An investigation into the effectiveness of the Dentron Biogunâ in killing strains of  Methicillin Resistant Staphylococcus aureus (MRSA) in a controlled laboratory environment.

 

A.P.Keen        Public Health Laboratory, West Wales Hospital, Carmarthen.

 Introduction

 The Dentron Biogun is an interesting piece of equipment designed to help reduce the numbers of microorganisms in dental caries (Burke et al. 1995). Some chiropodists have also found a use for the Biogun in the treatment of fungal skin & nail infections.

 The Biogun achieves its effect by delivering a stream of electrons which bind to the surrounding molecules of oxygen, forming the hydrated superoxide anion, (O₂-)(H₂O)n where n is between 4 and 8.  This anion radical is thought to act as a nucleophile on the phospholipid bi-layer of micro-organisms, causing de-esterification of fatty acids and weakening the cell membrane (Kellogg et al. 1979)

 Much work has been performed in establishing the bacteriocidal effect of the Biogun on populations of micro-organisms both in-vitro and in-vivo, including Streptococcus spp, lactobacilli, Actinomyces spp, Candida albicans and coagulase negative staphylococci (Shargawi et al. 1999, Kellogg et al. 1979, Rosenthal B. et al. 1979, Burke F.M. et al. 1995).  No data are available, however, to show the efficacy of the Biogun in killing populations of Methicillin Resistant Staphylococcus aureus (MRSA).  This study aims to investigate the effect of the Biogun in-vitro when applied to different strains of MRSA.

 MRSA is a term applied to strains of Staphylococcus aureus, which have become resistant to a range of antibiotics including all of the beta-lactam agents (penicillins, cephalosporins and carbapenems).  MRSA are also resistant to the beta-lactamase / beta-lactam combinations such as co-amoxiclavulanate and piperacillin-tazobactam.   Staphylococcus aureus is commonly isolated from infected wounds and abscesses.  MRSA strains are now widely distributed in UK hospitals. 

 Materials & Methods

 The Biogun was supplied by the manufacturer, Dentron Ltd, and set up as recommended.  When in use clinically, the patient and operator are ‘earthed’ by connecting them using metal wristbands wired to the generator.  For these experiments, the agar surfaces being treated were similarly ‘earthed’ using a wire connector embedded in the agar of each treated plate.

 

 The equipment is shown in                                    Photo 1

photo 1 to the right.  The

hand piece was held in a

clamp, and the distance

between each agar surface

being treated and the Biogun

emitter was measured and

set to 6mm, as shown in

detail in photo 2.  This

distance was determined

to be optimal, reflecting

actual use, where the emitter

to surface recommended

distance is between 2 – 10 mm.

An ammeter was connected into the circuitry to ensure that a good earth connection had been established with the agar, and that the current was regulated to a steady 100μA. 

  Photo 2 

 

 

 

 

 

 

                                                                   The agar plates (Oxoid Isosensitest, code CM471) were seeded with four strains of Staphylococcus aureus, as detailed in table A.  Two of these were National Type Culture strains and the other two were wild strains isolated from hospital patients.  These were grown up overnight in nutrient broth (Oxoid, code CM1), and diluted to give an opacity

equal to a 0.5 McFarland standard.  These suspensions were then diluted a further 100-fold to give the working dilutions.  Agar plates were dried at room temperature and then seeded with the working dilutions of test organisms using sterile cotton tipped applicator swabs and a rotary plater.

Table 1

 

 The seeded plates were allowed to dry at room temperature for a further 90 minutes before being treated with the Biogun. Each culture plate was then subjected to a timed burst of ionised air , and the test performed in triplicate on three separate areas of the each agar surface.  Intervals of 30, 60, 120 and 240 seconds treatment were used in different experiments to gauge how time dependant the effects would be.

 After treatment, all plates were incubated at 30⁰C for 24 hours to allow the viable organisms to grow, and plates read with a plate microscope to quantify the results.

 Results

 After incubation, all culture plates showed a semi-confluent growth of organisms as expected.  It was evident that after only 30 seconds of treatment with the Biogun some reduction of the numbers of organisms had been achieved, and that number of cells killed increased with time exposure to the negative air ions.  Using a plate

                                  Photo 3

 

 

 

 

 

 

microscope and a 10mm diameter

circular template, the numbers of

colonies for each organism were

counted within the template area

on areas of untreated agar surface,

and then each treated area was

counted using the same template

to determine the effect of the

Biogun.  Photo 3 shows the

three areas of treatment where

the bacterial cells have been

killed and failed to grow during the post-treatment incubation period.

 Table 2 gives the actual counts of the numbers of surviving organisms in the treated areas for each time band of treatment.