K.T. Holland

Erythromycin resistant propionibacteria in antibiotic treated acne patients: association with therapeutic failure

Erythromycin resistant (EmR propionibacteria were isolated from the skin surface of 51% of patients treated with oral erythromycin and 42% of patients treated with topical clindamycin compared with 3% of untreated control subject (P < 0.001). Amongst the topical clindamycin‐treated patients, there was a higher incidence of EmR propionibacterial carriage in those patients who had previously been treated with oral erythromycin (64%) than in patients with no known previous exposure to erythromycin (20%; 0.01 > P > 0.001). Patients responding to oral erythromycin treatment carried EmR propionibacteria less frequently (24%) than patients who were not responding or who had relapsed (70%; P < 0.001). These observations suggest that the use of oral erythromycin and/or topical clindamycin encourages the development of resistant propionibacteria and that the emergence of resistant strains is associated with therapeutic failure in erythromycin‐treated patients. In total 63 resistant isolates were obtained from 52 subjects. There were 42 strains of Propionibacterium acnes, 16 strains of Propionibacterium granulosum and five strains of Propionibacterium avidum. The majority of isolates were inducibly or constitutively resistant to macrolide (e.g. erythromycin), lincosamide (e.g. clindamycin) and streptogramin B type antibiotics. Therefore, the isolates are phenotypically indistinguishable from the majority of EmR bacteria in which resistance is due to methylation of 23S ribosomal RNA.

Proinflammatory cytokine production by human keratinocytes stimulated with Propionibacterium acnes and P. acnes GroEL.

Background  Keratinocytes form the first line of defence in the skin and alert the host to danger by the production of a number of cytokines and chemokines. However, the interaction of commensal microorganisms with keratinocytes has not been well studied.

The effects of acne treatment with a combination of benzoyl peroxide and erythromycin on skin carriage of erythromycin resistant propionibacteria

Summary Concomitant application of 5% w/w benzoyl peroxide and 3% w/w erythromycin has previously been shown to prevent the overgrowth, on the skin of acne patients, of crythromycin‐resistant coagulase‐negative staphylococci, which occurs when the antibiotic is used alone. Two in vivo studies were carried out to assess the ability of the same therapeutic combination to inhibit the growth of pre‐existing erythromycin‐resistant propionibacteria and to prevent the selection of resistant strains during treatment. A double‐blind clinical trial in 37 patients with mild to moderate acne vulgaris showed that the combination brought about a > 3 log10 c.f.u. reduction in total propionibacterial numbers/cm2 after 6 weeks therapy (P < 0.001, Wilcoxons matched pairs) and also significantly reduced the number of erythromycin‐resistant propionibacteria (P < 0.05). In contrast, erythromycin alone reduced the total propionibacterial count by < 1.5 log10 c.f.u./cm2 after 6 weeks (P < 0.05) and did not affect the number of erythromycin‐resistant strains. The combined formulation was significantly more effective at reducing total propionibacterial numbers at 6 (P < 0.01, Mann‐Whitney) and 12 weeks (P < 0.05) than erythromycin alone, although, after 12 weeks, the anti‐propionibacterial efficacy of both preparations was less marked. Five patients on combination therapy, and five treated with erythromycin alone, acquired erythromycin‐resistant strains de novo at week 6 or week 12. In an open study in 21 acne patients, who each carried > 103 c.f.u. erythromycin‐resistant propionibacteria/cm2 skin pretreatment, the combination of erythromycin and benzoyl peroxide reduced the total propionibacterial count by > 2.5 log10 and the number of erythromycin‐resistant strains by a similar amount (P < 0.001, Wilcoxon). This was accompanied by highly significant reductions in acne grade and lesion counts (P < 0.001). These data suggest that the combination of 5% w/w benzoyl peroxide and 3% w/w erythromycin has greater in vivo antipropionibacterial activity than 3% w/w erythromycin alone, and brings about significant clinical improvement in acne patients with high numbers of erythromycin‐resistant propionibacterial strains pretreatment.

Interaction of Propionibacterium acnes with skin lipids in vitro

Propionibacterium acnes is the predominant microbial resident within the pilosebaceous follicles of sebum-rich areas of human skin. This study investigated the effects of known hydrophobic components of sebum on the physiology and nutrition of this microorganism, grown anaerobically at 33 degrees C, under defined conditions using continuous culture techniques. The medium used was chemically defined, comprising eight amino acids, with glucose as the main carbon energy source, and the culture pH was maintained at 5.6. The range of sebum lipids assayed was based on the C18 monounsaturated fatty acid 9-cis-octadecenoic acid (oleic acid). Stock micronized solutions were aseptically pulsed into continuous cultures in the presence and absence of glucose, and nutritional effects monitored. None of the lipid substrates significantly affected P. acnes growth either in terms of maximum specific growth rate (mu max) or final culture biomass yield. Glycerol (3 mg ml-1) was found to be a poor carbon/energy source in comparison to glucose. Bacterial cells did, however, adhere with varying degrees, to the different lipid species, with maximum adherence occurring with the free fatty acid. This observation was confirmed by preliminary uptake experiments using [14C]oleic acid. The interactive site for cell adherence may be the lipid-fibrillar layer associated with the cell surface of P. acnes, as discerned in electron microscopical studies. The findings of this investigation suggest that one function of the P. acnes lipase may be to aid colonization within the pilosebaceous follicle, by promoting cell adherence to components such as oleic acid.

A randomized, double-blind comparison of a clindamycin phosphate/benzoyl peroxide gel formulation and a matching clindamycin gel with respect to microbiologic activity and clinical efficacy in the topical treatment of acne vulgaris

BACKGROUND One approach to suppressing the overgrowth of antibiotic-resistant bacteria is to develop combination products composed of active constituents with complementary but distinct mechanisms of antibacterial action. OBJECTIVE The purpose of this study was to compare the antimicrobial and clinical efficacy and tolerability of clindamycin phosphate 1%/benzoyl peroxide 5% gel formulation with matching clindamycin 1% gel in the treatment of acne vulgaris. METHODS This 16-week, single-center, double-blind, randomized, parallel-group study compared the combination gel with clindamycin monotherapy applied BID in patients 13 to 30 years of age with mild to moderate acne and facial Propionibacterium acnes counts > or = 10(4) colony-forming units per square centimeter of skin. RESULTS Seventy-nine patients were enrolled and randomly assigned to receive the combination gel (n = 40) or clindamycin monotherapy (n = 39). Seventy patients (50 males, 20 females; mean age, 18.2 years) were included in the intent-to-treat group. The combination gel treatment produced significantly greater reductions (P < or = 0.046) from baseline in total lesion counts and in numbers of inflammatory lesions and comedones compared with clindamycin monotherapy. Greater reductions in the severity of acne also were observed in the physicians and patients Clinical Global Improvement scale scores and in other secondary efficacy measurements. Reductions in clindamycin-resistant P acnes counts were observed relative to baseline in the combination gel group; in contrast, P acnes counts increased by >1,600% in the clindamycin monotherapy group at week 16 (P = 0.018 vs combination gel). Reductions in inflammatory (r2 = 0.31; P = 0.016) and total (r2 = 0.28; P = 0.027) lesions were correlated with decreases in clindamycin-resistant bacteria. Also, significant correlations were observed between the percent change from baseline in total lesion counts (r2 = 0.44; P < 0.001) and comedo counts (r2 = 0.50; P < 0.001) and the log10 change from baseline in total P acnes counts. CONCLUSIONS The total P acnes count (P = 0.002) and the clindamycin-resistant P acnes count (P = 0.018) were significantly reduced after 16 weeks of treatment with combination gel compared with clindamycin monotherapy. These reductions in total P acnes and clindamycin-resistant P acnes counts correlated with reductions in total acne lesions.

Molecular analysis and expression of the lipase of Staphylococcus epidermidis

Lipase of Staphylococcus epidermidis 9 was purified from culture supernatant fluid. Two polypeptides (51 and 43 kDa) were detected by SDS-PAGE, of which the 43 kDa polypeptide reacted with anti-lipase serum. The S. epidermidis 9 lipase gene (gehC) was cloned in Escherichia coli and localized to a 2.1 kb sequence by subcloning and transposon mutagenesis. The nucleotide sequence of gehC (2064 nucleotides) was determined and the predicted amino acid sequence of the encoded lipase (77 kDa) identified. A 97 kDa lipase was detected in extracts of E. coli harbouring gehC and in post-exponential-phase culture supernatant fluids of S. epidermidis 9. Data presented indicate that the lipase behaves anomalously during SDS-PAGE and that a pro-lipase is proteolytically processed in cultures of S. epidermidis 9 during growth.

Inhibition of erythromycin‐resistant propionibacteria on the skin of acne patients by topical erythromycin with and without zinc

Summary Propionibacteria resistant to high concentrations of erythromycin [minimal inhibitory concentration (MIC)≥0·5 mg/ml) are now commonly isolated from the skin of antibiotic‐treated acne patients. This double‐blind study was carried out to assess the ability of 4% w·v erythromycin with and without 1–2% w/v nine acetate to reduce the numbers of erythromycin‐resistant propionibacteria in vivo, and also to monitor the acquisition of resistant strains de novo during therapy. Under laboratory conditions, erythromycin‐resistant propionibacteria were shown to be as sensitive to zinc acetate as fully sensitive strains. In vivo, the erythromycin/zinc complex and erythromycin alone produced highly significant reductions in total propionibacteria (P<0·01) and in the number of erythromycin‐resistant strains (P<0·01 at 8 weeks). After 12 weeks, resistant propionibacteria were re‐acquired, or acquired de novo. by three patients treated with erythromycin alone and four patients treated with the erythromycin/zinc complex. In contrast, changes in numbers of Micrococcaceae were slight and. after 12 weeks, erythromycin‐resistant strains were predominant in both treatment groups. In vitro MIC determinations suggested that this finding might be explained by the exceptionally high degree of erythromycin resistance displayed by some staphylococcal strains (MIC>4 mg/ml) and by the relative insensitivity of all staphylococcal strains to zinc acetate. Krythromycin with and without zinc was clinically effective, and both preparations produced significant reductions in acne grade, and inflamed and non‐inflamed lesion counts (F<0·001). In particular, 11 of 12 patients who harboured >103 c.f.u. erythromycin‐resistant propionibacteria/cm2 skin pretreatment (seven on the erythromycin/zinc complex and five on erythromycin alone) showed clinical improvement, with a>50% reduction in acne grade and/or lesion count. These results show that topical 4% w/v erythromycin with and without zinc eradicates erythromycin‐resistant propionibac‐teria in vivo, and is thus therapeutlcally effective in patients who harbour such strains.

Differential innate immune responses of a living skin equivalent model colonized by staphylococcus epidermidis or staphylococcus aureus

Staphylococcus epidermidis is a commensal on skin, whereas Staphylococcus aureus is a transient pathogen. The aim was to determine whether the skins innate defence systems responded differently to these microorganisms. Differential gene expression of a human skin equivalent (SE) model was assessed by microarray technology, in response to colonization by S. epidermidis or S. aureus. Only a small number of transcripts were significantly (P<0.0001) increased (12) or decreased (35) with gene expression changes of >2-fold on SEs colonized with S. epidermidis compared with controls (no colonization). Expression of one innate defence gene, pentraxin 3 (PTX3), was upregulated, while psoriasin, S100A12, S100A15, beta defensin 4, beta defensin 3, lipocalin 2 and peptidoglycan recognition protein 2 were downregulated. In contrast, large numbers of transcripts were significantly increased (480) or decreased (397) with gene expression changes of >2-fold on SEs colonized with S. aureus compared with controls. There was upregulation in gene expression of many skin defence factors including Toll-like receptor 2, beta defensin 4, properdin, PTX3, proinflammatory cytokines tumour necrosis factor-alpha, IL-1 alpha, IL-1 beta, IL-17C, IL-20, IL-23A and chemokines IL-8, CCL4, CCL5, CCL20 and CCL27. These differences may partly explain why S. epidermidis is a normal skin resident and S. aureus is not.

Propionibacterium acnes, a resident of lipid-rich human skin, produces a 33 kDa extracellular lipase encoded by gehA

Five independent clones of the Propionibacterium acnes P-37 lipase gene (gehA) were obtained in Escherichia coli, and the gene was localized to a 2.75 kb Xhol fragment by subcloning. The five clones were shown to contain the same gene by Southern blotting with a DIG-labelled probe to gehA. The nucleotide sequence of gehA was determined, and shown to contain a single ORF of 1017 kb, encoding a protein of 339 amino acids. The predicted molecular mass was 36 kDa. A 33 kDa (PAGE) radiolabelled polypeptide was detected from E. coli minicell preparations harbouring gehA, which could correspond to GehA after cleavage of the putative 26 amino acid residue signal peptide. gehA was overexpressed in E. coli under the control of the bacteriophage T7 promoter, and the corresponding polypeptide was found to be present in insoluble aggregates. Active lipase was produced when the overexpressing strain was incubated at a reduced temperature in the presence of sucrose. Purification of lipase from P. acnes culture supernatant fluids confirmed the production of a 33 kDa (PAGE) lipase.

Superior antibacterial action and reduced incidence of bacterial resistance in minocycline compared to tetracycline-treated acne patients

Twenty‐five previously untreated acne patients were monitored throughout a 6‐month course of therapy with either tetracycline or minocycline for changes in the numbers of staphylococci, propionibacteria and yeasts of the genus Malessezia on the skin surface. Antibiotic resistant staphylococci and propionibacteria were also counted. Minocycline (50 mg b.d.) produced a 10‐fold greater reduction in propionibacterial numbers compared to tetracycline (500 mg b.d.) after 12 (P < 0.02, t‐test) and 24 weeks (P < 0.05) of therapy. As treatment progressed, propionibacteria were replaced by yeasts, numbers of which were significantly increased by week 12 (P < 0.02) in tetracycline‐treated patients and by week 24 (P < 0.01) in minocycline‐treated patients. This suggests that yeasts have no role in the pathogenesis of acne but may compete with propionibacteria for the same niche. Overgrowth of antibiotic resistant staphylococci prevented any decrease in staphylococcal numbers in tetracycline‐treated patients, but minocycline produced a significant and sustained reduction in staphylococcal numbers after 1 week of therapy (P < 0.001). An increase in the number of multiply resistant ( 3 resistances) staphylococci occurred in 67% of tetracycline‐treated and 33% of minocycline‐treated patients by the end of the treatment period. There was no evidence of propionibacterial resistance in either treatment group. This study shows that minocycline has much greater antibacterial activity in vivo against both staphylococci and propionibacteria and produces less staphylococcal antibiotic resistance than tetracycline.