numbers of propionibacteria on the skin, other modes of action may contribute to or explain their therapeutic efficacy. At a time when there is global concern that antibiotic resistance rates in common bacterial pathogens may threaten our future ability to control bacterial infections, practices which promote the spread of antibiotic-resistant bacteria must be fully justified. A thorough reappraisal of the role of propionibacteria in acne is overdue. It is likely that further experimental work is needed to confirm or refute that P. acnes is aptly named.

Topical antibiotic therapy: current status and future prospects.

As we enter a new decade, topical antibiotics are the subject of much renewed interest and are being used on a wider scale than ever before. The reasons for using topical rather than oral therapy for a variety of dermatoses include the reduced risk of systemic side effects, the avoidance of resistance selection in the gut microflora, the higher achievable concentration of antibiotic at the site of action and the overall usage of less drug. Somewhat surprisingly, treatment costs are not reduced by the use of topical therapy. The number of antibiotics licensed for topical use has increased in recent years and now includes representatives of the tetracycline, macrolide, lincosamide, aminoglycoside and peptide families of antibiotics in addition to fusidic acid, chloramphenicol and pseudomonic acid. Opinions regarding the clinical efficacy of topical antibiotics are conflicting, and for most indications alternative oral therapies are available. Topical antibiotics are the drugs of choice for the elimination of nasal carriage of Staphylococcus aureus and for the therapy of eye and external ear infections. They are also effective in the treatment of impetigo and other superficial pyodermas and in the management of localised infected eczema. Topical preparations of erythromycin, clindamycin and tetracycline are widely prescribed for the therapy of acne and are of clinical benefit in mild--moderate cases. However, they are no more effective against inflamed lesions than benzoyl peroxide and are less effective against non-inflamed lesions. They are not as effective as oral tetracycline in moderate to severe acne and should not be considered as a therapy for severe acne, for which 13-cis-retinoic acid is the drug of choice. It is well known that many antibiotics, when used topically, especially for prolonged periods, select for antibiotic-resistant staphylococci at the skin surface. Tetracyclines, erythromycin and clindamycin also select for resistant staphylococci on the surface of intact skin when delivered by the oral route. The contribution of topical antibiotic usage to the current high level of antibiotic resistance in coagulase-negative staphylococci, which are increasingly implicated in infections of compromised hosts, has not been quantified, although it is known that cutaneous staphylococci possess a large pool of transferable resistance genes.(ABSTRACT TRUNCATED AT 400 WORDS)

Temporal changes in sebum excretion and propionibacterial colonization in preadolescent children with and without acne.

BACKGROUND: It is generally accepted that the onset of sebum secretion occurs before puberty in boys and girls as a result of increasing androgen output during the adrenarche. Propionibacteria are part of the commensal skin flora and, in adults, are found in highest numbers in sebum-rich areas of skin such as the face and upper trunk. Previous studies investigating the association between sebum output and propionibacterial population densities have been cross-sectional and have been carried out mainly in adults. OBJECTIVES: The purpose of this study was to examine the association between the onset of sebum secretion and expansion of the propionibacterial flora in a population of early adolescent children aged between 5.5 and 12 years, and to evaluate the temporal relation between the two factors longitudinally. In addition, the study aimed to evaluate the change with age in sebaceous gland activity and propionibacterial colonization on the skin and in the nares between children who developed acne and those who did not. METHODS: Biannual examinations of volunteers included age, pubertal (Tanner) stage, weight and height, lesion counting on the face, propionibacterial colonization on the skin surface and in the nares and sebum secretion. A longitudinal analysis based on all observations of each subject throughout the study was applied to examine the change of sebaceous gland activity and propionibacterial colonization with age and pubertal stage. A generalized estimating equation was used with a 0.05 level of significance. RESULTS: The commencement of sebum production was asynchronous, with only a small number of follicles initially starting to secrete sebum onto the skin surface. The number of secreting follicles and the area of sebum increased with age and pubertal stage (P < 0.0001, P < 0.05, respectively). Numbers of propionibacteria on the skin tended to increase after the age of 9 years, but not significantly so. In contrast, numbers of propionibacteria in the nares increased significantly with age (P < 0.0001) but not with pubertal maturation. Children who developed acne had higher sebum output and propionibacterial densities with increasing age than children who did not develop acne. This effect was significant for the increase of total sebum area with age in pubertal children (P = 0.0023), the increase in number of secreting follicles with age (P = 0.020) in prepubertal children, and the increase in propionibacteria densities in the nares with age (P = 0.0005) in pubertal children. Sebaceous gland activity and propionibacterial numbers on the skin surface remained unchanged with increasing age in children who did not develop acne. Propionibacterial population densities in the nares increased with age regardless of the development of acne. CONCLUSIONS: Onset of sebum secretion and consequently expansion of the propionibacterial skin flora occur earlier in children who develop acne than in children of the same age and pubertal status who do not develop acne. These observations suggest that postponing the onset of sebum production or the expansion of the propionibacterial skin flora until after puberty may represent ways of preventing the disease or minimizing its severity. Determinants of propionibacterial colonization on the skin and in the nares may be different.

ABC proteins and antibiotic drug resistance: is it all about transport?

The precise mechanism of antibiotic-resistance-conferring ABC (ATP-binding-cassette) proteins (termed NBD2) remains open to debate. Currently, two hypotheses are recognized. In one, the NBD2 proteins are envisaged to act at the ribosome to impair antibiotic access to the target site on the 23 S rRNA. In the other, NBD2 proteins are believed to act as the components of ATP driven efflux pumps by associating with membrane spanning proteins capable of binding and transporting antibiotics. Pertinent data in support of these two hypotheses are discussed in this paper.

Efficacy of oral isotretinoin in the control of skin and nasal colonization by antibiotic-resistant propionibacteria in patients with acne.

BACKGROUND: Skin colonization by antibiotic-resistant propionibacteria is commonplace among acne patients globally. Increasing attention is now being paid to how resistance rates might be reduced to preserve the future efficacy of antibiotics, especially erythromycin and clindamycin in acne therapy. OBJECTIVE: To assess the efficacy of oral isotretinoin in the control of antibiotic-resistant propionibacteria. METHODS: Acne patients (72 in the U.K., 62 in the U.S.A.) colonized with high numbers of antibiotic-resistant propionibacteria were sampled before, during and 12 weeks after oral isotretinoin therapy. Propionibacterial samples were collected from five acne-prone skin surface sites using a detergent scrub method and from the anterior nares using moistened swabs. Total and antibiotic-resistant propionibacteria were enumerated by viable counting on media with and without selective antibiotics. RESULTS: After 16 weeks of oral isotretinoin therapy, mean population densities of viable propionibacteria and variants resistant to erythromycin, clindamycin or tetracycline had fallen by more than 90% at all skin sites and in the nares. The sole exception was a smaller reduction in tetracycline-resistant strains on the lower back. In general, greater reductions were observed on skin than in the nares. By the end of the treatment period only three patients (all in Philadelphia) yielded no antibiotic-resistant strains from any site. Post-treatment, propionibacterial counts remained well below pretreatment levels but had begun to recover on the face and in the nares. The recovering propionibacterial population included both susceptible and resistant strains. Changes during and post-treatment at the two centres were similar but not identical. CONCLUSIONS: Oral isotretinoin effectively reduced skin and nasal colonization by antibiotic-resistant propionibacteria. However, viable populations of resistant isolates persisted post-treatment at multiple sites. Novel methods are required to eradicate antibiotic-resistant propionibacteria completely, especially from the nasal reservoir.

The effect of benzoyl peroxide on cutaneous micro-organisms in vitro.

The survival curves of cutaneous micro-organisms in the presence of benzoyl peroxide were investigated. All the curves exhibited a shoulder prior to exponential cell death. Benzoyl peroxide was lethal to the cutaneous organisms tested and they varied in sensitivity increasing as follows: Propionibacterium acnes, Staphylococcus capitis, Staph. epidermidis, Staph. hominis, Prop. avidum, Prop. granulosum and Pityrosporum ovale.

An analysis of sebum excretion rate, bacterial population and the production rate of free fatty acids on human skin.

Bacteria were sampled using a "scrub" technique from the skin surface of the faces of forty-nine female subjects aged 18-21 years. The sebum excretion rate was determined by a gravimetric method and the level of free fatty acids by titration. The production rate of free fatty acids was calculated from the product of the concentration of free fatty acids in the sebum and the sebum excretion rate. The date was analysed using Kendall's rank correlation method. Positive correlations existed between the number of Micrococcaceae and the skin propionibacteria (P < 0.001) and between both groups of organisms and the production rate of free fatty acids (P < 0.001). There was no significant correlation between the size of the bacterial population and the sebum excretion rate. The results support the view that free fatty acids are produced as a result of bacterial action, that the Micrococcaceae and skin propionibacteria do not compete to the detriment of their respective populations, and that the size of the bacterial population is not dependent upon the sebum excretion rate.

Acne vulgaris: is the bacterial population size significant?

The levels of Propionibacterium acnes (P. acnes) and members of the Micrococcaceae were enumerated in two separate studies. The first investigation on the foreheads of thirty-five mild and thirty-five moderate acne patients showed no significant difference in the bacterial populations of the two groups. The second investigation of twelve patients on 250 mg tetracycline twice daily for 3 months showed no significant difference compared to pre-treatment data in the bacterial population during the successful treatment period. The data indicate that greater numbers of bacteria are not associated with increasing severity of acne and that the effectivenss of oral tetracycline in treating the disease can not be explained by a reduction in the number of viable bacteria.