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Antimicrobial activity of copper and silver Nano films on nosocomial bacterial species

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Irina Codita at Cantacuzino Institute
Irina Codita
Dana Magdalena Caplan
Dana Magdalena Caplan
Dana Magdalena Caplan
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Elena Carmina Dragulescu at Cantacuzino National Institute of Research
Elena Carmina Dragulescu
  • Cantacuzino National Institute of Research
Brindusa Elena Lixandru at Cantacuzino Institute
Brindusa Elena Lixandru
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Abstract and Figures

Contaminated surfaces are possible vehicles in infection transmission. It is known that both Copper (Cu) and Silver (Ag) efficiently inactivate microbes by direct contact. Aiming at using these metals for benefitting from their antimicrobial effect, but to avoid subsequent toxic effects, we evaluated the antimicrobial activity of nanometric thin Silver and Copper films covering less expensive materials. Using a modified version of the Japan Industrial Standard JIS Z 2801:2000, we demonstrated the antimicrobial activity of the surfaces covered with metal ions nanofilms on microorganisms possibly involved in nosocomial infections and on Bacillus anthracis, bacteria with possible implication in bioterrorist attacks. Copper covered surfaces proved to have better antimicrobial activity than Silver surfaces. Silver covered surfaces showed better activity on Gram negative bacteria than on Gram positive cocci. Going deeper with studies on antimicrobial effects using new methods with better direct and/or functional discriminatory capacity is needed in order to provide additional information on the mechanisms of Silver and Copper nanofilms antimicrobial activity.
The TVA plasma ignited in a) Ag and b) Cu vapors and samples; c) plates and d) spheres covered with Ag/Cu. Original images obtained at the National Institute of Research-Development for Lasers, Plasma and Radiation Physics, Bucharest
The TVA plasma ignited in a) Ag and b) Cu vapors and samples; c) plates and d) spheres covered with Ag/Cu. Original images obtained at the National Institute of Research-Development for Lasers, Plasma and Radiation Physics, Bucharest
… 
Schematic representation of the test used for antimicrobial activity invetigation of the stainless steel and metal ions nanofilm covered plates. All bacteria will be in contact with the plate surface, as the filter diameter is smaller than the plate dimensions. After Necula et al. (2009). a. The liquid component of the b. The inoculated filter is placed with the bacterial suspension is absorbed; side with retained bacteria in contact with bacteria are retained on the filter the metal plate
Schematic representation of the test used for antimicrobial activity invetigation of the stainless steel and metal ions nanofilm covered plates. All bacteria will be in contact with the plate surface, as the filter diameter is smaller than the plate dimensions. After Necula et al. (2009). a. The liquid component of the b. The inoculated filter is placed with the bacterial suspension is absorbed; side with retained bacteria in contact with bacteria are retained on the filter the metal plate
… 
Time dependent curves of antimicrobial activity of Copper and Silver nanofilm covered surfaces expressed by the number of CFU/ml recovered from the washing liquid, Staphylococcus aureus ATCC 25923. Comparison with the stainless steel uncovered surface
Time dependent curves of antimicrobial activity of Copper and Silver nanofilm covered surfaces expressed by the number of CFU/ml recovered from the washing liquid, Staphylococcus aureus ATCC 25923. Comparison with the stainless steel uncovered surface
… 
Time dependent curves of Copper and Silver nanofilm covered surfaces antimicrobial activity expressed by the number of CFU/ml recovered from the washing liquid, Staphylococcus aureus ATCC 6538 . Comparison with the stainless steel uncovered surface
Time dependent curves of Copper and Silver nanofilm covered surfaces antimicrobial activity expressed by the number of CFU/ml recovered from the washing liquid, Staphylococcus aureus ATCC 6538 . Comparison with the stainless steel uncovered surface
… 
Time dependent curves of Copper and Silver nanofilm covered surfaces antimicrobial activity expressed by the number of CFU/ml recovered from the washing liquid, Enterococcus faecium ATCC 35667 . Comparison with the stainless steel uncovered surface.
+5
Time dependent curves of Copper and Silver nanofilm covered surfaces antimicrobial activity expressed by the number of CFU/ml recovered from the washing liquid, Enterococcus faecium ATCC 35667 . Comparison with the stainless steel uncovered surface.
… 
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INTRODUCTION
Contaminated surfaces are possible vehicles in
infection transmission. This aspect stimulated scien-
tific research on materials with antimicrobial activity
in the last years, when antimicrobial resistance be-
came a public health concern. Different implications
in several areas of activity, including the medical
environment have been communicated (1-14).
It is known that both Copper and Silver effi-
ciently inactivate microbes by direct contact (13).
Due to this property, objects made of Silver have
been used since ancient times. Copper has not been
used much due to its known toxicity. Massive use of
these metals was not possible also due to their rela-
tively high cost. Aiming at using these metals in less
quantity while benefitting from their antimicrobial
effect, we evaluated the antimicrobial activity of
nanometric thin Silver and Copper films covering less
expensive materials. The final result of such an
approach would be the extensive use of Ag and Cu to
control infection in hospitals, schools, passenger
transportation vehicles and in every area of activity
where infection may occur.
IV.2. MATERIALS AND METHODS
Copper and Silver covered surfaces
50x50x2 mm stainless steel plates were covered
with Cu and also Ag nanolayers using the Thermoio-
nic Vacuum Arc (TVA) plasma source (14).
The TVA plasma is suitable for the deposition of
adherent, very thin metallic films under vacuum envi-
ronment, on different substrates: stainless steel, plas-
tics, textiles etc. Images of the TVA plasma ignited in
Ag and also in Cu vapors are presented in Fig. 1. The
substrates were placed above the plasma source, as
can be observed in this figure.
Apart from stainless steel plates, Silver and Co-
pper covered spherical shape samples were also used
to evaluate the effects of metal ions on the production
of staphylococcal thermonuclease.
Testing for antimicrobial activity was performed
in the Nosocomial Infections and Antimicrobial Re-
sistance Reference Laboratory or in the Zoonoses Refe-
rence Laboratory, “Cantacuzino” National Institute of
Research-Development for Microbiology and Im-
munology, Bucharest.
204
ABSTRACT
Contaminated surfaces are possible vehicles in infection transmission. It is known that both Copper
(Cu) and Silver (Ag) efficiently inactivate microbes by direct contact. Aiming at using these metals for
benefitting from their antimicrobial effect, but to avoid subsequent toxic effects, we evaluated the
antimicrobial activity of nanometric thin Silver and Copper films covering less expensive materials.
Using a modified version of the Japan Industrial Standard JIS Z 2801:2000, we demonstrated the
antimicrobial activity of the surfaces covered with metal ions nanofilms on microorganisms possi-
bly involved in nosocomial infections and on Bacillus anthracis, bacteria with possible implication
in bioterrorist attacks. Copper covered surfaces proved to have better antimicrobial activity than
Silver surfaces. Silver covered surfaces showed better activity on Gram negative bacteria than on
Gram positive cocci.
Going deeper with studies on antimicrobial effects using new methods with better direct and/or
functional discriminatory capacity is needed in order to provide additional information on the
mechanisms of Silver and Copper nanofilms antimicrobial activity.
ANTIMICROBIAL ACTIVITY OF COPPER AND SILVER NANOFILMS
ON NOSOCOMIAL BACTERIAL SPECIES
Irina Codiþã1*, Dana Caplan1, Elena-Carmina Drãgulescu1, Elena Branduºa Lixandru1, Luminiþa Coldea1,
Cerasella Dragomirescu1, Cristina Surdu-Bob2, Marius Badulescu2
1“Cantacuzino” National Institute of Research-Development for Microbiology and Immunology, Bucharest;
2National Institute of Research-Development for Lasers, Plasma and Radiation Physics, Bucharest
Key words: Silver, Copper, nanofilms, antimicrobial activity, nosocomial pathogens, Bacillus anthracis
*
Corresponding author:
Irina Codiþã, I.N.C.D.M.I. CANTACUZINO, Splaiul Independenþei 103, sector 5, 050096, Bucureºti, România,
email: adirina_2005@yahoo.com
Microbial strains
We used eight bacterial strains, from which
seven belonged to species known as being involved
in nosocomial infections.
One Bacillus anthracis nonpathogen strain, B.
anthracis 34 F2Sterne was introduced in the study
because of the possible use of this species as inten-
tional attack biological weapon.
Reference disc diffusion susceptible strains and
other strains recommended to be used for antimicro-
bial activity testing were introduced in the study, as
follows: S. aureus ATCC 25923, S. aureus ATCC
6538, E. coli ATCC 25922, E. coli ATCC 35218, Ente-
rococcus faecium ATCC 35667, Enterococcus faecalis
ATCC 51299, Pseudomonas aeruginosa 27853.
Antimicrobial activity testing
The antimicrobial activity of the metalic ions
nanofilm covered surfaces was tested using a proto-
col representing a modified version of the Japanese
Industrial Standard JIS Z 2801:2000, adapted after
Necula et al., 2009 (12).
We used Copper and Silver nanofilms covered
stainless steel plates, 50/50/2 mm and stainless steel
uncovered plates with the same profile, as negative
probes. All plates used in the experiment were
cleaned with 70% ethanol, rinsed with distilled water
and UV sterilised for 15 minutes, each side before
being inoculated with the bacterial suspension.
Bacterial suspensions were prepared in Trypticase
Soy Broth, corresponding to a 107UFC/ml concen-
tration (0.03 OD620). This concentration is equiva-
lent to a bacterial suspension obtained by preparing
a 1/10 dilution of a suspension corresponding to the
0.5 McFarland standard.
Nitrocellulose filters with 0.40 mporosity, 25 mm
Ø, UV sterilised were used to apply the bacterial
inoculum.
Antimicrobial activity of Copper and Silver nanofilms on nosocomial bacterial species
205
Figure 1. The TVA plasma ignited in a) Ag and b) Cu vapors and samples; c) plates and d) spheres
covered with Ag/Cu. Original images obtained at the National Institute of Research-Development
for Lasers, Plasma and Radiation Physics, Bucharest
These filters were firstly placed on the surface of
a blood-agar plate and a 40 ml volume of bacterial
suspension prepared as described was applied on
their upper surface. The liquid component of the sus-
pension was absorbed by the agar, whilst the bacte-
ria remained on the filter surface (Fig. 2).
A 40 ml 1% Tryptic Soy Broth (TSB) in 10 mM
phosphate volume was centrally placed on the Copper
or Silver covered plates or on the stainless steel
uncovered plates.
The filter inoculated with the microbial suspen-
sion was then carefully placed on the top of the TSB-
phosphate drop, with the initial upside down. Metal
plates were placed in Petri sterile plates and mentained
at 36 ± 1 oC, in a humid athmosphere, for different
time intervals in order to establish the needed time
for bacterial suspension inactivation.
After incubation, the filter-plate installation was
flooded with a 20 mL volume of physiologic saline
and washed by shaking for 60 seconds with a mecha-
nical shaker.
Decimal dilutions were prepared from the washing
liquid from -1 to -7, and 10 ml from each dilution where
then transferred on blood agar plates, in sectors. Tri-
plicate experiments were performed for each strain-
metal plate binome and for each incubation time.
After incubating the plates inoculated with wa-
shing liquid dilutions for 18 hours at 37oC, we counted
the colonies grown on the sectors with distinct colony
growth and calculated the medium number of
CFU/ml in the washing liquid.
Thermonuclease activity
Thermonuclease or heat stable nuclease is an
enzyme elaborated by S. aureus.
Testing of thermonuclease by Lachica method
was performed to detect possible effects on proteins
activity due to the action of metalic ions nanofilms
placed on metalic surfaces.
A series of spherical 2 mm diameter metal ions
nanofilm covered samples were placed in S. aureus
broth cultures and were incubated for 2 hours at 36
± 1oC and then tested for thermonuclease activity.
Lachica method principle is based on detecting
changes in nucleic acids-bromthymol blau comple-
xes caused by the thermonuclease activity, which are
resulting in turning of the complex purple color to a
pink color.
Semiquantitative evaluation of S. aureus thermo-
nuclease activity was done by appreciating the pink
diameter of nucleic acids lysis of each bacterial sus-
pension incubated in the presence of a different num-
ber of metalic ions nanofilm covered balls.
RESULTS
Antimicrobial activity of Copper or Silver nano-
film covered surfaces on bacterial strains with pos-
sible implication in nosocomial infections
Medium number of CFU/ml in the metal plate-
filter installation washing liquid obtained in triplicate
was used for tracing antimicrobial activity curves for
the stainleess steel uncovered and Copper or Silver
covered plates.
All results are presented in the following tables
and graphs.
206
CODIÞÃ et al.
Fig. 2. Schematic representation of the test used for antimicrobial activity invetigation of the stainless
steel and metal ions nanofilm covered plates. All bacteria will be in contact with the plate surface, as
the filter diameter is smaller than the plate dimensions. After Necula et al. (2009).
a. The liquid component of the b. The inoculated filter is placed with the
bacterial suspension is absorbed; side with retained bacteria in contact with
bacteria are retained on the filter the metal plate
Antimicrobial activity of Copper and Silver nanofilms on nosocomial bacterial species
207
Staphylococcus aureus ATCC 25923
Table 3. Antimicrobial activity of metal plates covered with different metal ions nanofilms
depending on the length of the contact time - Staphylococcus aureus ATCC 25923
Fig. 3. Time dependent curves of antimicrobial activity of Copper and Silver nanofilm covered surfaces
expressed by the number of CFU/ml recovered from the washing liquid, Staphylococcus aureus ATCC
25923. Comparison with the stainless steel uncovered surface
Staphylococcus aureus ATCC 6538
Table 4. Antimicrobial activity of metal plates covered with different metal ions nanofilms
depending on the length of the contact time - Staphylococcus aureus ATCC 6538
Fig. 4. Time dependent curves of Copper and Silver nanofilm covered surfaces antimicrobial activity
expressed by the number of CFU/ml recovered from the washing liquid, Staphylococcus aureus ATCC
6538. Comparison with the stainless steel uncovered surface
208
CODIÞÃ et al.
Enterococcus faecium ATCC 35667
Table 5. Antimicrobial activity of metal plates covered with different metal ions nanofilms
depending on the length of the contact time - Enterococcus faecium ATCC 35667
Fig. 5. Time dependent curves of Copper and Silver nanofilm covered surfaces antimicrobial activity
expressed by the number of CFU/ml recovered from the washing liquid, Enterococcus faecium ATCC
35667. Comparison with the stainless steel uncovered surface.
Enterococcus faecalis ATCC 51299
Table 6. Antimicrobial activity of metal plates covered with different metal ions nanofilms
depending on the length of the contact time - Enterococcus faecalis ATCC 51229
Fig. 6. Time dependent curves of Copper and Silver nanofilm covered surfaces antimicrobial activity
expressed by the number of CFU/ml recovered from the washing liquid, Enterococcus faecalis ATCC
51229. Comparison with the stainless steel uncovered surface
Antimicrobial activity of Copper and Silver nanofilms on nosocomial bacterial species
209
E. coli 25922
Table 7. Antimicrobial activity of metal plates covered with different metal ions nanofilms
depending on the length of the contact time - Escherichia coli ATCC 25922
Fig. 7. Time dependent curves of Copper and Silver nanofilm covered surfaces antimicrobial activity
expressed by the number of CFU/ml recovered from the washing liquid, Escherichia coli ATCC 25922.
Comparison with the stainless steel uncovered surface
E. coli ATCC 35218
Table 8. Antimicrobial activity of metal plates covered with different metal ions nanofilms
depending on the length of the contact time - E. coli ATCC 35218
Fig. 8. Time dependent curves of Copper and Silver nanofilm covered surfaces antimicrobial activity
expressed by the number of CFU/ml recovered from the washing liquid, E. coli ATCC 35218. Comparison
with the stainless steel uncovered surface
210
CODIÞÃ et al.
Pseudomonas aeruginosa ATCC 27853
Table 9. Antimicrobial activity of metal plates covered with different metal ions nanofilms
depending on the length of the contact time - Pseudomonas aeruginosa ATCC 27853
Fig. 9. Time dependent curves of Copper and Silver nanofilm covered surfaces antimicrobial activity
expressed by the number of CFU/ml recovered from the washing liquid, Pseudomonas aeruginosa ATCC
27853. Comparison with the stainless steel uncovered surface.
Silver or Copper covered surfaces antimicrobial activity on Bacillus anthracis 34 F2 Sterne strain
Table 10. Antimicrobial activity of metal plates covered with different metal ions nanofilms
depending on the length of the contact time - Bacillus anthracis 34 F2 Sterne
Fig. 10. Time dependent curves of Copper and Silver nanofilm covered surfaces antimicrobial activity
expressed by the number of CFU/ml recovered from the washing liquid, Bacillus anthracis 34 F2 Sterne.
Comparison with the stainless steel uncovered surface.
The effect of Copper and Silver nanofilm on S.
aureus thermonuclease activity
Semiquantitative results of S. aureus thermonu-
clease activity obtained by Lachica method (Fig. 11)
are presented in Table 11.
Preliminary results are leading to the hypothesis
that Copper and Silver are acting depending on the
doses to stimulate the enzyme production in low to
moderate doses, or to inhibit it, in high doses.
DISCUSSION
Possible mechanisms of metal ions antimicrobial
action have been put forward, like oligodynamic effect,
absorbtion in the electric field, catalysis process, dena-
turating action of Silver on the bacterial cell enzymes.
Interaction between a bacterial cell and the surface
is governed primarily by van der Waals and electrosta-
tic forces, depending on the physicochemical pro-
perties of the substratum and the bacterial surface, such
as hydrophobicity, free energy and surface charge (8).
The results obtained in our study demonstrated
the antimicrobial properties of surfaces covered with
metallic Silver or Copper nanofilms prepared using
plasma in vacuum.
Antimicrobial effects were better for Copper,
which has a rapid bactericidal effect on all tested bac-
terial species. With Silver we noticed differences de-
pending on the bacterial species, Gram negative rods
showing greater susceptibility than Gram positive
cocci. On the other hand, differences between Gram
positive cocci of the same Genus and species sugges-
ted possible acquired resistance mechanisms.
The screening study results are encouraging us to
go deeper by using Atomic Force Microscopy and ex-
tending the area of observation on the possible effects
of metal ions covered surfaces on bacterial enzyme
production and activity in potentially nosocomial mi-
croorganisms.
Preliminary results obtained in our study brought
positive proof for Copper nanofilm covered surfaces
use in nosocomial infection prevention and/or dimi-
nishing. These results confirm recent research results
communicated by international teams after the initia-
tion of our project (4).
CONCLUSIONS
1. The methods used in this study were able to
detect the antimicrobial activity of the surfaces covered
with metal ions nanofilms on microorganisms with
possible implication in nosocomial infections.
2. Our experiments demonstrated the antimicro-
bial activity of the surfaces covered with Copper and
Silver nanofilms obtained by TVA plasma on most
potentially nosocomial microorganisms as well as on
Bacillus anthracis, bacteria with possible implication
in bioterrorist attacks.
3. Copper covered surfaces proved to have bet-
ter antimicrobial activity than Silver surfaces.
4. Silver covered surfaces showed better activity
on Gram negative bacteria than on Gram positive
cocci.
5. Going deeper with studies on antimicrobial
effects by using Atomic Force Microscopy studies (8,
15, 16) and by extending our observations about the
metal ions effects on the proteic enzyme production
and activity could provide additional information on
the mechanisms of Silver and Copper nanofilms
antimicrobial activity.
Antimicrobial activity of Copper and Silver nanofilms on nosocomial bacterial species
211
Table 11. Semiquantitative results expressing
the thermonuclease activity appreciated by
measuring the diameter of the pink turned zones
Fig. 11. Checking the thermonuclease activity on
Lachica medium
Legenda: M - positive reference sample; 3 Ag - 3 Silver
balls; 6 Ag - 6 Silver balls; 12 Ag - 12 Silver balls; 24
Ag - 24 silver balls; 1 - 1 Copper ball; 2 - 2 Copper
balls; 4 - 4 Copper balls; 8 - 8 Copper balls
ACKNOWLEDGEMENTS
I.C., E-C.D. acknowledge the support of this work
from the Romanian Ministry of Research through
project PNII-42129/2008.
C.S-B is grateful for the financial support from
POSDRU/89/1.5/S/60746.
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CODIÞÃ et al.
... Compounds were produced as follows: First a master batch was fabricated by extrusion. Afterwards, test specimens (plates) were manufactured by injection molding utilizing a certain amount of the master [3], [4], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16] other microorganisms included: 8 [17], [18], [19], [20], [21], [22], [23], [24] additional microorganisms employed: 6 [4], [6], [7], [9], [10], [11] preparatory incubation of bacteria no information: 20 [3], [4], [6], [7], [8], [9], [10], [11], [12], [13], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24] inconsistent with standard: 1 [14] cell concentration of inoculum no information: 8 [3], [4], [6], [13], [15], [16], [20], [24] according to standard: 7 [8], [12], [14], [17], [18], [19], [21] higher than standard: 5 [7], [9], [10], [11], [22] lower than standard: 1 [23] solution for bacterial suspension no information: 16 [3], [4], [6], [7], [8], [13], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24] according to standard: 4 [9], [10], [11], [12] lower dilution factor: 1 [14] specimen no information: 7 [3], [4], [8], [17], [19], [22] according to standard: 5 [6], [7], [9], [12], [13] other dimensions (smaller, different shape): 9 ...
... Compounds were produced as follows: First a master batch was fabricated by extrusion. Afterwards, test specimens (plates) were manufactured by injection molding utilizing a certain amount of the master [3], [4], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16] other microorganisms included: 8 [17], [18], [19], [20], [21], [22], [23], [24] additional microorganisms employed: 6 [4], [6], [7], [9], [10], [11] preparatory incubation of bacteria no information: 20 [3], [4], [6], [7], [8], [9], [10], [11], [12], [13], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24] inconsistent with standard: 1 [14] cell concentration of inoculum no information: 8 [3], [4], [6], [13], [15], [16], [20], [24] according to standard: 7 [8], [12], [14], [17], [18], [19], [21] higher than standard: 5 [7], [9], [10], [11], [22] lower than standard: 1 [23] solution for bacterial suspension no information: 16 [3], [4], [6], [7], [8], [13], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24] according to standard: 4 [9], [10], [11], [12] lower dilution factor: 1 [14] specimen no information: 7 [3], [4], [8], [17], [19], [22] according to standard: 5 [6], [7], [9], [12], [13] other dimensions (smaller, different shape): 9 ...
... Compounds were produced as follows: First a master batch was fabricated by extrusion. Afterwards, test specimens (plates) were manufactured by injection molding utilizing a certain amount of the master [3], [4], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16] other microorganisms included: 8 [17], [18], [19], [20], [21], [22], [23], [24] additional microorganisms employed: 6 [4], [6], [7], [9], [10], [11] preparatory incubation of bacteria no information: 20 [3], [4], [6], [7], [8], [9], [10], [11], [12], [13], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24] inconsistent with standard: 1 [14] cell concentration of inoculum no information: 8 [3], [4], [6], [13], [15], [16], [20], [24] according to standard: 7 [8], [12], [14], [17], [18], [19], [21] higher than standard: 5 [7], [9], [10], [11], [22] lower than standard: 1 [23] solution for bacterial suspension no information: 16 [3], [4], [6], [7], [8], [13], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24] according to standard: 4 [9], [10], [11], [12] lower dilution factor: 1 [14] specimen no information: 7 [3], [4], [8], [17], [19], [22] according to standard: 5 [6], [7], [9], [12], [13] other dimensions (smaller, different shape): 9 ...
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Bactericidal materials gained interest in the health care sector as they are capable of preventing material surfaces from microbial colonization and subsequent spread of infections. However, commercialization of antimicrobial materials requires proof of their efficacy, which is usually done using in vitro methods. The ISO 22196 standard (Japanese test method JIS Z 2801) is a method for measuring the antibacterial activity of daily goods. As it was found reliable for testing the biocidal activity of antimicrobially active materials and surface coatings most of the laboratories participating in this study used this protocol. Therefore, a round robin test for evaluating antimicrobially active biomaterials had to be established. To our knowledge, this is the first report on inaugurating a round robin test for the ISO 22196 / JIS Z 2801. The first round of testing showed that analyses in the different laboratories yielded different results, especially for materials with intermediate antibacterial effects distinctly different efficacies were noted. Scrutinizing the protocols used by the different participants and identifying the factors influencing the test outcomes the approach was unified. Four critical factors influencing the outcome of antibacterial testing were identified in a series of experiments: (1) incubation time, (2) bacteria starting concentration, (3) physiological state of bacteria (stationary or exponential phase of growth), and (4) nutrient concentration. To our knowledge, this is the first time these parameters have been analyzed for their effect on the outcome of testing according to ISO 22196 / JIS Z 2801. In conclusion, to enable assessment of the results obtained it is necessary to evaluate these single parameters in the test protocol carefully. Furthermore, uniform and robust definitions of the terms antibacterial efficacy / activity, bacteriostatic effects, and bactericidal action need to be agreed upon to simplify communication of results and also regulate expectations regarding antimicrobial tests, outcomes, and materials.
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... Among metal nanoparticles, silver nanoparticles have notable potentials in the field of nanotechnology which has gained utmost interest due to their exceptional properties such as chemical stability, good conductivity, catalytic, and most important antibacterial, anti-viral, antifungal, and anti-inflammatory activities. They have widely employed the development of composite fibers, cryogenic superconducting, food packaging, fabrics, wound healing creams and pastes, cosmetic therapies, dental implants etc [4][5][6][7][8][9]. ...
... Nanoparticle synthesized by using Riccia plant extract assisted method is found to be effective against Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumoniae, Escherichia coli and are found to be effective against all the organism tested. Codiţă et al 2010 [5] reported that both Copper (Cu) and Silver (Ag) have efficient antimicrobial activities , this study also proved that antimicrobial activity was more in gram-positive bacteria than in negative bacteria. A study showed that more Antibacterial activity was observed when treated with.Cu Riccia Np than Ag Riccia Np .However, another study conducted by Mukherji et al 2012 [7] reported that Chemically synthesized Ag Nps have more activity than Cu-Nps. ...
Antimicrobial activity of copper and silver metal nanoparticles synthesized from Riccia fluitans
Article
Full-text available
  • Jul 2021
Riccia bryophyte, a genus of liverworts that comes under the family of Ricciaceae, order Marchantiales. The plants are primitive plant structure, that's not differentiated into root, stem, and leaf. The material utilized in this study was a Spp of a Riccia fluitans, that grows on damp soil or, less unremarkably, floating in ponds, and is usually utilized in aquariums. The nanoparticles utilized in this study exhibit potential medicinal drug activity. Advantages of its therapeutic potential can be utilized in a sizable number of fields like health care, cosmetics, biomedical, food and feed, drug-gene delivery, surroundings, health mechanics, optics, chemical industries, physical science, area industries, energy science, catalysis, lightweight emitters, single lepton transistors, nonlinear optical devices, and photo-electrochemical applications. This study aimed to gauge the medicinal drug activity of Riccian nanoparticles, copper-loaded nanoparticles, and silver-loaded nanoparticles against varied microorganisms. The result of the study showed that the Nanoparticle, Ag-NP and Cu-Np synthesized from Riccia is having good antimicrobial activity against tested organisms.
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... Metals have been also used for their antiseptic, wound healing and subsequently analgesic properties. The antiseptic and wound healing properties of copper were known since the time of Hippocrates; he used copper to disinfect ulcers from varicose veins and treated painful wounds and skin problems with copper and honey [4,8,[54][55][56][57]. Slaves rarely experienced rheumatic or arthritic problems and contaminated wounds because they were chained with copper strips in their wrists and ankles that protected them from such diseases [4,8,20,[54][55][56][57]. ...
... The antiseptic and wound healing properties of copper were known since the time of Hippocrates; he used copper to disinfect ulcers from varicose veins and treated painful wounds and skin problems with copper and honey [4,8,[54][55][56][57]. Slaves rarely experienced rheumatic or arthritic problems and contaminated wounds because they were chained with copper strips in their wrists and ankles that protected them from such diseases [4,8,20,[54][55][56][57]. ...
Pharmacies for pain and trauma in ancient Greece
Article
  • Nov 2018
Purpose To summarize pharmacies for pain and trauma in ancient Greece, to present several pharmaceutical/therapeutical methods reported in myths and ancient texts, and to theorize on the medical explanation upon which these pharmacies were used. Method A thorough literature search was undertaken in PubMed and Google Scholar as well as in physical books in libraries to summarize the pharmacies and pain practices used for trauma in ancient Greece. Results Archeological findings and historical texts have revealed that humans have always suffered from diseases and trauma that were initially managed and healed by priests and magicians. In early Greek antiquity, the term pharmacy was related to herbal inquiries, with the occupants called charmers and pharmacists. Additionally, apart from therapeutic methods, ancient Greeks acknowledged the importance of pain therapy and had invented certain remedies for both acute and chronic pain management. With observations and obtaining experience, they used plants, herbs, metals and soil as a therapeutic method, regardless of the cultural level of the population. They achieved sedation and central and peripheral analgesia with opium and cold, as well as relaxation of smooth muscle fibers and limiting secretions with atropina. Conclusion History showed a lot of experience obtained from empirical testing of pain treatment in ancient people. Experience and reasoning constructed an explanatory account of diseases, therapies and health and have provided for the epistemology of medicine.
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... The majority of antimicrobial agents identified carry a body of evidence supporting their mechanism and utility [45]. The antimicrobial activity of copper and silver, especially in nanoparticle form, is well documented, including towards coronaviruses [46][47][48]. Indeed, N-halamines, graphene and QACs have proved effective against a broad spectrum of microorganisms, with long-term stability and durability [49][50][51]. ...
Enhanced Medical and Community Face Masks with Antimicrobial Properties: A Systematic Review
Article
Full-text available
  • Sep 2021
Face masks help to limit transmission of infectious diseases entering through the nose and mouth. Beyond reprocessing and decontamination, antimicrobial treatments could extend the lifetime of face masks whilst also further reducing the chance of disease transmission. Here, we review the efficacy of treatments pertaining antimicrobial properties to medical face masks, filtering facepiece respirators and non-medical face masks. Searching databases identified 2113 studies after de-duplication. A total of 17 relevant studies were included in the qualitative synthesis. Risk of bias was found to be moderate or low in all cases. Sixteen articles demonstrated success in avoiding proliferation (if not elimination) of viruses and/or bacteria. In terms of methodology, no two articles employed identical approaches to efficacy testing. Our findings highlight that antimicrobial treatment is a promising route to extending the life and improving the safety of face masks. In order to reach significant achievements, shared and precise methodology and reporting is needed.
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... Many environmental parameters, such as humidity, pH, and temperature, also affect the toxicity of AgNPs. Studies have shown that silver nanoparticles when used alone or in combination with copper [41,42] or titanium dioxide [43,44], have a great biocidal effect on heterotrophic microorganisms. Effective results with AgNPs were obtained in diminishing the deterioration capability of many strains of bacteria and pathogenic fungi [45,46], moulds [47], algae such as Aspergillus niger [48], Cladosporium growth on gypsum [49], and termites [50]. ...
INTERNATIONAL JOURNAL OF CONSERVATION SCIENCE HISTORICAL PERSPECTIVE OF THE SULTANATE PERIOD MONUMENTS AND THEIR CONSERVATION USING BIOTECHNOLOGICAL TOOLS
Article
Full-text available
  • Mar 2021
Delhi, the capital of India, offers a treasure trove of many sacred groves, water bodies, structures, and precincts that are of historic, aesthetic, architectural, and cultural significance. Efforts have been made mostly to preserve the architectural concepts of the Mughal era, but Delhi has many splendid monuments of the Sultanate era, which need exigent restoration and repair from getting faded into oblivion. Sadly, only the Qutb complex and Nizamuddin Dargah have received much attention from both government and non-government organisations, but other structures have been left unattended. It is also evident that every monument cannot be given due importance, and scores of capital cannot be utilized for their maintenance, but science and technology can offer alternate methods to restore and repair damages to these lesser-known monuments, which can also be considered in the long-term interest of the society from economic, cultural, and environmental viewpoints. This review is an attempt to delve into the architectural marvels of the Delhi Sultanate period, the cause of their deterioration, and the means of their conservation by the use of biotechnological tools, that is, use of essential oils, nanotechnology, and bio-mineralization as an effective, low-cost, eco friendly option to be adapted for future restoration and conservation of our heritage culture.
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... The commercialization and public use of AgNP is due to the advancement of research on its use on biological systems (Lee et al. 2007). Experiments using AgNP on different strains of bacteria such as E. coli (Sondi and Salopek-Sondi 2004), termites (Kartal et al. 2009), fungi that are pathogenic to plant (Kim et al. 2006), Cladosporium growth on gypsum (Shirakawa et al. 2013), molds (Pietrzak et al. 2015a, b) and Aspergillus niger (Goffredo et al. 2017) have proven that AgNP when used as it is or in the combined compound state either with copper (Ruparelia et al. 2008;Codiţă et al. 2010) or with titanium dioxide (Hamal et al. 2010;Li et al. 2011), has great biocidal effect on heterotrophic microorganisms. Bacterial, fungal and animal cells can be penetrated by AgNP (Navarro et al. 2008;Vass et al. 2015). ...
Biotechnology to Restoration and Conservation
Chapter
  • Jun 2020
Biotechnology is a broad area of biology that involves the technological application of living cells, organisms and systems to make or develop products. It exploits cellular and bimolecular processes to innovate technologies and products that improve the standard of living and the planet’s health. Biotechnology has been essential in the betterment of medicine, industry and agriculture. It has great potential for the conservation and preservation of cultural monuments. With a rapid increase in population and pollution, cultural monuments are now being degraded and eroded away at a rapid pace. Some have blackened, some have lost their shine and some are left ruined by acid rain. Mechanical methods such as washing have not proven to be a success in the conservation of these monuments. Recent work using biotechnology has proven to be a better alternative in the conservation and preservation of monuments. Biotechnology research in restoration work of cultural monuments develops in two directions, one which focuses on the development of accurate diagnostic techniques for the identification and characterization of bio-deteriogens and alterations and the other which focuses on the development of innovative restoration methods by the employment of new products. Bacteria that reduce sulphur were used for black crust of marble from the Cathedral of Florence. The Desulfovibrio desulfuricans bacteria have been used for the removal of black patina which contains large amounts of sulphates. Denitrifying bacteria, for example, Pseudomonas stutzeri, have been used for the removal of nitrate pollutants.
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... -OH to benzene ring of thiosemicarbazone moiety increases the chelate stability and anticancer activity of copper(II) thiosemicarbazones. (11) Bimetallic structure of complex is found to have higher anticancer activity than that of monometallic structure. ...
Anticancer potency of copper(II) complexes of thiosemicarbazones
Article
Being a structural and catalytic cofactor in a number of biological pathways, copper accumulates in tumors owing to selective permeability of the cancer cell membranes. Copper(II) ion forms the active centers in a large number of metalloproteins. The coordination of Schiff's base ligands to the metal ion results in the high extent of increase in anticancer activity. The copper(II) complexes can cleave DNA through oxidative and hydrolytic pathways, cell apoptosis via intrinsic reactive oxygen species (ROS) mediated mitochondrial pathway due to excessive production of ROS and hence, are found more active than Ni and Pt complexes. Flexible Cu(I/II) redox behavior helps the copper complexes to form more potent, clinically effective and less toxic copper based antiproliferative drugs of lower IC50 value and higher growth inhibitory activity. Copper(II) complexes of thiosemicarbazones of Isatin, Pyridine, Benzoyl pyridine, Diacetyl/Dimethyl glyoxal, Acetophenone/Acetoacetanalide, Thiazole/Pyrazole, Quinoline, Carboxybenzaldehyde, Cinnamaldehyde/Cuminaldehyde, Citronellal, Chromone, Pyridoxal, 8-Ethyl-2-hydroxytricyclo (7.3.1.02,7) tridecan-13-one, Acyl Diazines, Naphthalene, Proline, 5-Formyluracil, 2-Hydroxy-8-propyltricyclo (7.3.1.02,7) tridecan-13-one, 9-cis-Retinal, Curcumin, Helicin (Salicylaldehyde-β-D-glucoside), Thiophene carboxaldehyde, Salicylaldehyde, Iminodiacetate, and 3-Formyl-4-hydroxy benzenesulfonic acid have been found to exhibit more anticancer activity toward HCT116, MCF7, A549, U937, HeLa, HepG2, SGC-7901, A2780 cell lines than that of their corresponding thiosemicarbazones and standard topoisomerase-II inhibitors.
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... To date, much research has been focused on various approaches to reduce the bacterial burden correlated to implants, which include: surface treatments [12], the use of photochemical reactions [13], alloying of silver to titanium [14], and recently, the alloying of copper to titanium [15][16][17]. The antibacterial effect of Cu additions was found to be superior compared to silver [18]. The Ag appears to be effectual only at elevated temperatures [19], whereas Cu was found-using in vitro tests-to have antibacterial ability at ambient conditions [20]. ...
Development of Antibacterial Ti-Cux Alloys for Dental Applications: Effects of Ageing for Alloys with Up to 10 wt% Cu
Article
Full-text available
  • Dec 2019
Peri-implantitis, a disease caused by bacteria, affects dental implants in patients. It is widely treated with antibiotics, however, with growing antibiotic resistance new strategies are required. Titanium-copper alloys are prospective antibacterial biomaterials, with the potential to be a remedy against peri-implantitis and antibiotic resistance. The aim of this study was to investigate Ti-Cux alloys, exploring how Cu content (up to 10 wt%) and ageing affect the material properties. Electron microscopy, X-ray diffraction, hardness testing, bacteriological culture, and electrochemical testing were employed to characterize the materials. It was found that alloys with above 3 wt% Cu had two phases and ageing increased the volume fraction of Ti2Cu. An un-aged alloy of 5 wt% Cu showed what could be Ti3Cu, in addition to the α-Ti phase. The hardness gradually increased with increased Cu additions, while ageing only affected the alloy with 10 wt% Cu (due to changes in microstructure). Ageing resulted in faster passivation of the alloys. After two hours the aged 10 wt% Cu alloy was the only material with an antibacterial effect, while after six hours, bacteria killing occurred in all alloys with above 5 wt% Cu. In conclusion, it was possible to tune the material and antibacterial properties of Ti-Cux alloys by changing the Cu concentration and ageing, which makes further optimization towards an antibacterial material promising.
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... Several technologies have proven antibacterial efficacy and these include hydrogen peroxide vapor [10][11][12][13][14], ultraviolet (UV) light decontamination [15,16], and copperand silver-coated surfaces [17][18][19]. However, their use is limited by high cost or high risk of recontamination. ...
Environmental disinfection with photocatalyst as an adjunctive measure to control transmission of methicillin-resistant Staphylococcus aureus: a prospective cohort study in a high-incidence setting
Article
Full-text available
Background Environmental disinfection with continuously antimicrobial surfaces could offer superior control of surface bioburden. We sought to decide the efficacy of photocatalyst antimicrobial coating in reducing methicillin-resistant Staphylococcus aureus (MRSA) acquisition in high incidence setting. Methods We performed prospective cohort study involving patients hospitalized in medical intensive care unit. A titanium dioxide-based photocatalyst was coated on high touch surfaces and walls. Five months of pre-intervention data were compared with five months of post-intervention data. The incidence rates of multidrug-resistant organism acquisition and the rates of hospital-acquired blood stream infection, pneumonia, urinary tract infection, and Clostridium difficile–associated diseases were compared using Cox proportional hazards regression analysis. Results In total, 621 patients were included. There was significant decrease in MRSA acquisition rate after photocatalyst coating (hazard ratio, 0.37; 95% confidence interval, 0.14–0.99; p = 0.04). However, clinical identification of vancomycin-resistant Enterococcus spp. and multidrug-resistant Acinetobacter baumannii did not decrease significantly. The hazard of contracting hospital-acquired pneumonia during the intervention period compared to baseline period was 0.46 (95% confidence interval, 0.23–0.94; p = 0.03). Conclusions In conclusion, MRSA rate was significantly reduced after photocatalyst coating. We provide evidence that photocatalyst disinfection can be an adjunctive measure to control MRSA acquisition in high-incidence settings. Trial registration ISRCTN Registry (ISRCTN31972004). Registered retrospectively on November 19, 2018. Electronic supplementary material The online version of this article (10.1186/s12879-018-3555-1) contains supplementary material, which is available to authorized users.
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... Another interesting application is the use of copper nanofilms to cover touch surfaces in hospitals and healthcare settings, where bacterial proliferation and resistance is becoming a serious problem. As a matter of fact, thin layers of copper on objects and surfaces are effective in inhibiting the growth of a number of nosocomial germs and Enterobacter species (Codita et al. 2010;Grass et al. 2011) exploiting an effect called 'contact killing'. These remarkable properties led to the approval of metallic copper as the first solid antimicrobial material by the U.S. Environmental Protection Agency. ...
Noble Metals in Pharmaceuticals: Applications and Limitations
Chapter
Full-text available
  • Feb 2018
Metal-based drugs like Cisplatin are still the most widely employed therapeutics in the treatment of cancer. Nevertheless, due to their severe drawbacks, such as extended toxicity, scarce bioavailability and drug resistance, the need of a valid alternative to Cisplatin and its derivatives has led to the synthesis and assessment as therapeutical agents of a series of complexes based on platinum and other noble metals, which showed not only anticancer activity but also antibacterial and antiprotozoal action with outstanding results. Here, the most used metals are discussed, together with their applications in medicine and limitations.
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Antibacterial Mechanism of Copper-bearing Antibacterial Stainless Steel against E. coli
Article
Full-text available
A preliminary study was made on the antibacterial mechanism of copper-bearing antibacterial stainless steels against E.coli through experiments of microbiology such as EDTA (ethylenediaminetetraacetic acid) complexing, DNA smearing and AFM (atomic force microscope) observation. It was measured that the antibacterial stainless steels showed excellent antibacterial functions with antibacterial rate to E.coli over 99.99%. The antibacterial rate was weak if the bacteria solution was complexed by EDTA, indicating that the copper ions play a dominant role in the antibacterial effect of the antibacterial stainless steels. The electrophoresis experiment did not show the phenomenon of DNA smearing for E.coli after contacting antibacterial stainless steels, which meant that DNA of E.coli was not obviously damaged. It was observed by AFM that the morphology of E.coli changed a lot after contacting antibacterial stainless steels, such as cell walls being seriously changed and lots of contents in the cells being leaked.
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Chemical characterization and antimicrobial activity of essential oils of Salvia L. species
Article
Full-text available
  • Dec 2009
In this work, the essential oils of S. officinalis, S. sclarea, S. lavandulifolia and S. triloba were chemically analyzed by gas chromatography coupled to a mass spectrometry detector (GC/MSD), and their antimicrobial activity was tested against 10 microorganisms using the disk diffusion method and the Minimum Inhibitory Concentration (MIC) technique. The following major compounds were identified in the essential oils: α - and β-thujone, camphor and 1,8-cineole, except in S. sclarea, where linalool, linalyl acetate and α-terpineol were the major constituents. The antimicrobial activity showed significant differences (p < 0.05) only when obtained by the MIC method. Gram-positive microorganisms presented larger sensitivity for the essential oils. The lowest MIC was observed when Staphylococcus aureus was exposed to 2.31 mg.mL-1 of S. lavandulifolia essential oil, while the highest MIC value was obtained when Shigella flexneri was exposed to 9.25 mg.mL-1 of the same essential oil, thus demonstrating that this essential oil may be effective as a bacteriostatic agent against Gram-positive microorganisms.
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General characteristics of the Thermoionic Vacuum Arc plasma
Data
Full-text available
  • Jan 2007
The paper presents the main characteristics of an original deposition method for the synthesis of quality thin films using anodic arc plasma, called Thermionic Vacuum Arc -TVA. This plasma was observed for the first time about 30 years ago, but only in the last three-four years research started being focused on the coating capabilities of this plasma. The work undertaken until now has demonstrated a great potential of this plasma to become a powerful thin film deposition tool for a large range of applications.
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Inhibitory action of some essential oils and phytochemicals on the growth of various moulds isolated from foods
Article
Full-text available
The aim of this study was to determine the sensitivity profile of mould strains isolated from foods to some essential oils and phytochemicals. The assayed mould strains were: Fusarium spp., Rhizopus spp., Aspergillus flavus, A. niger and Penicillium spp. According to results, Lippia alba N.E. Brown, Peumus boldus Molina, Lippia microphylla Phil., Citrus limon Risso and Cymbopogon citratus Stapf. essential oil and the phytochemicals citral, eugenol and mircene showed prominent antimould activity. Among the products that evidenced antimould activity, citral and eugenol showed the lowest minimum inhibitory concentrations, which was 1% and 4%, respectively, for the most of the tested mould strains.
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Antimicrobial and antioxidative activities of the essential oils and methanol extracts of Salvia cryptantha (Montbret et Aucher ex Benth.) and Salvia multicaulis (Vahl)
Article
Full-text available
The essential oils and methanolic extracts of Salvia cryptantha and Salvia multicaulis were examined for their potential antimicrobial and radical scavenging activities. No, or slight, activity was observed when the polar and non-polar subfractions of the extracts were tested, whereas essential oils exhibited antimicrobial activity. The essential oils isolated from S. cryptantha and S. multicaulis were analysed by GC–MS and 53 and 47 constituents were identified, respectively. Antioxidant activities of the polar subfraction and the essential oil were examined using 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl radical-scavenging and lipid peroxidation assays. The essential oils, in particular, and the non-polar subfractions of methanol extracts, showed antioxidant activity. In conclusion, the results indicate that the oils of S. cryptantha and S. multicaulis have the capacity to scavenge free radicals and to inhibit the growth of pathogenic microorganisms. Therefore they could be suitable for using as antimicrobial and antioxidative agents in the food industry.
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In vitro antibacterial activity of essential oil from Mediterranean Apiaceae, Verbenaceae and Lamiaceae against foodborne pathogens and spoilage bacteria
Article
  • Jan 2005
The aim of this work was to investigate the antimicrobial activity of twelve essential oils, against Gram-positive and Gram-negative bacteria, for a potential use in food industry. The antimicrobial activity of essential oils was determined by an agar diffusion method against foodborne pathogens and spoilage bacteria. Further, bacteriostatic and bactericidal concentrations were determined for each strain that evidenced sensitivity to the oils. All the oils showed bacteriostatic and bactericidal activity against Escherichia coli O157:H7 and Salmonella Typhimurium, while Brochotrix thermosphacta was inhibited by eight of 12 tested oils. Finally, the pathogenic microorganism Listeria monocytogenes and lactic acid bacteria strains were affected only by thyme, oregano and vervain oils. The essential oils considered in this research showed a satisfactory antimicrobial activity. The essential oils could be used for the development of novel systems for food preservation.
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Microstructure and antibacterial properties of AISI 420 stainless steel implanted by copper ions
Article
In order to study the effect of copper ion implantation on the antibacterial property of AISI 420 stainless steel (SS), specimens were implanted by copper ions in a dose ranging from 2 × 1016 to 5.0 × 1017ions cm− 2, using metal vapor vacuum arc source at an extracting voltage of 50 kV. The microstructure, phase compositions and Cu ions concentration profile in the implanted layer were revealed by glancing angle X-ray diffraction, transmission electronic microscopy (TEM) and Auger electronic spectroscopy, respectively. Film attachment method was adopted for evaluation of antibacterial property of specimens against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). With TEM, changes of bacterial appearance on the surface of un-implanted SS and annealed Cu-implanted SS were observed separately. Results showed that novel phases such as Fe4Cu3 and Cu9.9Fe0.1 were formed in the surface layer of the annealed Cu-implanted SS and that the antibacterial property resulted from the Cu-contained and Cu-rich phase which had a damaging effect on pericellular membrane and cell wall. Furthermore, the pericellular membrane was thickened and then the karyon was degraded, and finally, bacteria died. Annealed Cu-implanted SS not only obtains excellent antibacterial property, but also keeps good corrosion resistance which is equivalent to that of common AISI 420 SS.
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Behaviour of Listeria monocytogenes and autochthonous flora on meat stored under aerobic, vacuum and modified atmosphere packaging conditions with or without the presence of oregano essential oil at 5 degrees C
Article
The effect of aerobic, modified atmosphere packaging (MAP; 40% CO2/30% O2/30% N2) and vacuum packaging (VP) on the growth/survival of Listeria monocytogenes on sterile and naturally contaminated beef meat fillets was studied in relation to film permeability and oregano essential oil. The dominant micro-organism(s) and the effect of the endogenous flora on the growth/survival of L. monocytogenes were dependent on the type of packaging film. The fact that L. monocytogenes increased whenever pseudomonads dominated, i.e. aerobic storage and MAP/VP in high-permeability film, and even earlier than on sterile tissue, suggests that this spoilage group enhanced growth of the pathogen. Brochothrix thermosphacta constituted the major proportion of the total microflora in MAP/VP within the low-permeability film, where no growth of L. monocytogenes was detected either on naturally contaminated or sterile meat fillets. The addition of 0·8% (v/w) oregano essential oil resulted in: (i) an initial reduction of 2–3 log10 of the majority of the bacterial population, with lactic acid bacteria and L. monocytogenes indicating the most apparent decrease in all gaseous environments, and (ii) limited growth aerobically and survival/death of L. monocytogenes in MAP/VP, regardless of film permeability.
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