The Case for Antimicrobial Copper

breaking the chain of infection

Prof Bill Keevil PhD
Accounts for an additional ~$45 Billion
in health care costs in the USA

WHO suggest ~$80 Billion worldwide

"Unless we take global action, antimicrobial resistance will
become an even greater threat to mankind than cancer ..”

Kill as many as 10 million people by 2050, one every 3 seconds

Cost $100 trillion

Telegraph April 14, 2016

UN meeting tackles the 'fundamental threat' of antibiotic-
resistant superbugs

All 193 UN member states sign declaration agreeing to combat the

proliferation of drug-resistant infections, estimated to kill more than
700,000 people each year.

In two years, groups including UN agencies will provide an update on the

superbug fight to the UN secretary general

20th September 2016

 Why our Interest in Copper?
Copper Symbols since Antiquity

Eternal Life 銅
Chinese Medicine
increase the flow of ‘chi’
(life energy)

Egyptian Ankh
Alchemy
planetary symbol for Venus, protect
against evil
 Copper Used to Protect Human
Health in the Past
銅
 Egypt (2000 BC) - Sterilize drinking water and wounds

 Greece, Hippocrates (400 BC) – Treat leg ulcers related to varicose veins

 Aztecs – Copper oxide and malachite for skin conditions

 France (1850 AD) – Copper workers found to be immune during Cholera

epidemic

 USA, Phildadelphia Hospital Study (1983 AD) – Low E. coli count on brass
doorknobs

 Punjab, India (2005 AD) – E. coli eliminated in 24 hours in water-filled brass

containers (TamraJal used for 5000 years)
So why is Copper so good? Chemistry

Cu(0) 4s1 3d10

Cu(I) 4s0 3d10
Cu(II) 4s1 3d9

Good for redox reactions

 ANTIMICROBIAL COPPER
Water-borne pathogens in biofilms
• Legionella pneumophila, Helicobacter pylori
• E. coli O157
Food-borne pathogens on surfaces
• E. coli O157, Salmonella
• Listeria monocytogenes

Hospital-acquired pathogens
• MRSA, VRE, C. difficile,
• A. baumannii, CRE, K. pneumoniae NDM-1 etc

• Viruses - influenza H1N1, norovirus, adenovirus, coronavirus

• Fungi- Candida, Aspergillus (HVAC systems)
 Moist contact
_290343001182303604.jpg (JPEG Image, 543×360 pixels)
model
toilet_sneeze.jpg (JPEG Image, 160×126 pix
http://pileofphotos.com/pics/pic_290343001182303604.jpg
 MRSA on Stainless Steel ( ), C19700 ( ),
C24000 ( ) and C77000 ( ) at 20C

Moist test simulating coughs, sneezes etc (20 μL inoculum)

1.E+08

1.E+07
Stainless
1.E+06 *
* steel or
CFU per Coupon

1.E+05

* Silver for
1.E+04
24h
1.E+03 * *
1.E+02

1.E+01

1.E+00 * *** * * *
0 60 120 180 240 300 360
Time (mins)

Figure 1) Effect on MRSA viability of a 6h exposure to either stainless steel (  ),

C77000 ( *OIndicates p=<0.05
), C24000 (  ) compared to zero
or C19700 (  time
) at controls
22ºC. CouponsNoyce
(1 cm et
× al.,
1 cm)JHIwere
63, 289-297 (2006)
 Dry touch surface model
main-qimg-f0a341a110341f5a58a93b75b491448d (JPEG Ima... http://d2o7bfz2il9cb7.cloudfront.net/main-qimg-f0a341a1103...
 2
plaque forming units per cm
pfu per cm metal s
1e+4 1e+4

Norovirus inactivation on

2
1e+3 1e+3

copper surfaces 1e+2

1e+1
1e+2

1e+1

MOIST 1e+0
0 20 40 60
DRY 80 100 120
>267m infections p.a.
140
1e+0
0 2
1e+7
1e+7 Time of exposure to metal surface (minutes)
1e+7 Tim

plaque forming units per cm metal surface

1e+6
1e+6 1e+6 copper C11000
phosphor bronze C51000

pfu per cm metal surface

copper nickel C70600
pfu per cm metal surface

1e+5 1e+51e+5 cartridge brass C26000

nickel silver C75200

2
stainless steel S30400
1e+4 1e+41e+4

2
2

1e+3 1e+31e+3

1e+2 1e+21e+2

1e+1 1e+11e+1

1e+0 1e+01e+0
0 20 40 60 80 100 120 140 0 20 40
0 20 40 60 60 80 80100 120
100 140
120 140
Time of exposure to metal surface (minutes) Time of exposre to metal surface (minutes)
time of exposure to metal surface (minutes)

• for ‘wet’ inoculum rapid loss of viral infectivity occurs on copper and copper nickel
copper C11000
phosphor bronze C51000
copper nickel C70600

with significant reduction after 2 hours contact

cartridge brass C26000
nickel silver C75200
stainless steel S30400

• plus phosphor bronze for dry inoculum, with complete inactivation in several minutes
• nickel silver takes a little longer Warnes and Keevil, PLoS ONE 8, e75017 (2013)
 Rapid inactivation of dry challenge E. coli and
S. typhimurium on copper, brass and stainless steel

Warnes et al., Environ. Microbiol. 14, 1730–1743 (2012)

 Evolution of β-lactamase to carbapenamases e.g. blaNDM-1
December 2009, after unsuccessful treatments in hospitals in New Delhi, a Swedish national was referred back to a Swedish hospital,
where it was discovered that he had acquired an antibiotic-resistant bacterial infection during his stay in India; infected with Klebsiella
pneumoniae (Gram-negative bacterium found in the normal flora of the mouth, skin, and intestines) infection.
NDM-1 gene now found in India, Pakistan, Bangladesh, Australia, Canada, the Netherlands, United States, UK.
Carbapenamases hydrolyse carbapenems called 'antibiotics of last resort’.

1. Penicillins
- β-lactamases

2. Cephalosporins
- BS β-lactamases

3. Cephalosporins e.g. cefoxatime

- ESBL:
- CTX, OXA, TEM
NDM-1 SPREAD

3. Carbapenems e.g. meropenem 'antibiotics of last resort’

- KPC, NDM-1
 Survival of ESBL producing E. coli bla CTX-M-15 on metal
surfaces: ‘dry’ inoculum
Cells in bacteriological medium (BHIB) Cells in PBS
1e+8 1e+8

1e+7 1e+7
log 10 cfu per 1 cm2 metal surface

Log10 cfu per cm2 metal surface

1e+6 1e+6

1e+5 1e+5

1e+4 1e+4

1e+3 1e+3

1e+2 1e+2

1e+1 1e+1

1e+0 1e+0
0 10 20 30 40 50 60 70 0 5 10 15 20 25 30 35
Time of exposure to metal surface (minutes) Time of exposure to metal surface (minutes)
copper C11000 C11000
cartridge brass C26000 C26000
stainless steel S30400 S30400

Cells in PBS die very quickly on copper and copper alloy surfaces.
As for ‘wet’ inoculum the death-rate is reduced if cells are inoculated in complex matrix
particularly at lower copper concentrations Warnes et al. mBio 3, e00489-12 (2012)
E. coli CTX-M plasmid

M. Gołębiewski et al. Antimicrob. Agents Chemother. 2007;51:3789-3795

 Destruction of plasmid DNA of E. coli bla CTX-M-15
following exposure to copper at room temperature
Plasmid preparations of E. coli NCTC 13441 (CTX-M-15, ST 131, ESBL producer containing multi-
antibiotic-resistance plasmid pEK499 (117,536 bp Woodford, 2009))

Cells exposed to stainless steel for 0, 60 and

120 minutes (lanes 4, 5,6 respectively) have
intact plasmid DNA

Cells exposed to copper surfaces for 0, 60 and

120 minutes (lanes 7, 8, 9) demonstrate
progressive denaturation of plasmid DNA over
time.

Lanes 10, 3 untreated cells

Lane 11 is heated cells

0h 1h 2h 0h 1h 2h

Warnes et al. mBio 3, e00489-12 (2012)

 Direct detection of the CTX-M-15 bla gene in the same
plasmid preparations using quantitative PCR (qPCR)
Copy number of beta lactamase gene in antibiotic resistant E. coli
(untreated cells or those exposed to copper and stainless steel surfaces
at room temperature: 'wet' inoculum)

6
If the cT values are converted to
5
actual gene copy number per cell it
can be seen that copy number has
bla copy number per cell

4 depleted over time when exposed

to copper surfaces.
3

0
cell
s T0 T60 l t12
0 T0 T60 T12
0
ated teel e el stee
per per per
re e ss s s s st s co p co p cop
unt Sta
i n l in l e nl es
Sta stai

sample Warnes et al. mBio 3, e00489-12 (2012)

 Can antibiotic resistance genes be transferred by natural
conjugation on surfaces?
Pathogen containing E. coli RECIPIENT
antibiotic resistance gene strain, resistant to
on plasmid (green) sodium azide but
e.g. K. pneumoniae NDM-1 Bacteria sensitive to antibiotic
and E. coli CTX-M-15 mixed
DONOR, sensitive to together on
sodium azide surface

Transconjugants selected for growth on

medium containing antibiotic (e.g. cefotaxime,
meropenem)
AND sodium azide
 Detection of bla CTX-M-15 in possible transconjugants
(selected by ability to grow on medium containing cephalosporin and sodium azide)
Frequency of transfer of beta lactamase gene to recipient strains on
metal surfaces

3.5e-5

3.0e-5
conjugation frequency

2.5e-5

2.0e-5

1.5e-5

1.0e-5

5.0e-6

0.0 www.flickr.com
1 2 3 4 5 6
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Cu prevents transfer
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Conjugation frequency = no. transconjugants /no. donor cells Warnes et al. mBio 3, e00489-12 (2012)
 Survival of bla NDM-1 producing K. pneumoniae on metal surfaces:
‘dry’ inoculum
Cells in bacteriological medium (BHIB)
1e+8 1e+8
Cells in PBS
1e+7 1e+7

log 10 cfu per 1 cm2 metal surface

log 10 cfu per cm2 metal surface

1e+6 1e+6

1e+5 1e+5

1e+4 1e+4

1e+3 1e+3

1e+2 1e+2

1e+1 1e+1

1e+0 1e+0
0 5 10 15 20 0 5 10 15 20

Time (minutes) Time of exposure to metal surface (minutes)

copper C11000 copper C11000
phosphor bronze C51000 phosphor bronze C51000
copper nickel C70600 copper nickel C70600
cartridge brass C26000 cartridge brass C26000
nickel silver C75200 nickel silver C75200
muntz metal C28000 muntz metal C28000
stainless steel S30400 stainless steel S30400

Warnes et al. mBio 3, e00489-12 (2012)

 K. pneumoniae NDM-1 plasmid

Carries 12 resistance genes, including blaNDM-1, armA, and qnrB1, confers resistance to the
three key antimicrobial classes (carbapenems, aminoglycosides, and fluoroquinolones).
Yohei Doi et al. Antimicrob. Agents Chemother 2014;58:5947-5953
Exposure to copper or cartridge brass degrades plasmid DNA of MDR-
Klebsiella pneumoniae (‘dry’ touch contamination)
Degradation of K. pneumoniae plasmid DNA
occurs on copper (lanes 8, 9: 5 and 10
minutes contact respectively) and cartridge
brass (lanes 6, 7 :5 and 10 minutes contact)
but not on stainless steel (lane 5: 10
minutes).
Degraded DNA appears as a ‘smear’ of
multi-sized fragments. This can be seen
clearly in the small 1.5Kbp plasmid which is
evident on untreated, heat-killed and cells
exposed to stainless steel for 10 minutes but
not on copper or alloy (although faint band
can be seen after 5 minutes contact on
alloy)
10’ 5’ 10’ 5’ 10’

SS Brass Cu
Warnes et al. mBio 3, e00489-12 (2012)
 Horizontal transfer of K. pneumoniae bla NDM-1 occurs in
suspension and on stainless steel surfaces
Frequency of transfer of bla NDM-1 to recipient cells on surfaces or in
suspension.

3.5e-6

3.0e-6
SS
conjugation frequency

2.5e-6
Cu
2.0e-6

1.5e-6
Suspension
1.0e-6

5.0e-7
www.flickr.com
0.0
0 2

Time of contact of donor and recipient (hours) at room temperature

Cu prevents transfer
cells in suspension
copper surface
stainless steel surface
Warnes et al. mBio 3, e00489-12 (2012)
P. aeruginosa collapse on Cu

Stainless steel 1h Copper 1h

Gram-positive VRE DNA content and respiration on
stainless steel (inoculum 106 cfu per cm2; 4h)
Dry test simulating hand contact (1 μL inoculum)

E. faecalis

E. faecium

SYTO9 CTC Warnes and Keevil, AEM 77, 6049-59 (2011)

Destruction of VRE DNA and respiration on copper
(inoculum 106 cfu per cm2; 10 min)

E. faecalis

E. faecium

SYTO9 CTC Warnes and Keevil, AEM 77, 6049-59 (2011)

 100% Cu 70% Cu 0% Cu
Rapid breakdown of bacterial DNA
on copper (A) and alloy (C26000)
(B) surfaces compared to stainless
steel (C) occurs as part of killing
mechanism in Gram-positive VRE
or MRSA.

minutes

Warnes et al., Environmental Microbiology 14, 1730-43 (2012)

 E. faecalis : DNA & Respiration Damage Protection
10 min contact with copper
Tiron SYTO 9 EDTA SYTO 9 BCS SYTO 9

Tiron CTC EDTA CTC BCS CTC

Superoxide Cu(II) Cu(I)

Warnes and Keevil, AEM 77, 6049-59 (2011
 A. baumannii : Rapid Dry Assay on copper
Survival of Acinetobacter baumannii outbreak strain
on copper surfaces
1 microlitre inoculum
Dry test simulating hand contact Expt.9 160709

1e+7
1. ROS 2. Cu I/II
1e+6

• Significant short

log 10 cfu per 1 cm2 surface

1e+5
term protection with
D- Mannitol and 1e+4

Tiron (up to 2.5 1e+3

minutes contact )
1e+2

• Chelator protection
1e+1

continues and is still 1e+0

0 2 4 6 8 10
detected for EDTA Time of exposure to copper surface (minutes)
at 30 minutes PBS
20 mM D-Mannitol
20 mM Tiron
20 mM EDTA
20 mM BCS

Warnes et al., Environmental Microbiology 14, 1730-43 (2012)

ROS generation by A. baumannii on copper surfaces

• EDIC image reveals the

location of bacterial cells on
the ridges of the copper
coupon.

• After 15 minutes contact

ROS-positive staining cells
detected in the surface
ridges = Metabolic Suicide

• No staining on stainless
steel

Warnes et al., Environmental Microbiology 14, 1730-43 (2012) H2DFFDA

 Bacterial metabolic suicide on Cu

Cu overwhelms the cell

Cu (I) + H2O2 → Cu (II) + O2 + OH- + OH.

• Rapid 200,000x uptake of Cu(I) in seconds and attack
• Generation and attack by Reactive Oxygen Species
 Conclusions
• Contact surfaces are hitherto unrecognised reservoir for rapid HGT and emergence
of superbugs
• Copper alloys kill rapidly, particularly on dry contact
• Continuous activity 24/7 through Cu(I)/(II) and ROS

• Rapid destruction of genomic and plasmid nucleic acid could:

• prevent mutational resistance developing
• help reduce the spread of antibiotic resistance genes to receptive and
potentially more virulent organisms
• as well as genes responsible for virulence and toxin production.

• Combination of effective cleaning regimes and contact surfaces containing copper

could be invaluable to prevent spread of viable pathogens and AMR.
 INTERVENTION WITH COPPER

Schmidt et al., JCM 50, 2217-2223 (2012

 9,244

688
3,579
17,336

4,501

250/100cm2 < Benign

When we look, the risk is omnipresent!

M. Schmidt
 Risk was Significantly Lower with Copper

366
6,474

16 rooms sampled weekly for a period of 21 months, n=1012 Schmidt et al., JCM 50, 2217-2223 (2012)
Link between environmental bioburden and acquisition
of HCAIs reported

89% of HCAIs
occurred among
patients in rooms
with a bioburden
> 500 cfu/100cm2

Salgado et al., ICHE 34, 479-486 (2013)

 Ward Trials Worldwide

>58% infection reduction >90% bacterial reduction

HCAIs: 8.43% 58.1% reduction HCAIs: 3.4%

(p= 0.013)
 Summary
• Superbugs are killed on copper alloy surfaces and antibiotic resistance
gene transfer, which can easily occur, is abolished.

• Influenza, coronavirus and norovirus survive for extended periods on

contemporary materials but are rapidly inactivated on copper alloy
surfaces.

• epic3 guidelines recognise high-touch surfaces made of antimicrobial

copper alloys harbour 80–90% fewer bacteria than equivalent, non-
copper surfaces in busy wards undergoing routine cleaning worldwide.

• USA studies in 3 hospitals have demonstrated a 58% reduction in

infection rate in ICUs
 Copper alloy touch surfaces are an additional
infection prevention measure
Works 24/7

>400 Cu alloys now registered with US EPA with

an antimicrobial claim – being deployed in
Copper reduces bioburden and infection healthcare, public buildings, public transportation
Saves lives, saves £££ etc
New build hospital, payback in 3 months
 Acknowledgements
• University of Southampton:
– Sarah Warnes, Callum Highmore
– Sandra Wilks, Jonathan Noyce, Louise Weaver, Emma Goode

• Copper Alliance
– Harold Michels