A novel approach to antibiofilm susceptibility testing using a thermo-reversible matrix.

OBJECTIVE: Biofilm microorganisms are known to have a much higher tolerance to antimicrobials compared to their planktonic equivalents. Therefore, traditional antimicrobial susceptibility testing may not extrapolate to clinical treatment of infections of biofilm origin, and as a result, there is a need to not only develop antimicrobials with antibiofilm activity, but also suitable in vitro testing methods for their evaluation. In this study, we report on a novel method of antibiofilm testing using a thermo-reversible matrix (poloxamer 407), coupled with live/dead staining of bacteria cultured from the matrix. METHOD: Pseudomonas aeruginosa (NCIMB 8626) was cultured in medium containing poloxamer 407 at 37°C for 24 hours to generate biofilms. The preparation was cooled to liquefy the poloxamer and allow recovery of the biofilm cells, which were then stained with SYTO9 to determine viability following exposure to four antimicrobials: polyhexanide, octenadine dihydrochloride, povidone-iodine and silver carbonate. Over an 8-minute time period, fluorescence levels were spectrophotometrically measured and compared with bacterial controls, cultured in the absence of poloxamer and without antimicrobial. RESULTS: Untreated cells showed no reduction in viability over this period. Importantly, planktonic cells were more susceptible to test agents compared with those of a 'biofilm' phenotype cultured in poloxamer. Antibiofilm activity was evident for all of the test agents, with highest relative activity seen with octenadine dihydrochloride. CONCLUSION: In summary, a novel and relatively rapid approach to screen compounds for antibiofilm activity has been described. The method uses standard laboratory equipment and can be readily adapted to test a wide range of microorganisms and other antibiofilm compounds. DECLARATION OF INTEREST: This research was, in part, supported by Advanced Medical Solutions in the form of a Knowledge Transfer Project. Mr J. Nosworthy was employed by Advanced Medical Solutions. There are no other conflicts of interests to declare.

Antimicrobial activity of Citrox bioflavonoid preparations against oral microorganisms.

BACKGROUND: Citrox is a formulation of soluble bioflavonoids obtained from citrus fruits. The non-toxic and antimicrobial properties of natural bioflavonoids are well documented, and consequently there has been interest in the therapeutic application of these substances. OBJECTIVE: To determine the antimicrobial activity of two Citrox formulations (BC30 and MDC30) with different bioflavonoid combinations against a range of oral microorganisms. METHODS: The antimicrobial activity of both formulations was tested against 14 bacterial species and six Candida species. The two Citrox formulations (dilution range 0.007-8% v/v) were firstly evaluated by determining the in vitro Minimal Inhibitory Concentration (MIC) against planktonic microorganisms in a broth microdilution assay. Secondly, the ability of the same serial dilutions to inhibit microbial growth was assessed in a modified microtitre biofilm assay. RESULTS: Both Citrox formulations exhibited antimicrobial activity. The BC30 formulation demonstrated greater activity than MDC30 and significantly inhibited growth of all bacterial species and most candidal species tested at a concentration of 1% (v/v) in both the broth and the biofilm assay. CONCLUSION: Bioflavonoid preparations of Citrox have a broad-spectrum of antimicrobial activity against oral microorganisms, and as such have the potential to be used within therapeutic preparations for the control of the oral microflora.

A pilot study of bioaerosol reduction using an air cleaning system during dental procedures.

BACKGROUND: Bioaerosols are defined as airborne particles of liquid or volatile compounds that contain living organisms or have been released from living organisms. The creation of bioaerosols is a recognized consequence of certain types of dental treatment and represents a potential mechanism for the spread of infection. OBJECTIVES: The aims of the present study were to assess the bioaerosols generated by certain dental procedures and to evaluate the efficiency of a commercially available Air Cleaning System (ACS) designed to reduce bioaerosol levels. METHODS: Bioaerosol sampling was undertaken in the absence of clinical activity (baseline) and also during treatment procedures (cavity preparation using an air rotor, history and oral examination, ultrasonic scaling and tooth extraction under local anaesthesia). For each treatment, bioaerosols were measured for two patient episodes (with and without ACS operation) and between five and nine bioaerosol samples were collected. For baseline measurements, 15 bioaerosol samples were obtained. For bioaerosol sampling, environmental air was drawn on to blood agar plates using a bioaerosol sampling pump placed in a standard position 20 cm from the dental chair. Plates were incubated aerobically at 37°C for 48 hours and resulting growth quantified as colony forming units (cfu/m³). Distinct colony types were identified using standard methods. Results were analysed statistically using SPSS 12 and Wilcoxon signed rank tests. RESULTS: The ACS resulted in a significant reduction (p = 0.001) in the mean bioaerosols (cfu/m³) of all three clinics compared with baseline measurements. The mean level of bioaerosols recorded during the procedures, with or without the ACS activated respectively, was 23.9 cfu/m³ and 105.1 cfu/m³ (p = 0.02) for cavity preparation, 23.9 cfu/m³ and 62.2 cfu/m³ (p = 0.04) for history and oral examination; 41.9 cfu/m³ and 70.9 cfu/m³ (p = 0.01) for ultrasonic scaling and 9.1 cfu/m³ and 66.1 cfu/m³ (p = 0.01) for extraction. The predominant microorganisms isolated were Staphylococcus species and Micrococcus species. CONCLUSION: These findings indicate potentially hazardous bioaerosols created during dental procedures can be significantly reduced using an air cleaning system.

The role of gonadectomy and testosterone replacement on thymic luteinizing hormone-releasing hormone production.

We and others have identified luteinizing hormone-releasing hormone (LHRH) in cells of the immune system in both animals and humans. LHRH is an immunostimulant, and testosterone is an immunosuppressant. Because testosterone is known to modulate the concentrations of hypothalamic LHRH, we wondered whether testosterone might also alter the concentrations of rat thymic LHRH. Two weeks after castration or sham castration, adult male rats were implanted with either vehicle or testosterone capsules. All animals were killed 4 days after capsule implantation. Thymic LHRH concentration increased significantly in castrated animals. Testosterone replacement prevented this increase. The concentration of the LHRH precursor, proLHRH, decreased significantly, but testosterone replacement prevented this decrease. Steady-state concentrations of LHRH mRNA were not changed by castration or by hormonal replacement. In contrast to the post-castration increase in thymic LHRH, LHRH content of the hypothalamus decreased significantly. Whereas concentrations of LHRH were lower in the thymus than in the hypothalamus, proLHRH concentrations were much greater in the thymus. These data suggest that gonadal manipulation modulates LHRH molecular processing and its tissue concentration in the thymus in addition to those in the hypothalamus, and that the regulation of LHRH molecular processing by testosterone in the hypothalamus is different from that in the thymus.

The effect of castration on steady state levels of luteinizing hormone-releasing hormone (LHRH) mRNA and proLHRH processing: time course study utilizing semi-quantitative reverse transcription/polymerase chain reaction.

Many studies have consistently shown that castration induces a prompt increase in serum levels and pituitary content of the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), as well as a concomitant rise in steady state levels of the messenger RNAs directing their synthesis. The reports of effects of castration on the overall physiology of hypothalamic luteinizing hormone-releasing hormone (LHRH)--steady state levels of LHRH mRNA, post-translational processing and secretion--have, however, not been consistent. The goal of the studies reported here was to provide the first analysis of the effect of castration, at multiple postoperative time points, on steady state levels of LHRH mRNA and on the levels of hypothalamic proLHRH. All these data are correlated with hypothalamic levels of the mature LHRH decapeptide and with serum and pituitary levels of immunoreactive LH and FSH. Adult male rats were either castrated or sham-castrated (controls) and then sacrificed at 1, 3, 5, 7, 14, 21 or 28 days postoperatively. As expected, there was a prompt and sustained rise in serum immunoreactive LH and FSH in castrates compared with sham-operated animals. Intra-pituitary LH levels rose above levels in the sham-operated animals by 14 days post castration. Intra-pituitary FSH showed a biphasic response, first falling significantly below control levels, then rising above control levels at 21 days. Steady state levels of LHRH mRNA in castrates, measured by reverse transcription/polymerase chain reaction, were increased about 2-fold above control levels by 1 day postoperatively, but were virtually identical to control levels at each of the other time points despite marked changes in the gonadotropins. ProLHRH content in castrates was 1.8-times that seen in controls at 1 day post castration (P<0.05), concomitant with the rise in steady state levels of LHRH mRNA at that time point. However, proLHRH content in castrates was no different from that seen in controls at each of the later time points examined. LHRH content was unchanged through 7 days after castration, but then fell significantly to 57% of control levels in hypothalami from animals gonadectomized 14 to 21 days previously (P<0.001 vs control), and to 54% of sham-operated levels at 28 days postoperatively (P<0.001). We conclude that: (1) changes in steady state levels of LHRH mRNA after castration are small and transient and (2) increased proLHRH coupled with unchanged LHRH levels at 1 day post castration, and castrate animal proLHRH at control levels coupled with falling LHRH at later post-castration time points indicate that the effect of gonadectomy on post-translational processing of proLHRH to LHRH is, likewise, small and transient. In aggregate our data suggest that most of the increase in serum LH and FSH seen in male rats after castration is not mediated at the hypothalamic level.

Intra-epidermal carcinoma of the eyelid margin.

Twenty-two cases of intra-epidermal carcinoma of the ciliary margin have been diagnosed in a 15-year survey. The clinical appearance is variable, resembling in some instances a benign warty lesion, and in others a fully developed squamous cell carcinoma. As the lesion grows it produces keratotic plugging of the lash follicles and nodules on the lid margin; in due course this results in loss of the related cilia. When dermal invasion occurs the resultant squamous cell carcinoma is potentially dangerous because of the involvement of the adnexal structures, which on the ciliary margins are particularly large and penetrate deeply into the lid substance. Adequate biopsy which includes at least one lash follicle is essential for accurate diagnosis, and treatment requires complete excision with a reasonable margin for safety. Of the twenty-two patients, nineteen were male and three female; fourteen of the men involved had handled oils and greases for prolonged periods.