Minimum Inhibitory Concentrations (MIC) and Minimum

The bacterial isolates used in MIC testing are listed in Table 3.4 and consisted of reference strains from the National Type Culture Collection (NTCC) (Public Health England) and clinical isolates obtained by the Leeds General Infirmary (LGI), following ethical approval.

Bacterial isolate Source

S. aureus NTCC29213 NTCC

S. aureus patient 1 LGI

S. aureus patient 2 LGI

P. aeruginosa NTCC27853 NTCC

P. aeruginosa patient 1 LGI

P. aeruginosa patient 2 LGI

E. faecalis NTCC29213 NTCC

E. faecium patient 1 LGI

E. faecium patient 2 LGI

Media and Diluents

The media and diltuents used in this method are listed in Table 3.5. All media and diluents were prepared according to the manufacturer’s instructions, unless otherwise stated.

Medium/diluent Supplier

Mueller Hinton Broth Oxoid

0.9% saline Sigma Aldrich

Preparation of MGO MIC broths

MGO 40 wt % from Sigma Aldrich was diluted in sterile distilled water to achieve a working stock solution of 4096 mg L-1. A double strength dilution series was then prepared by diluting the working stock solution in Mueller Hinton broth to achieve a doubling dilution series from 2

Table 3.4: Bacterial isolates.

mg L-1 to 2048 mg L-1. The dilution series was dispensed across all rows of a 96-well microtitre tray (Fisher Scientific) in 50 µl amounts, as seen in Fig. 3.5.

Preparation of bacterial inocula

Each bacterial isolate was inoculated on to fresh blood agar (FBA) plates and incubated at 37oC for 24 h. Single colonies of each bacterial isolate were removed from the FBA and resuspended in 5 ml Mueller Hinton Broth to a 0.5 MacFarland turbidity equivalent.

Test procedure

Starting with the non-MGO-containing growth control, and then working from the lowest to the highest MGO containing broth, duplicate rows of the 96 well MGO MIC plate were inoculated with each bacterial isolate. Inoculation of the MGO MIC plates occurred within 15 minutes of inoculum preparation. Lids were placed on each MGO MIC 96-well plate, and these were then incubated at 37oC for 24 h.

Reading and interpretation of MIC results

The MIC was defined as the lowest concentration of MGO that completely inhibited the growth of the bacterial isolates, as detected by the unaided eye. This is assessed by placing a clear black line underneath the wells in the microtitre tray. If the black line is visible under the well, the concentration of MGO in that well shows an inhibitory effect. If the black line is not visible, this is due to the growth of bacteria in that well, creating a turbid solution. An example of this can be seen in Fig. 3.6A.

the highest concentration starting at the left and finishing with the lowest concentration at the right. The final well is left blank as the control.

In order to determine whether the growth inhibition at any particular dilution was bactericidal or bacteriostatic, triplicate 20 µl aliquots were inoculated onto each of four quarters of a FBA plate and spread over the surface of the agar quarter with a sterile inoculating loop. Inoculated FBA plates were then incubated at 37oC for 24 h. The MBC was defined as the lowest concentration at which there was no visible bacterial growth upon FBA. Fig. 3.6B shows an example of this.

(A) and determination of MBC via the regrowth of bacteria on FBA plates (B).

MGO concentration

Highest Lowest Growth _control

(no MGO)

B

A

Figure 3.6: Determination of MIC via visibility of black line under the well in the microtitre tray Figure 3.5: Preparation of the 96-well microtitre tray showing the placement of 50 µl of MGO, with

High performance liquid chromatography was used in this study to determine the release profile of MGO from the electrospun fibres.

High performance liquid chromatography (HPLC) is a physical technique used to separate a liquid sample into individual components (317, 318). The separation occurs by the interaction of the sample with a mobile and stationary phase. The mobile phase is the phase that moves in a definite direction and consists of the liquid sample in a solvent. When the mobile phase passes through the column the sample interacts with the stationary phase and is separated. There are many combinations of stationary and mobile phases that can be employed when separating a mixture. Based on each components affinity in the mobile phase, the components will migrate through the column at different rates. For example, if the components in the mobile phase are of different polarities, one component will migrate through the column faster than the other based on that component’s affinity for either the mobile or stationary phase (317). Several modes of liquid chromatography exist. In this study reversed-phase liquid chromatography (RPLC) was employed. In RPLC the column is non-polar and the mobile phase is polar; this is the opposite to normal phase chromatography (319).

To detect the presence of MGO after the dispersion of the PVA/MGO fibres in a distilled water solution, an Agilent 1290 Infinity with a Diode UV Array detector was used. The machine was fitted with an Agilent Eclipse XDB-C8 column, measuring 150 mm x 4.6 mm and having a 5 um particle size. The flow rate was set at 0.5 ml min-1. A binary pumping system allowed the mixing of two solvents as the mobile phase. Acetonitrile (solvent A) and water (solvent B) both containing 0.1% Trifluroacetic acid (TFA) were used. The acetonitrile and water were mixed by the pump in a linear gradient starting with 5% solvent A and 95% solvent B, increasing to 95% solvent A and 5% solvent B over 15 minutes. Peaks were detected by UV-absorbance at 280 nm.