An in vitro investigation of meticillin resistant Staphylococcus aureus (MRSA) decolonisation treatment failure Dr Sue Lang.

An in vitro investigation of meticillin resistant Staphylococcus aureus (MRSA) decolonisation

treatment failure

Dr Sue Lang

Staphylococcus aureus infections in Scotland

• Large reductions in MRSA bacteraemia since 2006, MSSA rates have remained largely unchanged

Healthcare Associated Infection Annual Report 2013

A need to decolonise?• Decolonisation; treatment to eradicate S. aureus colonisation• An infection prevention measure:

– Nasal colonisation with MRSA is the single most important determinant of subsequent MRSA infections (Nguyen et al., 1999)

– The risk of infection in nasal carriers of MRSA is estimated to be 15 times higher than in those who are not colonised (HPS Pathfinder 2009)

– Reduce cross transmission

• Pre-surgical decolonization has lead to reduction in infection of:– Surgical patients eg cardiothoracic: 58% decrease– Haemodialysis and peritoneal dialysis patients: decrease up to

80%

Decolonisation strategy

• Intra nasal application of mupirocin alone or in combination with use of antiseptic soaps or systemic antimicrobial agents– 2% mupirocin for nasal carriage and

chlorhexidine wash for skin carriage for 5 days

– If skin lesions present add in systemic antibiotic for 7 days

• Elimination from nares (VN) results in disappearance from other sites

S. aureus colonisation• 30% of healthy adults are

colonised with S. aureus– 20% persistently colonised– 30% intermittently

colonised– 50% non-carriers

• High carriage in:– Hospitalised patients– IVDA– Insulin-dependant diabetics– HIV positives– Haemodialysis patients

Decolonisation strategy• Short term decolonisation may be sufficient

– Prior to surgery• Post-operative S. aureus infection rate of 3.4%

(decolonised of S. aureus) vs 7.7% in control group (Bode et al., 2010)

• Mupirocin ointment has been shown to be 97% effective in temporarily decolonising S. aureus nasal carriers (Wertheim, 2005)

• Long term desirable– Patients frequently attending healthcare facilities,

eg undergoing dialysis

Mupirocin• Mupirocin available as a nasal ointment, Bactroban • Mixture of pseudomonic acids produced by

Pseudomonas fluorescens• Consists of 90% pseudomonic acid A and structurally

different to any other clinically relevant antibiotic

• Prevents bacterial protein synthesis by inhibiting the bacterial isoleucyl-tRNA synthetase (IleRS)

Resistance to mupirocin

• Low level resistance (LLR); MIC of 8-256mg/L– Conferred by mutations in native

isoleucine synthetase– Implicated in relapse after

treatment (Lee et al., 2011)

• High level resistance (HLR); MIC >512mg/L– Conferred by mupA; encodes an

additional isoleucine synthetase (IRS) enzyme

– mupA carriage has been associated with multi-drug resistance and decolonisation failure

LLR HLR

EUCAST database 07.11.14

Resistance to mupirocin

• 2009 – 2012; mupirocin use has slightly decreased• Resistance rates have not yet been seen to increase

Healthcare Associated Infection Annual Report 2013

Shortcomings of mupirocin decolonisation

• The current decolonisation regime may not be fit for purpose– The median length of stay is 3-4 days for all MRSA

colonised patient admissions (HPS, 2011)

– Only 3.1% of patients completed decolonisation treatment (HPS, 2011)

– 10 days median time for successful decolonisation (Ammerlan et al., 2011)

Shortcomings of mupirocin decolonisation

• MRSA decolonisation isn’t always successful:– Successful for 62% of patients with mupirocin sensitive

strains (mupS), 29% of cases with LLR, 24% of cases with HLR

• One eradication attempt may not be sufficient– Eradication achieved in 60% of patients with only one

decolonisation attempt– Increased to 80% with multiple attempts (Ammerlan et al.,

2011)

• Success rate is over estimated– Relapse is thought to occur at high rate– 38% re-colonisation after 6 months (Coates et al., 2009)

Shortcomings of mupirocin decolonisation

• Driver for the development of resistance– Patients previously exposed to mupirocin have a

substantially increased risk of mupirocin resistant MRSA

• Resistance to mupirocin complicates decolonisation– After initial success, only 25% of patients with LLR

remained culture-negative at 4 weeks (Coates et al., 2009)

– When patients do present with resistant strains often the correct treatment is not administered (Howard and Morris, 2012)

– LLR and qacA/B has been associated with decolonisation failure at 12 months

Aim of the study

To investigate mupirocin decolonisation treatment failure

1. Biofilm-related tolerance2. Low level resistance (LLR) to

mupirocin (Mup)3. Antibiotic induced stress

(Stringent Response)

1. Efficacy of Mup on biofilm-associated cells

LIVE/DEAD® staining of MupS 177 biofilms. A) without Mup treatment, B) 30 min exposure to 200mg/l Mup

Control

1 x

MIC

4 x

MIC

10 x

MIC

100

x M

IC

200m

g/l0

50

100

150

n=12error bars SEMMup concentration

Cel

l sur

viva

l (%

of

untr

eate

d co

ntro

l)

A B

• Despite exposure to concentrations of Mup greatly in excess of the MIC, a substantial proportion of cells remained metabolically active

189

190

194

195

196

0

25

50

75

1004 x MIC10 x MIC

200 mg/l

MSSA

Ce

ll S

urv

ival

(%

of

con

tro

l)

1. Efficacy of Mup on biofilm-associated cells

• Tolerance of biofilm-associated S. aureus cells was consistent between strains

Error bars SEM

1. Efficacy of Mup on biofilm-associated cells

• MupS (mean 14%; range 3-41%)

• LLR (mean 16%; range 2-94%)

• Not statistically significant

• Raised the issue that LLR might underlie decolonisation failure

Comparison of cell viability of surface-attached S. aureus strains after 24 h exposure to Mup 200mg/l. MupS (n = 14), LLR; (n = 20), error bars SEM

MupS LLR0

20

40

60

80

100

Mup tolerance in MupSand LLR S. aureus

Cells exposed to 200 mg/l Mup for 24 h

10 MSSA, 4 MRSA 20 LLR MRSACe

ll S

urv

iva

l (%

of

un

tre

ate

d c

on

tro

ls)

2. Efficacy of Mup on LLR strains• 35 clinical isolates of S. aureus

– 14 MupS (4 MRSA, 10 MSSA) and 21 LLR (20 MRSA, 1 MSSA)• S. aureus attached to wells coated with 0.25% mucin to mimic

colonisation– 1 x 105 cfu/mucin coated well incubated for 4 h at 37C on a rocking

platform to allow attachment• Exposure to Mup 200mg/l

– (at least 6-fold above the MIC for all strains) for 1, 4, 6, 12 and 24 h• Cell viability determined by incubation with the metabolic dye

resazurin– Fluorogenic product recorded as an indirect measure of cell viability

• Antibiotic induced inhibition and cell recovery evaluated over a time period of 24 h– Viability of untreated and treated surface-attached cells compared in

triplicate

MRSA 5

0

MRSA 1

44

MRSA 1

77

MSSA 1

87

MSSA 1

88

MSSA 1

890

20

40

60

Ce

ll s

urv

iva

l (%

of

un

tre

ate

d c

on

tro

ls)

1 h4 h6 h12 h24 h

2. Exposure of MupS strains to Mup

• All the MupS strains showed a loss in cell viability proportionate to exposure time

MupS cell viability as a percentage of untreated controls after exposure of surface-attached cells to Mup 200 mg/l for 1, 4, 6, 12, or 24 h, error bars represent SEM - 6 representative strains shown

0

20

40

60

12 h

1 h4 h6 h

24 h

Ce

ll v

iab

ilit

y (

% o

f u

ntr

ea

ted

co

ntr

ol)

2. Exposure of LLR strains to MupOf the 21 LLR strains;• 9/21 showed a decrease in

viability comparable to the MupS isolates (not shown)

• 6/21 LLR showed a significant recovery in cell number after 12 h exposure following an initial reduction (p <0.05) (LLR mean 36% cell survival; range 28 – 44% vs. MupS mean 24% cell survival, range 12 – 22%)

LLR cell viability as a percentage of untreated controls after exposure of surface-attached cells to Mup 200 mg/l for 1, 4, 6, 12, or 24 h, error bars represent SEM

2. Exposure of LLR strains to MupOf the 21 LLR strains;• 6/21 LLR strains, there

was a statistically significant cell recovery observed after 24 h exposure to Mup; LLR mean 105%; 20 – 286% vs. MupS mean 14%, 3 – 41% (p<0.05)

• The observed tolerance to Mup was genotype independent (data not shown)

LLR 100

LLR 205

LLR 206

LLR 4

LLR 13

LLR 15

0

20

40

60

100150200250300

1 h

4 h

6 h

12 h

24 h

Cel

l Su

rviv

al (

% o

f u

ntr

eate

d c

on

tro

l)

LLR cell viability as a percentage of untreated controls after exposure of surface-attached cells to Mup 200 mg/l for 1, 4, 6, 12, or 24 h, error bars represent SEM

2. Repeated exposure mimicking treatment

• Three surface adherent strains challenged with 200 mg/L Mup for 1 h twice a day for 5 days mimicking treatment

• After treatment cells were allowed to recover in antibiotic free broth for 11 h prior to the next treatment

• Cell viability was recorded and the extent of recovery between treatments determined

T = sampling point after 1 h Mup exposure, every 12 h for 5 days

T0T1 T2 T3 T4 T5 T6 T7 T8 T9

T101.0105

1.0106

1.0107

1.0108

1.0109

Mupirocin (Exposure time points)

Nu

mb

er o

f ce

lls (

cfu

/ml)

T0T1 T2 T3 T4 T5 T6 T7 T8 T9

T101.0105

1.0106

1.0107

1.0108

1.0109

Mupirocin (Exposure time points)

2. Repeated exposure mimicking treatment

S. aureus cell numbers as a percentage of untreated controls after repeated exposure of surface-attached cells to Mup 200 mg/l, black untreated, blue treated, error bars SEM

• After 5 days the viable cell number of the MS strain was reduced to 15% of the control, whereas for the two LLR strains 20% and 39% of cells remained compared to untreated cultures, though this did not reach statistical significance

MupS 177 LLR 17 LLR 206

T0T1 T2 T3 T4 T5 T6 T7 T8 T9

T101.0105

1.0106

1.0107

1.0108

1.0109

Mupirocin (Exposure time points)

3. Antibiotic induced stress - Stringent Response

• Antibiotic tolerant bacteria can resist cell killing but are not resistant

• Environmental stresses can provoke a stress phenomenon in bacterial cells termed the Stringent Response

• Hallmark of response – small molecule alarmones, pppGpp and ppGpp

• Bifunctional RelA synthetase (S)/hydrolase (H) enzyme

3. Antibiotic induced stress - Stringent Response

Global cellular reprogramming

↓ rRNA synthesis ↓ anabolic processes

↑ amino acid biosynthesis

↑ stress survival genes

U A C

Bacteriostatic

Pi Pi

GTPATP

RelA synthetase

ppGpp

GDP

pppGpp

RelA hydrolase PPi

GTP poolsDe-represses CodY

(transcriptional repressor)

Ribosome

E P A

3’

5’

SH

SH

RelA

Summary• Binding of S. aureus to a mucin-coated surface confers

tolerance to mupirocin

• LLR conveys an increased level of tolerance after single dose and repeated exposures to mupirocin

• The in vitro tolerance to mupirocin observed in LLR strains might underlie the higher rate of relapse following decolonisation therapy

• The impact of a mupirocin-induced Stringent Response on decolonisation therapy remains to be fully determined

Acknowledgements

Andrew AnyakwoDr Lesley PriceDr Kirsty Skinner

Dr Liz Dickson, Scottish MRSA Reference Laboratory, GRI