Microbial colonization dynamics of the axillae of an individual over an extended period.
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Actinomycetales↗
Biomedical subjects
Keith T Holland
Publications and source records attributed to Keith T Holland.
The hyaluronate lyase of Staphylococcus aureus - a virulence factor?
The hyaluronate lyase (HL) gene of Staphylococcus aureus 8325-4 (hysA) was inactivated in vitro with the insertion of the erythromycin determinant, ermC, from plasmid pE194. The hysA : : ermC mutation was introduced into S. aureus via a temperature-sensitive shuttle vector, where it underwent homologous recombination with the wild-type (w.t.) allele. The insertion of ermC in the chromosomal hysA locus was confirmed by Southern blot hybridization and the loss of HL activity was demonstrated macroscopically by a plate assay. The importance of HL for pathogenicity was assessed by comparing the virulence of the HL(-) mutant strain to that of the w.t. in an established mouse abscess model of S. aureus infection. A significantly higher cell recovery was obtained from lesions infected with the w.t. strain compared to the lesions infected with the HL(-) strain (P =0.01). Although the lesion areas from both groups were not significantly different (P=0.9) they were of different morphology. A colorimetric assay was used to measure HL activity from culture supernatants of the S. aureus 8325-4 strains w.t., WA250 (agr) and PC1839 (sar) grown in a chemically defined medium. HL activity reached a maximum in the w.t. strain during mid-exponential phase (t=5 h) and while it showed a 16-fold decrease in the agr mutant it increased 35-fold in the sar mutant background. These results strongly suggest that HL is a virulence factor which is important in the early stages of subcutaneous infections.
Abscess↗
The effect of cell density and specific growth rate on accessory gene regulator and toxic shock syndrome toxin-1 gene expression in Staphylococcus aureus.
A continuous culture technique utilising a variant of Staphylococcus aureus 8325-4 containing transcriptional gene fusions was used to investigate the relationships between cell density (OD(600)), steady-state specific growth rate (mu) and expression of both agr (accessory gene regulator) and tst (toxic shock syndrome toxin-1). The expression of these genes was assessed by two single-copy independently arranged chromosomal-based reporter systems, beta-galactosidase agr-P3 promoter fusion and a lux-tst promoter fusion. Cell density and specific agr expression were found to be positively correlated. In the model, the minimum cell density predicted to promote specific agr expression was an OD(600) of 0.14, equivalent to 1.2x10(8) CFU ml(-1). No direct relationship between cell density and specific tst expression was detected. Specific expressions of agr and tst were not correlated with specific growth rate and there appeared to be no direct link between agr and tst specific expression. The results support the hypothesis that agr is a functional unit of quorum sensing and that the amount of specific expression of tst is modulated independently of agr.
Antigens, Bacterial↗
Is the GehD lipase from Staphylococcus epidermidis a collagen binding adhesin?
The opportunistic human pathogen Staphylococcus epidermidis is the major cause of nosocomial biomaterial infections. S. epidermidis has the ability to attach to indwelling materials coated with extracellular matrix proteins such as fibrinogen, fibronectin, vitronectin, and collagen. To identify the proteins necessary for S. epidermidis attachment to collagen, we screened an expression library using digoxigenin-labeled collagen as well as two monoclonal antibodies generated against the Staphylococcus aureus collagen-adhesin, Cna, as probes. These monoclonal antibodies recognize collagen binding epitopes on the surface of S. aureus and S. epidermidis cells. Using this approach, we identified GehD, the extracellular lipase originally found in S. epidermidis 9, as a collagen-binding protein. Despite the monoclonal antibody cross-reactivity, the GehD amino acid sequence and predicted structure are radically different from those of Cna. The mature GehD circular dichroism spectra differs from that of Cna but strongly resembles that of a mammalian cell-surface collagen binding receptor, known as the alpha(1) integrin I domain, suggesting that they have similar secondary structures. The GehD protein is translated as a preproenzyme, secreted, and post-translationally processed into mature lipase. GehD does not have the conserved LPXTG C-terminal motif present in cell wall-anchored proteins, but it can be detected in lysostaphin cell wall extracts. A recombinant version of mature GehD binds to collagens type I, II, and IV adsorbed onto microtiter plates in a dose-dependent saturable manner. Recombinant, mature GehD protein and anti-GehD antibodies can inhibit the attachment of S. epidermidis to immobilized collagen. These results provide evidence that GehD may be a bi-functional molecule, acting not only as a lipase but also as a cell surface-associated collagen adhesin.
Adhesins, Bacterial↗
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 physician's and patient's 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.
Acne Vulgaris↗
Cosmetics: what is their influence on the skin microflora?
Human skin has a resident, transient and temporary resident microflora. This article considers the possibilities of topical products influencing the balance of the microflora. The resident micro-organisms are in a dynamic equilibrium with the host tissue and the microflora may be considered an integral component of the normal human skin. The great majority of these micro-organisms are gram-positive and reside on the skin surface and in the follicles. The host has a variety of structures, molecules and mechanisms which restrict the transient and temporary residents, as well as controlling the population and dominance of the resident group. These include local skin anatomy, hydration, nutrients and inhibitors of various types. The resident microflora is beneficial in occupying a niche and denying its access to transients, which may be harmful and infectious. Also, the residents are important in modifying the immune system. In the healthy host the microflora causes few and temporary problems. Therefore, it is of interest that topical products have little or no effect on the ecology of the microflora. A range of mechanisms by which long-term use of cosmetics may influence the microflora are considered. Although the risks associated are low, it is argued that it is necessary to monitor these changes in ecology and use technologies of modeling and bioinformatics to predict outcomes, whether good, neutral or of concern.
Cosmetics↗
Identification of a second lipase gene, gehD, in Staphylococcus epidermidis: comparison of sequence with those of other staphylococcal lipases.
The identification and molecular characterization of a previously unidentified lipase, gehD, from the human cutaneous commensal Staphylococcus epidermidis is reported. A lipase-GehC-deficient but otherwise isogenic mutant of S. epidermidis 9 was constructed by allele replacement. However, the mutant was found to retain 50% of the wild-type lipase activity in liquid culture. Rescreening of a genomic library revealed the presence of a second lipase gene, gehD, which was subsequently mapped and sequenced. In common with other staphylococcal lipases, GehD appeared to be translated as a 650-700 amino acid precursor which is processed post-translationally to an extracellular mature lipase of 360 amino acids with a size of approximately 45 kDa. Comparison of the amino acid sequence of GehD with those of other staphylococcal lipases revealed a high level of conservation between the mature lipase domains of different species. By hybridization studies, both gehC and gehD genes were found to be present in S. epidermidis isolates from both clinical and non-clinical backgrounds, but neither hybridized to DNA isolated from other staphylococcal strains. Construction of a phylogenetic tree and calculation of amino acid sequence homologies between mature lipases, however, suggested that the lipases of S. epidermidis may be more closely related to those of Staphylococcus aureus than to each other.
Amino Acid Sequence↗
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.
Amino Acid Sequence↗
Acne and Propionibacterium acnes.
The involvement of microorganisms in the development of acne has a long and checkered history. Just over 100 years ago, Propionibacterium acnes (then known as Bacillus acnes) was isolated from acne lesions, and it was suggested that P. acnes was involved in the pathology of the disease. The 1960s saw the use of antibiotics to treat acne, and the consequent clinical success combined with reductions in P. acnes gave new impetus to the debate. Over the past two decades, the inevitable emergence of antibiotic-resistant strains of P. acnes as a consequence of acne therapy not only has reopened the debate as to the role of P. acnes in acne, but also has created some serious health care implications.
Acne Vulgaris↗
Clinical and bacteriological evaluation of nadifloxacin 1% cream in patients with acne vulgaris: a double-blind, phase III comparison study versus erythromycin 2% cream.
The aim of this double-blind, multinational, phase III study was to investigate the clinical and bacteriological efficacy of nadifloxacin 1% cream compared with erythromycin 2% cream in 474 European patients with predominantly inflamed slight-to-moderate acne vulgaris. During 12 weeks of treatment both nadifloxacin and erythromycin caused significant reduction in the number of inflamed papulo-pustular lesions (66.7% and 64.7%, respectively) and open and closed comedones. The microbiological evaluation showed a significant reduction of coagulase negative staphylococci (CNS) only in the nadifloxacin group, while Propionibacterium acnes was significantly reduced by both formulations. A significantly higher resistance and the extent of resistant of P. acnes and CNS against erythromycin compared to nadifloxacin were also evidenced. All adverse events reported were minor in both groups. This pivotal erythromycin-controlled study has demonstrated that nadifloxacin 1% cream was as efficacious and safe as erythromycin 2% and extremely low numbers of nadifloxacin-resistant microorganisms were detected in the treatment period.
Acne Vulgaris↗