James G. McGovern

The concomitant development of poly(vinyl chloride)-related biofilm and antimicrobial resistance in relation to ventilator-associated pneumonia.

Ventilator-associated pneumonia is a major cause of death in intensive care patients and the endotracheal tube, commonly fabricated from poly(vinyl chloride) (PVC), is acknowledged as a significant factor in this. Bacteria colonise the biomaterial, thereby adopting a sessile mode of growth that progresses to the establishment of an antibiotic-resistant biofilm by the accretion of a protective glycocalyx. This study examined the sequential steps involved in the formation of biofilm on PVC by atomic force microscopy and the concomitant development of resistance to an antibiotic (ceftazidime) and to a non-antibiotic antimicrobial agent (hexetidine). Staphylococcus aureus and Pseudomonas aeruginosa isolated from ET tube biofilm were employed. The surface microrugosity of bacteria growing in sessile mode on PVC decreased significantly (p < 0.05) over the period 4, 24, 48 h and 5 d. The progressive accretion of bacterial glycocalyx was readily visualised in micrographs leading to a smoother surface topography with time. The minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) for ceftazidime and hexetidine against planktonic (suspension) S. aureus were lower than for Ps. aeruginosa. Furthermore, sessile populations of S. aureus and Ps. aeruginosa on PVC exhibited greater resistance to both ceftazidime and hexetidine when compared to planktonic bacterial growth. The efficacy of the agents, determined by kill kinetics, against sessile bacteria was dependent on age, with established biofilms (> or = 24 h) significantly more resistant (p < 0.05) than early sessile populations (< or = 4 h). Importantly, for practice, even newly colonised bacteria (1 h) were significantly more resistant to antibiotic than planktonic bacteria. Hexetidine was significantly more active (p < 0.05) than ceftazidime on biofilms of both isolates, irrespective of age, with total kill within 24 h treatment. Hexetidine may offer promise in the resolution of infection associated with PVC endotracheal tubes.

Role of physiological conditions in the oropharynx on the adherence of respiratory bacterial isolates to endotracheal tube poly(vinyl chloride)

Pneumonia is a major problem in intensive care patients and can be induced by pathogenic bacteria adhering to poly(vinyl chloride) (PVC) endotracheal (ET) tubes. This study examines the influence of surface properties on the adherence of the respiratory isolates Staphylococcus aureus and Pseudomonas aeruginosa to PVC. In particular, the influence of respiratory tract physiological conditions, 5% CO2 and saliva, on adherence was investigated. In general, decreased adherence to PVC was observed when bacteria were grown in CO2. When these CO2-grown bacteria were treated with saliva their adherence to PVC significantly increased; however, their adherence was significantly reduced to saliva-treated PVC. Treatment of both bacterial isolates with saliva decreased their negative zeta potential, a factor which may directly contribute to the observed increased microbial (saliva pretreated) adherence to PVC. Cell surface hydrophobicity (CSH) was evaluated by measuring the initial rates of microbial removal from a buffered aqueous phase, to ensure the absence of electrostatic interactions, to an organic phase (xylene). Under physiological conditions, CSH did not appear to be a dominant factor in biomaterial adherence as the CSH of S. aureus was decreased by saliva treatment but was unchanged for Ps. aeruginosa. Additionally, CSH also differed for the two isolates when grown in CO2, significantly decreasing with S. aureus but remaining unaltered with Ps. aeruginosa. Saliva treatment of PVC also decreased the advancing and receding contact angles of the biomaterial and its surface roughness, which may be a factor in the decreased adherence of saliva-treated bacteria to this surface. Alternative biomaterials or surface modifications appear necessary for the desired improvements in ET tube effectiveness. This study highlights the influence of physiological conditions on biomaterial and bacterial surface characteristics and subsequent interactions. It is imperative that the physiological conditions predominating in the clinical area of biomaterial use be considered when investigating device biocompatibility.

The effects of hexetidine (Oraldene) on the adherence of Candida albicans to human buccal epithelial cells in vitro and ex vivo and on in vitro morphogenesis.

AbstractPurpose. This study reports the effects of hexetidine (Oraldene™) on two virulence attributes of Candida albicans, namely,in vitro and ex vivoadherence of yeast cells to buccal epithelial cells (EEC) and in vitro morphogenesis. Methods. The effects of hexetidine treatment of either yeast cells (stationary and exponential phases) or BEC on Candidal adherence, in terms of viable and non-viable adherent yeast cells, were evaluated using an acridine orange stain in conjunction with fluorescence microscopy. Ex vivoanti-adherence effects were determined by rinsing BEC in vivo with hexetidine (0.1%), removal of BEC after defined periods and inclusion in the adherence assay. The effects of hexetidine on morphogenesis were evaluated using light microscopy. Yeast cell viability following exposure to a range of concentration of hexetidine (0.005-0.1 % v/v) for defined periods was determined following serial dilution and enumeration on solid media. Results. Treatment of stationary and exponential phase yeast cells or BEC with hexetidine (0.1%) for a range of times (10−300 s) or, alternatively, with a range of concentrations of hexetidine (0.005−0.1 %) for a fixed time (30s) significantly decreased the resultant Candidal/ epithelial adhesion. No correlations were observed between reduced adherence and either time of treatment or hexetidine concentration. In vivotreatment of BEC with hexetidine (0.1%) for 30s resulted in prolonged and significant reductions in the ex vivo adherence of both viable and non-viable yeast cells for periods of up to (and including) four hours post-rinsing. Treatment of C. albicans blastospores with hexetidine (0.05, 0.1% v/v) for 10s and 30s totally inhibited Candida morphogenesis, whereas treatment with lower antiseptic concentrations significantly reduced the extent of Candida morphogenesis and the rate of hyphal development. The effects of hexetidine on yeast cell viability were both concentration and time-dependent. Conclusions. The reduced adherence of C. albicans to BEC and the modification or inhibition of morphogenesis following exposure to hexetidine suggests a clinical role for hexetidine in the prophylaxis of both superficial candidosis and the systemic complications resulting from invasion of sub-epithelial tissue.

Conditioning film and environmental effects on the adherence of Candida spp. to silicone and poly(vinylchloride) biomaterials.

The reported incidence of colonization of oropharyngeal medical devices with Candida spp. has increased in recent years, although few studies that have systematically examined the adherence of yeast cells to such biomaterials, the primary step in the process of colonization. This study, therefore, examined the effects of oropharyngeal atmospheric conditions (5% v/v carbon dioxide) and the presence of a salivary conditioning film on both the surface properties and adherence of Candida albicans, Candida krusei and Candida tropicalis to PVC and silicone. Furthermore, the effects of the salivary conditioning film on the surface properties of these biomaterials are reported. Growth of the three Candida spp. in an atmosphere containing 5% v/v CO2 significantly increased their cell surface hydrophobicity and reduced the zeta potential of C. albicans and C. krusei yet increased the zeta potential of C. tropicalis (p<0.05). Furthermore, growth in 5% v/v CO2 decreased the adherence of C. tropicalis and C. albicans to both PVC and silicone, however, increased adherence of C. krusei (p<0.05). Pre-treatment of the microorganisms with pooled human saliva significantly decreased their cell surface hydrophobicity and increased their adherence to either biomaterial in comparison to yeast cells that had been pre-treated with PBS (p<0.05). Saliva treatment of the microorganisms had no consistent effect on microbial zeta potential. Interestingly, adherence of the three, saliva-treated Candida spp. to saliva-treated silicone and PVC was significantly lower than whenever the microorganisms and biomaterials had been treated with PBS (p<0.05). Treatment of silicone and PVC with saliva significantly altered the surface properties, notably reducing both the advancing and receding contact angles and, additionally, the microrugosity. These effects may contribute to the decreased adherence of saliva-treated microorganisms to these biomaterials. In conclusion, this study has demonstrated the effects of physiological conditions within the oral cavity on the adherence of selected Candida spp. to biomaterials employed as oropharyngeal medical devices. In particular, this study has ominously shown that these materials act as substrates for yeast colonization, highlighting the need for advancements in biomaterial design. Furthermore, it is important that physiological conditions should be employed whenever biocompatibility of oropharyngeal biomaterials is under investigation.

CONDITIONING FLUID INFLUENCES ON THE SURFACE PROPERTIES OF SILICONE AND POLYURETHANE PERITONEAL CATHETERS: IMPLICATIONS FOR INFECTION

Catheter-related infection remains a considerable problem in continuous ambulatory peritoneal dialysis (CAPD). This study examined the adherence of clinical isolates of Staphylococcus epidermidis to commercially available polyurethane and silicone peritoneal catheters in the presence and absence of a proteinaceous conditioning film. In addition, the effects of the conditioning film on the surface properties (advancing and receding contact angles, and surface rugosity) of these biomaterials were investigated. Bacterial adherence to polyurethane and silicone catheters, pre-treated with phosphate-buffered saline (PBS) or artificial spent peritoneal dialysate (ASD) for 1 h at 37°C, was examined using a radiometric (2-3H-adenine) adherence assay. The advancing and receding contact angles and the surface rugosity of ASD- and PBS-treated biomaterials were examined using a dynamic contact angle analyser and an atomic force microscope, respectively. The bacterial isolates were selected to represent high and low cell surface hydrophobicity. The hydrophobic isolate exhibited both a significantly greater rate and a significantly greater extent of adherence than the hydrophilic isolate to both catheter materials, independent of pre-treatment. In general, pre-treatment of the catheter materials with ASD significantly decreased the subsequent adherence of both isolates owing to the deposition of a conditioning film on the surface of the biomaterial. ASD treatment also decreased both the advancing and receding contact angles and the surface rugosity of both catheter materials. This study highlights the influence of both bacterial cell surface hydrophobicity and biomaterial surface conditioning films on bacterial adherence to CAPD catheters. In addition, it is recommended that the effects of proteinaceous conditioning films on biomaterial surface properties should be considered when assessing materials for medical devices and products.

Modification of biomaterial surface characteristics by body fluids in vitro

Abstract In practice, polyvinyl chloride endotracheal tubes and polyurethane urinary catheters are located in areas where they are exposed to the conditioning fluids saliva and urine, respectively. Samples of both biomaterials were incubated in these conditioning fluids and, following treatment, dynamic contact angle measurement and surface roughness assessment by atomic force microscopy were used to analyse surface characteristics. Over a 24 h period of contact with the conditioning fluids, the surface of both materials became significantly more hydrophilic (p

Frequency distribution of Candida albicans blastospores adhered to mucosal epithelial cells in-vitro

Although several methods are available for examination of microbial adherence to epithelial cells, these do not distinguish between adherence of viable and non‐viable micro‐organisms. This study reports the use of acridine orange‐stained blastospores of Candida albicans in conjunction with direct epifluorescence microscopy to determine viable (orange‐fluorescing) and non‐viable (green‐fluorescing) blastospore adherence to buccal epithelial cells. The method was also employed to examine the effects of chlorhexidine treatment at sub‐minimum inhibitory concentrations on the adherence of viable and non‐viable blastospores.

Determination of the salivary retention of hexetidine in-vivo by high-performance liquid chromatography.

The non‐antibiotic antimicrobial agent hexetidine is widely used at a concentration of 0–1% w/v as an oral rinse to reduce the number of viable microorganisms within the oral cavity. However, following use, the available concentration of hexetidine in the oral cavity declines with time, thus compromising the resultant antimicrobial activity. It is, therefore, desirable to determine the persistence of the agent in the oral cavity by quantification of the drug concentration in saliva, thus enabling prediction of its antimicrobial activity in the oral environment.

The influence of a non-ionic surfactant on the adherence of Candida albicans to human buccal epithelial cells

The initiation of oral candidosis involves adherence of the yeast Cnndidn nlhrcnns t o human buccal epithelial cells (BECs). Antimicrobial agents, such as hexetidine, in the form of an oral rinse can reduce yeast adherence to BECs and may prevent oral infection (Jones et a1 1997). The successful formulation of a mouth rinse incorporating an antimicrobial agent of limited solubility may require the inclusion o f a surfactant, one such non-ionic surfactant being polyoxyethylene sorbitan monooleate (Tween 80). In order to investigate whether the inclusion of surfactant in oral antimicrobial formulations influences yeast-BEC interaction, this study examined the effect of Tween 80 on the in vitro adherence of C. albicnns to BECs. using Wilcoxon signed rank test, p < 0.05 denoting significance.

Physical characterization of non-aqueous gels incorporating tetracycline hydrochloride for treatment of periodontitis

Periodontitis is & inflammatory disease of the periodontal tissues supporting the teeth. The management of the condition generally involves the removal of dental plaque in conjunction with use of antimicrobial agents, e.g. tetracycline (Jones el a1 1996). Drug delivery systems designed for use in the periodontal pocket include non-aqueous antimicrobial gels. In this study, the rheological and drug release properties of novel viscoelastic non-aqueous gels containing tetracycline hydrochloride (TH), designed for the treatment of periodontitis, were evaluated.