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The antimicrobial activity of copper and copper
alloys against nosocomial pathogens and
Mycobacterium tuberculosis isolated from
healthcare facilities in the Western Cape:
an in-vitro study
S. Mehtar
a,
*, I. Wiid
b
, S.D. Todorov
c
a
Academic Unit for Infection Prevention and Control, Department of Community Health, University
of Health Sciences, Faculty of Health Sciences, Tygerberg, Western Cape, South Africa
b
Division of Molecular and Cellular Biology and Human Genetics, University of Stellenbosch,
Faculty of Health Sciences, Tygerberg, Western Cape, South Africa
c
Department of Microbiology, University of Stellenbosch, Stellenbosch, Western Cape, South Africa
Received 6 June 2007; accepted 9 October 2007
Available online 11 December 2007
KEYWORDS
Copper; Alloys;
Mycobacterium
tuberculosis; Bactec
12B growth medium;
In-vitro activity; MRSA;
Acinetobacter
baumannii;Klebsiella
pneumoniae;
Candida albicans
Summary Clinical isolates of meticillin-resistant Staphylococcus aureus
(MRSA), Klebsiella pneumoniae,Pseudomonas aeruginosa,Acinetobacter
baumannii, Candida albicans and Mycobacterium tuberculosis (MTB) were
tested against copper (Cu) and its alloys. Stainless steel and polyvinylchlor-
ide (PVC) were used as controls. The amount of Cu required to inhibit test
isolates at room temperature (24 C) and at 4 C was determined. At room
temperature, Cu, DZR Brass (Cu 62%, Pb 2.5%, arsenate 0.13% and Zn
22.5%) and Brass 70/30 (Cu 70% and zinc 30%) inhibited C. albicans and
K. pneumoniae at 60 min; nickel silver (NiAg) inhibited C. albicans at
60 min and K. pneumoniae at 270 min. P. aeruginosa was inhibited by Brass
70/30 and nickel silver (NiAg) at 180 min and at 270 min by Cu and DZR. Cu
and DZR inhibited A. baumannii at 180 min while the other alloys were ef-
fective at 360 min. Stainless steel and PVC showed little or no inhibitory ac-
tivity. Two M. tuberculosis strains, one isoniazid resistant (R267) and the
other multidrug resistant (R432), demonstrated growth inhibition with Cu
of 98% and 88% respectively compared with PVC; the other alloys were less
* Corresponding author. Address: Academic Unit for Infection Prevention and Control, Department of Community Health, University
of Health Sciences, Faculty of Health Sciences, Tygerberg 7505, Western Cape, South Africa. Tel.: þ27 21 938 5051; fax: þ27 21 938
5065.
E-mail address: smehtar@sun.ac.za
0195-6701/$ - see front matter ª2007 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.jhin.2007.10.009
Journal of Hospital Infection (2008) 68,45e51
A
vailable online at www.sciencedirect.com
www.elsevierhealth.com/journals/jhin
active. Time to positivity (TTP) for R267 was >15 days with Cu and 11 days
for the other alloys; with R432 it was 5 days. Effective inhibition of noso-
comial pathogens and MTB by Cu and alloys was best when the Cu content
was >55%.
ª2007 The Hospital Infection Society. Published by Elsevier Ltd. All rights
reserved.
Introduction
An alarming increase in antibiotic resistance
among hospital pathogens has revived interest in
alternative methods of reducing bioburden in
healthcare facilities, focusing on the environment
within hospitals.
1
Copper is known to have activity against bac-
teria and fungi. Its natural ability to reduce the
bioburden of environmental microbes is exploited
in water purification, paint and building material,
and the textile industry. The activity of Cu against
Gram-positive cocci such as meticillin-resistant
Staphylococcus aureus (MRSA) and Gram-negative
bacilli causing food-associated disease, such as
Escherichia coli O157 Campylobacter jejuni and
Salmonella spp., has been reported.
2
More recently
multidrug-resistant (MDR) and extremely drug re-
sistant (XDR) Mycobacterium tuberculosis (MTB)
in South Africa has drawn attention to the spread
of tuberculosis in hospitals.
3,4
The aim of this study was to establish the in-vitro
activity of Cu and its alloys against highly multiple-
antibiotic-resistant nosocomialpathogens, yeast and
MTB isolated from South African patients. Test
strains were selected from the currently prevalent
nosocomial isolates in healthcare facilities and clin-
ical isolates of MTB from the Western Cape, and used
to establish the minimum quantity of Cu required in
an alloy that would produce sterilisation.
One of the drawbacks of Cu is discoloration
when exposed to oxygen. This study also aimed to
establish the Cu content in alloys with low tarnish-
ing properties which could be applicable to health-
care facilities with sufficient antimicrobial activity
to reduce the environmental bioburden.
Methods
Metal alloys
Metal coupons (1 cm 1 cm) of Cu and its alloys
(supplied by the International Copper Association)
were tested against multiple-antibiotic-resistant
clinical isolates. The coupons were made of de-
oxided phosphorus high (DPH) Cu containing 99.9%
Cu, Brass 70/30 (Cu 70%, zinc 30%), copper nickel
(CuNi) (Cu 90% and Ni 10%), nickel silver (NiAg)
(Cu 55%, Ni 18%, Zn 27%), dezincification resistant
(DZR) Brass (Cu 62%, Pb 2.5%, arsenate 0.13%, Zn
22.5%). Stainless steel (SS) and polyvinylchloride
(PVC) were included as controls. Each set of cou-
pons was allocated a code which was broken at
the end of the study. The coupons were sterilised
by autoclaving followed by flaming with 70% etha-
nol, and stored in sterile Petri dishes; the differ-
ence in the inhibitory effect of Cu and its alloys
by the two methods of sterilisation was noted.
Bacterial and fungal strains
Candida albicans,Pseudomonas aeruginosa,Kleb-
siella pneumoniae and meticillin-resistant Staphy-
lococcus aureus (MRSA) were isolated from blood
cultures, wounds and endotracheal sites of pa-
tients admitted to the intensive care unit. The
strain of Acinetobacter baumannii used was a mul-
tiple-antibiotic-resistant isolate from a patient in
the burns unit. Two clinical strains of MTB, Strain
R267 resistant to isoniazid alone (>0.1 mg/ml)
and Strain R432 multidrug resistant (isoniazid >
0.1 mg/ml, rifampicin >2.0 mg/ml, streptomycin >
2.0 mg/ml, and ethambutol >2.5 mg/ml), were
tested against Cu and its alloys, stainless steel
(SS) and PVC (control) at both room temperature
(25 C) and 4 C.
Killing curves
Non-MTB strains
2
A single colony of each test strain was transferred
to Sabouraud agar for C. albicans, and Brain Heart
Infusion (BHI, Biolab, South Africa) for K. pneumo-
niae,S. aureus,P. aeruginosa and A. baumannii
and incubated at 37 C overnight. After purity
had been checked by Gram stain, 0.1 ml of C. albi-
cans was inoculated into 15 ml of Sabouraud and
similarly K. pneumoniae,P. aeruginosa,A. bau-
mannii and S. aureus were inoculated in 15 ml of
46 S. Mehtar et al.
BHI and incubated at 37 C overnight to yield
w510
8
cfu/ml.
Each coupon was inoculated with 20 ml of culture
(2.5 10
7
cfu) and spread using the tip of a sterile
pipette. The coupons were then incubated at
either room temperature or at 4 C for the desig-
nated exposure period (i.e. 15, 30, 45, 60, 75, 90,
180, 270 and 360 min). After incubation, the coupons
were placed in sterile disposable screw-capped
bottles containing 10 ml sterile phosphate-buffered
saline (PBS) with w20 glass beads (2 mm in diame-
ter), and centrifuged for 30 s at 300 g. One hundred
microlitres were removed and serially diluted
through four 10-fold dilutions in sterile PBS. Nutri-
ent agar plates were inoculated with 100 ml of each
dilution and spread evenly over the surface of the
agar with a sterile glass rod. After incubation at
37 C for 18 h the counts from each coupon were
recorded. Each set of coupons was tested in tripli-
cate from each dilution at both temperatures; the
mean was taken as the final result. The controls
were sampled at time zero and the initial number
of viable cells determined.
MTB strains
All mycobacterial work was carried out in a Level
III Biosafety laboratory. MTB strains were grown on
LowensteineJensen culture medium. After incu-
bation at 37 C for 21 days, purity of the MTB
strains was ascertained by acid-fast staining and
then suspended in 7H9 mycobacterial growth me-
dium. The bacterial suspension was added to a Bac-
tec vial containing the radiolabelled Bactec 12B
growth medium (Becton Dickinson, Franklin Lakes
NJ, USA). To obtain an approximately standard in-
oculum, vials were checked every 24 h until
a growth inhibition (GI) index of 300e500 was
achieved; this index is a quantitative determina-
tion of radioactive CO
2
on a scale from 0 to 999.
The bacterial suspension was added to a Bactec
vial containing Bactec 12B. Growth was monitored
every 24 h until a GI-value of 300e500 was
reached; 0.1 ml of this culture was added to
a new Bactec vial and the growth monitored every
24 h until a GI-value of 500 was reached. This pri-
mary culture was used for testing bacterial viabil-
ity against a variety of metal alloys. The metal
coupons were aseptically arranged in a sterile plas-
tic 150 mm Petri dish which contained a 2 mm
layer of sterile 7H11 mycobacterial agar to prevent
drying and disc movement during handling. One
hundred microlitres of the primary culture was
added to each disc using a 1 ml tuberculin syringe.
Each 100 ml aliquot was gently agitated to eventu-
ally form an 8 mm circular spread on the disc. The
Petri dishes were covered and sealed.
After 72 h at 37 C (three doubling times) cul-
tures were aspirated from the individual coupons
and added to a fresh Bactec vial which was then in-
cubated at 37 C and the GI index monitored every
24 h for up to 15 days.
The inhibitory effect of the coupons on myco-
bacterial growth was evaluated by the time
taken for the culture to become detectable by
Bactec with a growth index of >10 [time to
positive (TTP)].
Results
Killing curves showing the antimicrobial activity of
the metal alloys at room temperature are illustrated
while the activity at 4 C is described in the text.
Overall, better antimicrobial activity was noted for
all the metal alloys at room temperature than 4 C.
Candida albicans
The activity of Cu and related alloys was better
at room temperature (Figure 1)thanat4
C. Cu
was equally active against C. albicans at both
temperatures, resulting in a reduction from 10
7
colony-forming units (cfu)/ml to zero at 60 min.
The antifungal activity of NiAg improved from
360 min at 4 C to 60 min at room temperature.
Other alloys such as DZR, Brass 70/30 and
CuNi showed a 100-fold reduction at 4 Cafter
260 min and total killing at60 min and 90 min respec-
tively at room temperature (Figure 1). It was note-
worthy that at 360 min C. albicans was not isolated
from SS; a 100-fold reduction was noted with PVC.
Klebsiella pneumoniae
This was completely inhibited by Cu at both
temperatures by 60 min. A 100-fold reduction was
noted for DZR and NiAg at 4 C after 360 min. At
room temperature the antibacterial activity of
Cu, DZR and Brass 70/30 against K. pneumoniae
was complete within 60 min and with NiAg total in-
hibition was evident at 270 min (Figure 2). Neither
SS nor PVC demonstrated a lethal effect on
K. pneumoniae at either temperature.
Pseudomonas aeruginosa
Copper and its alloys showed little or no effect
against P. aeruginosa at 4 C; however, at room
temperature, total inhibition was achieved with
Brass 70/30 and NiAg at 180 min and with Cu and
DZR at 270 min (Figure 3). A 100-fold reduction
was noted with CuNi but not with either SS or PVC.
Copper and alloys against bacteria, fungi and MTB 47
Acinetobacter baumannii
Copper and all its alloys inhibited A. baumannii af-
ter 180 min at room temperature (Figure 4); at 4 C
DZR demonstrated total inhibition of the strain at
180 min, whereas the other metals demonstrated
the same effect only after 360 min. There was no
effect with either SS or PVC.
Meticillin-resistant Staphylococcus aureus
(MRSA)
There was little or no inhibition of MRSA at 4 C
when exposed to any of the metal alloys. However,
at room temperature, inhibition was found with
NiAg and Brass 70/30 at 180 min and with Cu and
DZR at 270 min (Figure 5). A 100-fold reduction
8
7
6
5
4
3
2
1
0
cfu/ml
15 30 45 60 75 90 180 270 360
Time (min)
SSDZR CuNi 10
Cu NiAg
Br70/30
PVC
Figure 1 Killing curves for C. albicans (intensive care unit, room temperature). C. albicans was killed after 60 min
exposure to copper (Cu), DZR Brass, copper nickel (CuNi) and at 90 min with Brass (Br) 70/30 and nickel silver (NiAg).
PVC, polyvinylchloride; SS, stainless steel.
9
8
7
6
5
4
3
2
1
0
15 30 45 60 75 90 180 270
Time (min)
cfu/ml
360
Figure 2 Killing curves for K. pneumoniae (intensive care unit, room temperature). K. pneumoniae was killed after
60 min exposure to copper and its alloys except NiAg (270 min). No killing effect was noted by polyvinylchloride (PVC)
or stainless steel (SS) on prolonged exposure. For key, see Figure 1.
48 S. Mehtar et al.
was noted with CuNi at room temperature; neither
PVC nor SS demonstrated any antimicrobial effect.
Overall, the antimicrobial activity of Cu and its
alloys was much less at 4 C than at room tempera-
ture except for Cu against C. albicans and K. pneu-
moniae; at room temperature, total killing within
the 360 min of the experiment was noted against
all strains tested except MRSA (Figures 1e5). DZR
and NiAg were equally active against the various
test isolates, except K. pneumoniae for which
DZR was better. Brass 70/30 was active against P.
aeruginosa, C. albicans, K. pneumoniae and MRSA.
Mycobacterium tuberculosis
Two clinical strains of MTB, one resistant to isoniazid
alone (R267) and the other multidrug resistant
(R432), were tested against Cu and its alloys for
inhibitory activity.
Growth inhibition
Growth inhibition, relative to the control, was
calculated when both cultures reached GI >300.
Strain R267 was inhibited by Cu (98% inhibition)
and remained so after 15 days of incubation. Com-
pared with the control (PVC), growth inhibition by
DZR (78%), Brass 70/30 (83%), CuNi (81%) and NiAg
(64%) was superior (Table I).
The MDR clinical isolate of MTB (R432) became
culture positive within 24 h of incubation. R432
showed maximum inhibition by Cu alone [88%
growth inhibition, followed by DZR (76%)]. Growth
inhibition by the other alloys such as Brass 70/30,
CuNi and NiAg averaged 29.7% inhibition. Compared
with R267 where the average growth inhibition was
76.1% for the above alloys, R432 demonstrated less
growth inhibition. Stainless steel did not affect the
growth rate of strain R432 (3%).
9
8
7
6
5
4
3
2
1
0
15 30 45 60 75 90 180 270 360
Time (min)
cfu/ml
Figure 3 Killing curves for P. aeruginosa (burns unit, room temperature). P. aeruginosa was killed by between 180
and 270 min after exposure to copper and its alloys; no killing effect was noted with stainless steel (SS) or polyvinyl-
chloride (PVC). For key, see Figure 1.
8
9
7
6
5
4
3
2
1
0
15 30 45 7560 90 180 270 360
Time (min)
cfu/ml
Figure 4 Killing curves for A. baumannii (burns unit, room temperature). A. baumannii was killed by all concentra-
tions of Cu when exposed for 180 min. PVC, polyvinylchloride; SS, stainless steel. For key, see Figure 1.
Copper and alloys against bacteria, fungi and MTB 49
Time to positivity (TTP)
When strain R267 was exposed to Cu and its alloys,
the time for the culture to become Bactec positive
(GI >10) in the presence of Cu was >5 days
whereas for other Cu-containing alloys it was 11
days, Brass 70/30 demonstrating the highest inhibi-
tion (>11 days). Compared with SS (five days) and
PVC (eight days), the antimicrobial activity of Cu
and its alloys was at least twice as much (Table I).
TTP for R432 was less notable. Cu and its alloys
demonstrated inhibition of up to five days com-
pared with 24 h for SS and three days for PVC.
Overall, the more sensitive strain of MTB was in-
hibited by Cu and its alloys compared with R432.
Discussion
Copper and its alloys demonstrated good antimi-
crobial activity against multiple-antibiotic-resistant
nosocomial bacteria and C. albicans isolated from
a Western Cape tertiary hospital. Stainless steel is
widely used in the healthcare environment be-
cause it is corrosion resistant to most cleaning
materials and always appears clean; our studies
show that there is no inherent antimicrobial
advantage to using this metal. Native PVC is used
in the manufacture of most disposable laboratory
and hospital plastic ware and has been reported to
permit the formation biofilms.
5
Hand hygiene remains the single most effective
strategy for preventing cross-contamination yet
compliance among healthcare workers remains
poor, resulting in the inevitable contamination of
the surrounding hospital environment.
6,7
Door han-
dles, in particular, may be important secondary
reservoirs for cross-contamination in healthcare
facilities.
8
Boyce et al. found that when patients
with MRSA in a wound or urine were admitted to
the ward, the environmental contamination in-
creased from 27% to 36%. Environmental contami-
nation occurred in the rooms of 73% infected
patients and 69% colonised patients.
9
It is well rec-
ognised that touch surfaces in a healthcare facility
are a potential source of transmission, particularly
for MRSA.
K. pneumoniae is well known not only to cause
serious nosocomial infections but also as a source
9
8
7
6
5
4
3
2
1
0
15 30 45 60 75 90 180 270 360
Time (min)
cfu/ml
Figure 5 Killing curves for meticillin-resistant Staphylococcus aureus (MRSA; intensive care unit, room tempera-
ture). MRSA was killed within 180 min of exposure to NiAg and Brass 70/30, and at 270 min of exposure to Cu and
DZR. For key, see Figure 1.
Table I Growth inhibition (GI) index and time to
positivity (TTP) for two clinical isolates of Mycobac-
terium tuberculosis (MTB) when exposed to copper
(Cu), Cu alloys and stainless steel (SS)
Alloy MTB strain R432 MTB strain R267
% GI TTP (days) % GI TTP (days)
Primary culture 0 4
Copper 87.9 w5 98.0 >15
DZR Brass 75.9 w5 78.5 11
Brass 70/30 37.0 4 83.0 >11
CuNi 27.7 4 80.8 11
NiAg 24.5 4 64.5 11
SS 3 1 0 ~5
PVC (control) 0 3 0 8
PVC was used as control with no growth inhibition.
Percentage GI was calculated at the time when the GI of the
polyvinylchloride (PVC) culture was >300. At this time the GI
values of the inocula from the other materials were calcu-
lated as proportions of the PVC culture, and were taken as
the growth inhibition. TTP is expressed in days before an
MTB strain becomes culture positive in Bactec 12B growth
medium (GI >10).
50 S. Mehtar et al.
of antibiotic resistance genes including extended-
spectrum b-lactamases; these have been identified
in South Africa.
10
P. aeruginosa has been reported
to colonise the environment and equipment sur-
rounding cystic fibrosis patients.
11
Copper and its alloys showed a marked inhibitory
effect on MTB, despite the strains being drug re-
sistant. Growth of both strains showed inhibition by
Cu (88e98% inhibition). The monodrug-resistant
strain (R267) was inhibited for a longer period
(15 days) than the multidrug-resistant strain R432
(w6e7 days). Multidrug resistance in MTB could also
reflect reduced susceptibility to other substances
such as Cu. It is possible that multidrug resistance in
MTB may also involve other genes rendering MTB
more resistant to the inhibitory effect of heavy
metals such as Cu. This may be an explanation for
the difference between R267 and R432 in their
response to the alloys in that they have different
drug profiles. Copper resistance has been demon-
strated in E. faecium mediated by the tcr genes.
12
From this study we conclude that the minimum
concentration of Cu to be an effective antimicro-
bial agent is >55% for bacteria excluding MTB and
for yeasts. The incorporation of Cu in healthcare
facilities may well assist in the reduction of the
environmental bioburden and would be a useful
adjunct to the current infection prevention and
control armamentarium.
The findings for MTB suggest that whereas Cu
has activity against the strains tested here, in-
cluding the MDR-TB strain, higher concentrations
of Cu are required to produce a satisfactory out-
come. Based on these and other studies, applica-
tion of Cu touch surfaces in healthcare facilities
should be evaluated.
Acknowledgements
We are grateful for the support from Professor
Leon Dicks and Professor Paul van Helden towards
undertaking this study.
Conflict of interest statement
None declared.
Funding sources
Support received from the International Copper
Association.
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Copper and alloys against bacteria, fungi and MTB 51
