Introduction:

Staphylococcus aureus is an opportunistic pathogen found on the skin of between 70-90% people who have atopic dermatitis, this is much higher than the rates at which it is found on people without atopic dermatitis , approximately 5%. As a result people with atopic dermatitis often have bacteria infection within the atopic dermatitis lesions. S. aureus are the most common cause of bacterial super infection in atopic dermatitis and this is in part due to the production of virulence factors which can cause damage to the host and exacerbate inflammation. Treatment of atopic dermatitis is complicated and in many cases is not resolved by conventional medication, antibiotics and topical creams. Figure 1 shows the link between the increase in S. aureus, the corresponding decrease in bacterial diversity on skin and the flares in atopic dermatitis symptoms.

Honey represses virulence and viability in Staphylococcus aureus from atopic dermatitis

Suhaila al Kindi, Rose Cooper and Rowena Jenkins Cardiff Metropolitan University, Department of Biomedical Sciences, Cardiff CF5 2YB

Email: rojenkins@cardiffmet.ac.uk, Twitter @jenkins7ro

Introduction:

Figure 2: (A) Apis mellifera foraging on manuka (Leptospermum scoparium). (B) Different types of honey available. Honey is known to have a broad spectrum antimicrobial activity, which has been demonstrated both in vitro and in clinical practice (Figure 2). Studies into the effect of honey has also established that it can elicit an immune modulatory effect on human tissue. As previous genomic analysis has shown that honey is capable of modulating gene expression in bacteria such as S. aureus and P. aeruginosa, and can down regulate genes associated with virulence there is potential for honey to be used as an anti virulence agent in addition to its current role as an antimicrobial agent. (1-3)

Combination antibiotic therapy is currently used to improve efficacy against bacteria which potentially could display antibiotic resistance, and to lower the doses used reducing the likelihood of problematic side effects such as toxicity. If honey could act in synergy with antibiotics it could be considered for use as an adjuvant therapy.

Aims: • To determine the ability of honey to inhibit S. aureus isolated from atopic dermatitis. • To ascertain whether sub inhibitory levels of honey can reduce the expression of virulence

factors.

• To evaluate the ability of honey to enhance the activity of antibiotics.

Figure 1: Diagram highlighting link between presence of S. aureus and flares in atopic dermatitis. Heidi H. Kong et al. Genome Res. 2012;22:850-859

A B

Methods

Seven S. aureus isolates were tested. The minimum inhibitory concentration, minimum bactericidal concentration and minimum biofilm inhibition concentration of honey were determined using adapted EUCAST microbroth dilution technique.

The effect of sub inhibitory manuka honey on virulence production was determined for haemolysin, protease, lecthinase and DNase activity using 5% sheep blood agar, 5% skim milk agar and DNase agar with and without 5% or 10% (w/v) manuka honey (Figures 3 and 4).

Antibiotic susceptibility testing was carried using EUCAST disk diffusion methodology. Mupirocin (MUP), fusidic acid (FD), ciprofloxacin (CIP), gentamicin (CN), erythromycin (ERY), tetracycline (TET) and cefoxitin (FOX) with and without sub-lethal concentrations of manuka honey 5% and 10%( w/v) were tested.

All tests were carried out at least in triplicate and a paired students T test was used to compare the groups.

Methods

Cells streaked onto agar then sub cultured onto specialist media to screen for virulence.

Cells were grown with and without sub MIC honey

Honey represses virulence and viability in Staphylococcus aureus from atopic dermatitis

Suhaila al Kindi, Rose Cooper and Rowena Jenkins Cardiff Metropolitan University, Department of Biomedical Sciences, Cardiff CF5 2YB

Email: rojenkins@cardiffmet.ac.uk, Twitter @jenkins7ro

Figure 3: Method by which virulence phenotype was established.

Figure 4: Method by which virulence expression was tested (control, 5 and 10% (w/v) honey).

Honey represses virulence and viability in Staphylococcus aureus from atopic dermatitis

Suhaila al Kindi, Rose Cooper and Rowena Jenkins Cardiff Metropolitan University, Department of Biomedical Sciences, Cardiff CF5 2YB

Email: rojenkins@cardiffmet.ac.uk, Twitter @jenkins7ro

Results: All isolates were inhibited by honey at ≤ 14 % (w/v) and had an minimum bactericidal concentration of ≤ 18% (w/v) honey.

All the virulence factors measured were significantly down regulated when isolates were treated with sub MIC honey.

Zones of inhibition were significantly increased when antibiotics were used in conjunction with sub MIC honey; indicating an increase in susceptibility.

Bacterial strain MIC (%w/v) MBC (%w/v) MBIC (%w/v)

NCTC6751 14 18 16

4/0/C 14 18 16

4/0/T 14 16 16

6/0/T 14 18 16

6/7/T 14 16 16

12/0/T 14 16 16

14/0/T 14 18 16

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Manuka honey % (w/v)

Bacterial strain

Virulence characteristic

Haemolysis Protease Lecthinase DNase Agglutination

NCTC 6751 β + + + +

4/0/T β + + + +

4/0/C β - + + +

6/0/T β - - + +

6/7/C β - - + +

12/0/T β - + + +

14/0/T β - + + +

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Manuka honey (% w/v)

Figure 7: Virulence profiles of all isolates tested. Figure 5: Table displaying MIC, MBC and MBIC values for all isolates.

Figure 6: Isolate 4/o/T MIC demonstrating good growth at both 5 and 10% (w/v) honey.

Figure 9: Isolate 4/0/T showing a significant decrease (p = 0.014) in protease activity when treated with sub MIC honey.

88.5

99.510

10.511

11.512

12.513

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Manuka honey (% w/v)

Figure 8: Isolate 4/0/T showing a significant decrease (p = 0.020, p =0.007) in haemolytic activity when treated with sub MIC honey, 5 and 10 % (w/v) respectively.

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Figure 10: Isolate 4/0/T showing a significant decrease (p = 0.003) in lecithinase activity when treated with sub MIC honey .

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1012141618

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Figure 11: Isolate 4/0/T showing a significant decrease (p = 0.04, 0.011 ) in DNase activity when treated with sub MIC honey, 5 and 10 % (w/v) respectively.

Results Conclusions

Manuka honey effectively inhibits S. aureus at low concentrations, clinically achievable concentrations.

Sub lethal concentrations of manuka honey significantly reduced the expression of virulence in S. aureus , this could suggest a role for S. aureus in treatment of atopic dermatitis as it could help alleviate symptoms. The use of low concentrations of honey might also minimise disruption to beneficial microflora found in treatment sites.

Sub inhibitory concentrations of honey in combination with antibiotics increased sensitivity of all isolates to antibiotics tested. As almost all of the isolates were already sensitive (in line with EUCAST breakpoints) it would be interesting to see if the same effect is recorded when antibiotic resistant isolates are used.

Potentially for honey could be used as a treatment S. aureus associated with for atopic dermatitis. Further clinical trials are needed to see if honey could be incorporated into clinical practice as part of a treatment regimen.

Thanks to: Professor Rose Cooper and Suhaila al Kindi Derma Sciences for providing the honey used throughout this study, and for sponsoring my

attendance at this conference.

References: 1. Jenkins RE, Burton NF, Cooper RA (2014) Proteomic and genomic analysis of methicillin-resistant

Staphylococcus aureus (MRSA) exposed to manuka honey in vitro demonstrated down-regulation of virulence markers. Journal of Antimicrobial Chemotherapy, 69 (3) 603 – 615.

2. Jenkins RE, Cooper RA. (2012) Improving antibiotic activity against wound pathogens with manuka honey in In Vitro. PLOS one, 7 (9) 1-9 e45600.

3. Roberts AE, Maddocks SE & Cooper RA. (2012) Manuka honey is bactericidal against Pseudomonas aeruginosa and results in differential expression of oprF and algD. Microbiology, 158 (12), 3005-3013.

Honey represses virulence and viability in Staphylococcus aureus from atopic dermatitis

Suhaila al Kindi, Rose Cooper and Rowena Jenkins Cardiff Metropolitan University, Department of Biomedical Sciences, Cardiff CF5 2YB

Email: rojenkins@cardiffmet.ac.uk, Twitter @jenkins7ro

Figure 12: Zones of inhibition from a range of antibiotics against multiple S. aureus strains with and without 5 and 10 (% w/v ) honey.

Zone of inhibition (mm)

Bacterial strain MUP (200) FD (10) CIP (5) CN (10) ERY (15) TET (30) FOX (30)

NCTC 6751 30.5 29.4 28 21.3 27.2 26.2 11.8

4/0/T 34.5 30.3 27.2 21.6 0 26.15 30.75

4/0/C 37.1 32.1 29 23.7 0 25.7 31.6

6/0/T 35.7 31.4 27.7 23.3 25.4 27 24.9

6/7/C 34.2 30.8 28.3 22.7 26 26.9 28.55

12/0/T 41 37.9 33.1 29.8 24.3 28.5 29.35

14/0/T 34.2 31.3 28.1 23 27.4 29.2 28.25

Zone of inhibition (mm) + 5% (w/v) honey

MUP (200) FD (10) CIP (5) CN (10) ERY (15) TET (30) FOX (30)

NCTC 6751 39 41.2 34.7 33.5 31.6 37.6 22

4/0/T 39.2 33.5 29.7 27.8 0 25.2 30

4/0/C 39.5 36.8 28.8 27.7 0 36 30

6/0/T 39.5 38.7 33.3 28.2 34 35 34.5

6/7/C 41 40.2 31.9 25 32.3 33.8 32

12/0/T 39.8 36.5 29.5 25.8 32.3 35.5 30

14/0/T 38.8 35.2 29.2 25.3 29.5 34.7 30

Zone of inhibition (mm) + 10 % (w/v) honey

MUP (200) FD (10) CIP (5) CN (10) ERY (15) TET (30) FOX (30)

NCTC 6751 45.8 46 35 34 32 41 24.3

4/0/T 41.5 41.5 35.2 38.8 34.2 40.2 32.4

4/0/C 42.5 47 32.7 32.3 33.7 41 34.3

6/0/T 42.8 47.7 33.3 33.7 33.7 39.3 37

6/7/C 41 40.8 31.2 31.2 34.2 38.5 33.6

12/0/T 40.7 43 32.5 33.2 36.3 39 31.4

14/0/T 40.7 42.7 32 32.3 36.3 40.5 31.8