Geoff Hanlon

Action of Disinfectant Quaternary Ammonium Compounds against Staphylococcus aureus

ABSTRACT Mode-of-action studies concluded that alkyldimethylbenzylammonium chloride (ADBAC) (a blend of C12, C14 and C16 alkyl homologues) and didecyldimethylammonium chloride (DDAC) are both membrane-active agents, possessing subtly different modes of action reflecting early cell interactions against Staphylococcus aureus. ADBAC and DDAC exhibited similar MIC behaviors from 0.4 ppm to 1.8 ppm over an inoculum range of 1 × 105 to 1 × 109 CFU/ml at 35°C. For ADBAC and DDAC, an increased rapidity of killing against S. aureus (final concentration, 2 × 109 CFU/ml) was observed at 35°C compared to 25°C. Concentration exponents (η) for killing were <2.5 for both agents, and temperature influenced the η value. Examination of leakage and kill data suggested that a single leakage marker was not indicative of cell death. ADBAC and DDAC possessed Langmuir (L4) and high-affinity (H3/4) uptake isotherms, respectively. ADBAC molecules formed a single monolayer of coverage of cells at the end of primary uptake, and DDAC formed a double monolayer. Rapid cell leakage occurred at bactericidal concentrations, with total depletion of the intracellular potassium and 260-nm-absorbing pools released in this strict order. Autolysis was observed for ADBAC and DDAC at concentrations of 9 μg/ml (0.0278 mM and 0.0276 mM, respectively) and above, together with the depletion of approximately 30% of the internal potassium pool. Autolysis contributed to ADBAC and DDAC lethality, although high biocide concentrations may have inhibited autolytic enzyme activity.

The synergistic effect of EDTA/antimicrobial combinations on Pseudomonas aeruginosa

Aims:  To demonstrate that the nonlinear concentration‐dependent inhibition of Pseudomonas aeruginosa to EDTA can be used to successfully model and predict the potentiation of antimicrobials by EDTA.

Triclosan-tolerant bacteria: changes in susceptibility to antibiotics

There is no clear consensus regarding the effect of biocide tolerance on antibiotic susceptibility. In this work, triclosan-tolerant strains of Escherichia coli, Staphylococcus aureus and Acinetobacter johnsonii were compared with sensitive strains in order to ascertain their susceptibility to a range of antibiotics. The minimum inhibitory concentrations of triclosan were measured using broth- and agar-dilution techniques. Antibiotic susceptibilities were determined using the British Society for Antimicrobial Chemotherapy guidelines. No triclosan-tolerant strains were resistant to antibiotics, and there was no overall tendency for triclosan-tolerant strains to have significantly smaller zones of inhibition compared with counterpart strains. Triclosan-tolerant strains of E. coli were significantly more susceptible to aminoglycoside antibiotics. The mechanism by which E. coli develops tolerance to triclosan appears to be linked to aminoglycoside susceptibility. It is proposed that changes in outer membrane, or the loss of plasmids, may be responsible for this relationship.

The emergence of multidrug resistant Acinetobacter species: a major concern in the hospital setting

Acinetobacter spp. have emerged in recent years as a major cause of nosocomial infections that are associated with significant morbidity and mortality. Developing resistance patterns have prompted the suggestion that we are closer to the end of the antibiotic era with Acinetobacter than with methicillin‐resistant Staphylococcus aureus (J Hosp Infect58, 2004, 167).

A rapid method for assessing the suitability of quenching agents for individual biocides as well as combinations

Aims: To develop a novel, rapid method for testing the ability of quenching agents to neutralize disinfectants.

Long-term persistence of a single Legionella pneumophila strain possessing the mip gene in a municipal shower despite repeated cycles of chlorination

The ability of Legionella pneumophila to colonise domestic water systems is a primary cause of outbreaks of Legionnaires disease in humans. World Health Organization guidelines recommend that drinking water is chlorinated to between 0.2 and 1mg/L [Chlorine in drinking-water. Guidelines for drinking-water quality, 2nd edn. Geneva: World Health Organization; 1996], but L. pneumophila is repeatedly isolated from chlorinated water systems, indicating that this treatment is not effective at preventing colonisation. Current UK guidelines recommend a one-off treatment of 20-50mg/L of free chlorine to remove the bacteria. In this study we report on the persistence of L. pneumophila serogroup 1 in a domestic shower system despite repeated cycles of chlorination at 50mg/L for 1h exposure time, over the course of two and a half years. Persisting isolates were subjected to in-vitro phenotypic analyses and polymerase chain reaction analysis for the toxin-encoding mip gene. Random amplified polymorphic DNA typing was also performed to determine whether the isolates recovered on different occasions were the same strain. We found that seven isolates of L. pneumophila recovered over a two-and-a-half year period are the same genetically defined strain, indicating that the bacteria can persist despite repeated cycles of chlorination after each successive isolation.

The development of in vitro biocompatibility tests for the evaluation of intraocular biomaterials.

Recent developments in ocular implant technology require the in vitro evaluation of ocular compatibility in early stage development programs. This requires an understanding and appreciation of the biological interactions which occur in the ocular environment and their relevance with respect to the clinical complications associated with surgical implantation of devices. This paper describes the development of a series of clinically reflective in vitro assays for assessing the potential ocular compatibility of novel intraocular lens materials. Staphylococcus epidermidis attachment, fibrinogen adsorption, mouse embryo fibroblast 3T3 adhesion and proliferation, primary rabbit lens cell adhesion, human peripheral blood macrophage adhesion and granulocyte activation tests were employed to evaluate two widely used intraocular biomaterials poly(methyl methacrylate) (PMMA) and silicone, and a novel biomimetic phosphorylcholine-based coating (PC). The performance of these materials in the in vitro assays was compared to their ability to reduce postoperative inflammation in vivo in a rabbit model. The results demonstrated that the in vitro assays described here are predictive of in vivo ocular compatibility. These assays offer a more relevant means of assessing the ocular compatibility of biomaterials than those presently required by the authorities for regulatory approval of medical devices and implants.

Elucidating the genetic basis of crystalline biofilm formation in Proteus mirabilis.

ABSTRACT Proteus mirabilis forms extensive crystalline biofilms on urethral catheters that occlude urine flow and frequently complicate the management of long-term-catheterized patients. Here, using random transposon mutagenesis in conjunction with in vitro models of the catheterized urinary tract, we elucidate the mechanisms underpinning the formation of crystalline biofilms by P. mirabilis. Mutants identified as defective in blockage of urethral catheters had disruptions in genes involved in nitrogen metabolism and efflux systems but were unaffected in general growth, survival in bladder model systems, or the ability to elevate urinary pH. Imaging of biofilms directly on catheter surfaces, along with quantification of levels of encrustation and biomass, confirmed that the mutants were attenuated specifically in the ability to form crystalline biofilms compared with that of the wild type. However, the biofilm-deficient phenotype of these mutants was not due to deficiencies in attachment to catheter biomaterials, and defects in later stages of biofilm development were indicated. For one blocking-deficient mutant, the disrupted gene (encoding a putative multidrug efflux pump) was also found to be associated with susceptibility to fosfomycin, and loss of this system or general inhibition of efflux pumps increased sensitivity to this antibiotic. Furthermore, homologues of this system were found to be widely distributed among other common pathogens of the catheterized urinary tract. Overall, our findings provide fundamental new insight into crystalline biofilm formation by P. mirabilis, including the link between biofilm formation and antibiotic resistance in this organism, and indicate a potential role for efflux pump inhibitors in the treatment or prevention of P. mirabilis crystalline biofilms.

The rapid and specific real-time detection of Legionella pneumophila in water samples using Optical Waveguide Lightmode Spectroscopy

The detection of Legionella pneumophila in water samples using standard microbiological culture techniques is both prolonged and problematic. The bacterium is slow-growing and nutritionally fastidious, such that other indigenous species can out-compete the Legionella even when using antibiotic supplemented media. Optical Waveguide Lightmode Spectroscopy (OWLS) is a real-time analytical system whereby a change to a higher coupling angle where the refractive index of a bacterial cell is higher than that of the covering medium. In this study an aqueous suspension of L. pneumophila was passed across the surface of waveguides functionalised with a specific anti-Legionella antibody. The binding between the bacterial cells and the antibody specific for that cell resulted in an increase in the refraction indices of the transverse electric and transverse magnetic photoelectric currents. We report the optimisation of a rapid and sensitive (1.3 x 10(4) CFU mL-1) detection method for L. pneumophila contamination in a water sample in less than 25 min. This is a significant reduction in the time taken to determine the presence of the bacterium which with conventional techniques normally takes up to fourteen days. In addition, the specificity of the technique to L. pneumophila was demonstrated. The OWLS results were validated by conventional microbiology screening and atomic force microscopy of the surface of the waveguide, showing its species specificity and potential applications in environmental and clinical analysis.

Dosing regimen of meropenem for adults with severe burns: a population pharmacokinetic study with Monte Carlo simulations

OBJECTIVES To develop a population model to describe the pharmacokinetics (PK) of intravenous meropenem in adult patients with severe burns and investigate potential relationships between dosage regimens and antimicrobial efficacy. PATIENTS AND METHODS A dose of 1 g every 8 h was administered to adult patients with total body surface area burns of ≥15%. Doses for subsequent courses were determined using results from the initial course and the patients clinical condition. Five plasma meropenem concentrations were typically measured over the dosage interval on one to four occasions. An open, two-compartment PK model was fitted to the meropenem concentrations using NONMEM and the effect of covariates on meropenem PK was investigated. Monte Carlo simulations investigated dosage regimens to achieve a target T>MIC for ≥40%, ≥60% or ≥80% of the dose interval. RESULTS Data comprised 113 meropenem concentration measurements from 20 dosage intervals in 12 patients. The parameters were CL (L/h) = 0.196 L/h/kg × [1 - 0.023 × (age - 46)] × [1 - 0.049 × (albumin - 15)], V1 = 0.273 L/kg × [1 - 0.049 × (albumin - 15)], Q = 0.199 L/h/kg and V2 = 0.309 L/kg × [1 - 0.049 × (albumin - 15)]. For a target of ≥80% T>MIC, the breakpoint was 8 mg/L for doses of 1 g every 4 h and 2 g every 8 h given over 3 h, but only 4 mg/L if given over 5 min. CONCLUSIONS Although 1 g 8 hourly should be effective against Escherichia coli and CoNS, higher doses, ideally with a longer infusion time, would be more appropriate for empirical therapy, mixed infections and bacteria with MIC values ≥4 mg/L.