2014-03 Val edward Jones Kytocel report - AllWeCare · MelBec Microbiology Ltd 1 ... or...

Professor Val Edwards-Jones

MelBec Microbiology Ltd

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Background

KytocelTM is a new dressing primarily focused on its absorbent and

haemostatic properties. Initial studies and previous publications have

demonstrated that the material within KytocelTM, Chitosan, has

antimicrobial properties. There are a number of methods that can be

used to demonstrate antimicrobial activity of dressings but the most

accepted methods are zone of inhibition (Thomas and McCubbin 2003),

Log reduction assay (Gallant-Behm et al 2005) and challenge testing

(Thomas and McCubbin 2003). These following studies were undertaken

to assess the dressing for antimicrobial activity.

1.1 Zone of inhibition assay

The basis of the zone of inhibition test is to place a dressing of known

size on the surface of a Petri dish containing a suspension of a

microorganism (at approx 106 cfu/ml) and incubate overnight at 37oC.

The zone of inhibition surrounding the dressing is measured. The effect

underneath the dressing should also be assessed visually for growth and

also sub cultured onto a sterile culture medium.

1.2.1 Method

1.2.1 Dressings for testing

The dressings tested were:-

Dressing 1 Tricotex

Dressing 2 Aquacel

Dressing 3 Kytocel

Organisms used

Staphylococcus aureus ATCC 6538P (methicillin resistant Staphylococcus

aureus (MRSA)

Pseudomonas aeruginosa ATCC 9027

Candida albicans NCTC 1363

Esherichia coli ATCC 8739



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1.2.2. Method

Petri dishes containing a 5mm layer of Tryptone Soy Agar (TSA, LabM,

LAB011) for bacteria and Sabour aud Dextrose Agar (SDA, LabM,

LAB009) for the yeast were inoculated with 0.2ml of a log phase broth

culture of each test organism. The suspension was distributed uniformly

on the surface of the plate and dried for 15minutes. Portions of each

dressing measuring 40mm X 40mm were then placed on the agar, with

the wound contact surface downwards.

Each test was performed in triplicate and a similar piece of non-woven

gauze fabric (Tricotex) was used as a control. The plates were incubated

for 24hrs at 370C and the plates for fungi at 370C for 48hrs.

1.2.3. Bacteriocidal / Bacteriostatic activity

Following removal of the dressing, the original plate was re-incubated

for a further 24hrs if the area under the dressing after removal, showed

no microbial growth. After incubation, the area under the dressing was

sub-cultured onto a sterile TSA or SDA plate and examined for residual

growth to test for bacteriocidal/bacteriostatic or yeasticidal activity.

1.3.1 Results

There was no zone of inhibition observed with any of the dressings

tested. This demonstrated that there was no antimicrobial substance

diffusing laterally from the product.

1.3.2 Bacteriocidal/bacteriostatic and Yeasticidal activity

Following removal of Tricotex and Aquacel dressings from the 24hr

incubated plates, there was some evidence of reduced growth where

the dressing had been placed on the plate. When examined closely,

following a further 24hr incubation, this effect had disappeared and

there was visual growth under the Tricotex and Aquacel dressings.

However, under the Kytocel dressing there was no visible growth (see

figure 1) and there was no growth on sub culture. This demonstrated

that the Kytocel dressing was killing the organisms underneath the

dressing (bacteriocidal and Yeasticidal). However, there was evidence of



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residual dressing on the plate where it had been co-cultured with

Candida albicans) (see figure 2)

Kytocel demonstrated antimicrobial activity against all four

microorganisms tested and killed the organisms on the surface of the

culture plate at a concentration of 106 cfu/ml.

1.4 Discussion

This experiment demonstrated that the antimicrobial activity observed

with the Kytocel dressing was not due to a diffusible antimicrobial

substance but an interaction with the organism and the dressing

material. The retention of the dressing on the surface of the agar plate

in the presence of Candida albicans is not easily understood. This may

have been due to an interaction of the culture medium and the dressing

or alternatively this could have been due to an unknown interaction with

the organism, dressing and culture medium.

Previous studies have shown that there is an interaction between

positively charged chitosan molecules and negatively charged microbial

cell membranes, altering cell permeability which leads to leakage of

intracellular constituents (Cheung et al 2004). There have been other

interactions described with intracellular components including DNA and

mRNA and inhibition of protein synthesis (Cheung et al 2004).

1.5 Key points Zone of Inhibition Assay

The antimicrobial effect of the dressing is not dependent on the release

of a soluble/diffusible compound but the effect is based upon the

interaction with the organism. This is probably due to an interaction of

the cationic charged dressing and negatively charged membrane.

The relevance of the dressing retention on the surface of the culture

plate in the presence of Candida albicans is interesting and further work

would have to be undertaken to fully understand this phenomenon.



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Figure 1 The area underneath the dressing following removal of Kytocel dressing showing killing of the organisms.

Key: from left to right MRSA, Candida albicans, E.coli and Pseudomonas aeruginosa



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Figure 2 Residual Kytocel dressing following testing with Candida albicans



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2.0 Challenge testing

2.1 Principle of the test

A known number of organisms are added to a fluid that will maintain

numbers without allowing growth, and added to a piece of dressing at

time 0hrs. This is then incubated for 24hrs at 37 oC and at 2, 4 and 24hrs

the numbers of viable organisms are accurately counted following

vigorous mixing (to loosen organisms form the dressing that may have

adhered to it). This test helps to determine the speed of kill and the

time taken to reduce numbers or kill all organisms added to a dressing

within a 24hr period.

Objective:

To assess the time taken to kill four microorganisms using a modified

challenge test as described by Thomas and McCubbin 2003.

2.2 Materials and Methods

2.2.1 Dressings for testing

The dressings tested were :-

Dressing 1 Tricotex

Dressing 2 Aquacel

Dressing 3 Kytocel

Organisms used

Staphylococcus aureus ATCC 6538P (MRSA)




2.2.2 Method

1ml of a dilution of a log phase culture of each organism (approx.

106colony forming units (cfu)/ml)) was added to portions of the dressing

measuring 2.5 x 2.5cm (see figure 1). (Note; 1ml fluid in total was added



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because the dressings absorbed approximately this amount and

remained moist up to 24hrs incubation).

The inoculated dressings were incubated at 370C in a sealed container

for 2,4,and 24hrs then transferred to 9ml of Letheen broth (Lab 184, Lab

M. UK) to neutralize any antimicrobial inhibitors) and vigorously vortex

mixed to remove any viable organisms remaining in the dressings. 0.1ml

of this was added to 9.9ml of PBS (Phosphate Buffered Saline) and the

number of viable organism present in each was determined using a

standard surface counting technique.

Each dressing was tested in triplicate and serial dilutions were

performed in triplicate on each extract (n=9) for each dressing.

The time kill kinetics was calculated up to a twenty four hour period.

2.3.1. Results

The results of the challenge tests are shown in tables 1-4. These are the

mean values for nine estimations of viable counts.

All the microorganisms grew in the test system and there was no marked

decrease in organism numbers compared to the original numbers in the

original inoculum.

Dressing 1 – Tricotex (Control Dressing)

This dressing showed no antimicrobial activity against any organism

tested.

Dressing 2 – Aquacel

This dressing showed no antimicrobial activity against any organism

tested.

Dressing 3 – Kytocel

All microorganisms were killed within a 24hr period.

MRSA was killed within two hours.



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Ecoli was killed within two hours.

Pseudomonas aeruginosa was killed within 24hrs

Candida albicans was killed within 24hrs.

Table 1. Microbial challenge test; recovery of S. aureus. The results are

the mean of three repeat samples (nine replicate counts)

2hrs 4hrs 24hrs

No of

organisms

applied

(cfu’s)

No of

organisms

recovered

(cfu’s)

No of

organisms

recovered

(cfu’s)

No of

organisms

recovered

(cfu’s)

Tricotex 3.06 x 106 5.0 x 105 9.94 x 105 1.83 x 106

Aquacel 3.06 x 106 4.4 x 106 4.1 x 106 2.34 x 106

Kytocel 3.06 x 106 NG NG NG

Control

(inoculum)

3.06 x 106 2.52 x 106 3.15 x 106 2.51 x 106

Key: cfu’s – colony forming units

Table 2. Microbial challenge test; recovery of Ps.aeruginosa. The

results are the mean of three repeat samples (9 replicate counts)

2hrs 4hrs 24hrs

No of

organisms

applied (cfu’s)

No of

organisms

recovered

(cfu’s)

No of

organisms

recovered

(cfu’s)

No of

organisms

recovered

(cfu’s)

Tricotex 1.82 x 105 1.27 x105 5.7 x 105 2.26 x106

Aquacel 1.82 x 105 1.76 x104 5 x104

4.5 x105

Kytocel 1.82 x 105 5.5 x104 6 x103 NG

Control

(inoculum)

1.82 x 105 0.9 x105 1.55 x105

4.83 x106




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Table 3. Microbial challenge test; recovery of Candida albicans. The


2hrs 4hrs 24hrs

No of

organisms

applied

(cfu’s)

No of

organisms

recovered

(cfu’s)

No of

organisms

recovered

(cfu’s)

No of

organisms

recovered

(cfu’s)

Tricotex 2.58 x 105 2.9 x 105 1.2 x 106 2.3 x 106

Aquacel 2.58 x 105 2.2 x 105 7.3 x 105 7.3 x 105

Kytocel 2.58 x 105 1.2 x 105 2.9 x 103 NG

Control

(inoculum)

2.58 x 105 5.7 x105 9.2 x 105 1.9 x 106


Table 4. Microbial challenge test; recovery of Escherichia coli. The


2hrs 4hrs 24hrs

No of

organisms

applied

(cfu’s)

No of

organisms

recovered

(cfu’s)

No of

organisms

recovered

(cfu’s)

No of

organisms

recovered

(cfu’s)

Tricotex 1.06 x 105 1.27 x 105 5.77 x 105 2.11 x 106

Aquacel 1.06 x 105 1 43 x 105 3 x 105 5.77 x 105

Kytocel 1.06 x 105 NG NG NG

Control

(inoculum)

1.06 x 105 1.33 x 105 1.27 x 105 9.68 x 105




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2.4 Discussion

Tricotex and Aquacel did not show any antimicrobial activity. This was

expected as no previous studies have demonstrated any antimicrobial

effect of the dressings. The model test system was appropriate as there

was no marked increase or decrease in cell numbers in the inoculum

control over the twenty four hour period, even when incubated at 37oC,

the ideal temperature for the organisms tested. The PBS contained no

nutrients but a buffered salt solution to maintain osmolality and

maintain pH. The dressings over the twenty four hour period showed a

slight increase in cell numbers but this was not markedly different from

the inoculum control, implying that the dressing itself had not been used

as a source of nutrients for any of the organisms tested.

Kytocel showed total kill within 24hrs for the three bacteria tested and

the fungal strain. Some strains had a quicker speed of kill with MRSA and

E.coli being killed within a two hour period. The yeast (Candida albicans)

and P. aeruginosa showed a marked reduction in numbers at four hours

and total kill at 24hrs. In order to calculate the exact time of kill, further

time points would have had to be included.

2.5 Key Points Challenge test

The importance of the speed of kill of an antimicrobial agent depends

upon the application. The dressings tested are applied to a wound to

absorb excess exudate and the number of times they are changed daily

would depend upon strikethrough. If a dressing is applied to a wound to

help reduce bacterial numbers and help resolve infection or colonization

then it would be important to have the quickest speed of kill as possible.

Most wounds are cleaned and may also be debrided. It is at this point

that the numbers of microorganisms in a wound will be reduced and

there is a known therapeutic window when an antimicrobial agent has

its maximum effect. Kytocel killed over one million cells of two

organisms within a two hour period (MRSA and E.coli) and the

remaining two organisms within a 24 hr period with a marked reduction

in cell numbers in 4hrs, demonstrating a good speed of kill for the

application as a dressing.



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3. Log reduction assay

3.1 Principle of the test

A known number of organisms are added to a fluid that will maintain

numbers without allowing growth, and added to a piece of dressing at

time 0hrs. This is then incubated for 30minutes and 2hrs and the

numbers of viable organisms are accurately counted following vigorous

mixing (to loosen organisms from the dressing that may have adhered to

it). The reduction in organism numbers (expressed as log10) between

30minutes and 2hrs are recorded. This test helps determine how quickly

the antimicrobial substance acts against microorganisms and the level of

activity.

As defined by Gallant–Behm et al ., (2005) low antimicrobial activity was

considered to be less than 1 log reduction, moderate activity between 1

and 3 log reduction and high antimicrobial activity as greater than 3 log

reduction.

3.2 Materials and method

3.2.1 The dressings tested were:-

Dressing 1 Tricotex

Dressing 2 Aquacel

Dressing 3 Kytocel

Organisms used

Staphylococcus aureus ATCC 6538P




3.2.2 Method

1ml of a dilution of a log phase culture of each organism (approx

106colony forming units (cfu)/ml)) was added to portions of the dressing

measuring 2.5 x 2.5cm (see figure 1). (Note; 1ml fluid in total was added

because the dressings absorbed approx this amount and remained moist

throughout the incubation time).



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The inoculated dressings were incubated at 370C in a sealed container

for 30minutes and 2hrs then transferred to 9ml of Letheen agar (Lab

184, Lab M. UK) to neutralize many antimicrobial inhibitors) and vortex

mixed to remove any viable organisms remaining in the dressings. 0.1ml

of this was added to 9.9ml of PBS and the number of viable organism

present in each was determined using a standard surface counting

technique.

Each dressing was tested in triplicate and serial dilutions were

performed in triplicate on each extract (n=9) for each dressing.

3.3 Results

Aquacel did not show any antimicrobial activity against any organism

compared to the control dressing (Tricotex) or the inoculum control.

Kytocel showed a high level of antimicrobial activity against MRSA with a

3log reduction at 30minutes and 6 log reduction after 2hrs.

Kytocel showed a high level of antimicrobial activity against E.coli with a

2 log reduction at 30minutes and a 5 log reduction after 2hrs.

Kytocel showed a low level of antimicrobial activity against

Pseudomonas aeruginosa and Candida albicans at 30minutes and 2hrs

with less than one log reduction for both organisms at both time

periods.

Table 2 showing log reduction (30minutes/120 minutes) induced by the

dressings under test compared to the Tricotex control.

Microorganism Aquacel Kytocel Tricotex

Control

MRSA 0.35/-0.14 3.61/6.47 0/0

E. coli 0.20/-0.06 1.95/5.02 0/0

P. aeruginosa 0.03/-0.39 0.66/0.13 0/0

C. albicans 0.07/0.11 -0.33/0.46 0/0



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Discussion

Kytocel showed a high level of antimicrobial activity against two

common organisms found in wounds, MRSA and E.coli within a two hour

period showing a total kill within two hours equivalent to a 5-6 log

reduction. This would be extremely advantageous in wound care,

especially if there had been a biofilm on a chronic wound. Previous work

has shown that there is a small therapeutic window following

debridement when an antimicrobial dressing would have a beneficial

effect to help disrupt a biofilm and reduce numbers of common

colonisers in a wound.

Further Recommendations

The zone of inhibition assay showed Kytocel did not release any

diffusible antimicrobial properties but there was an interaction with the

dressing and the organisms on the surface of the culture plate as there

was total kill of the organisms underneath the dressing. This was not due

to absorbency, as Aquacel, which had similar absorbency properties to

Kytocel, think sentence needs finishing?

Kytocel has a rapid speed of kill and a 5-6 log reduction of common

pathogens, with a total kill observed within 24hrs on planktonic cells

(cells in suspension).

Therefore because Kytocel demonstrates kill of organisms on the surface

of a culture plate and also with planktonic cells in suspension, it would

be beneficial to test this dressing in a single organism and mixed

organism biofilm model to determine whether the mixed absorbent,

haemostatic and antimicrobial properties of the dressing would prevent

and inhibit a biofilm.

I recommend two further experiments:

1. Prevention of a biofilm formation in an explant pork model.

2. Eradication of a biofilm in an explant pork model.



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Gallant-Behm C.L. et al., 2005 Comparison of in vitro disc diffusion and

time kill assays for the evaluation of antimicrobial wound dressing

efficiency Wound Repair and Regeneration 13; 4. 412-417.

Thomas and McCubbin 2003 A comparison of the antimicrobial effects of

four silver-containing dressings on three organisms J of Wound Care

2003; 12; 3; 101-107)

Ying-chien CHUNG,Ya-ping SU, Chiing-chang CHEN, Guang JIA ,

Huey-lan WANG, J C Gaston WU, Jaung-geng LIN 2004 Relationship

between antibacterial activity of chitosan and surface characteristics of

cell wall Acta Pharmacol ;25): 932-936.

2014-03 Val edward Jones Kytocel report - AllWeCare · MelBec Microbiology Ltd 1 ... or...

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