Letters in Applied Microbiology ISSN 0266-8254
ORIGINAL ARTICLE
A long-term study examining the antibacterial effectiveness of Agion silver zeolite technology on door handles within a college campus B.A. Potter1, M. Lob1, R. Mercaldo1, A. Hetzler1, V. Kaistha1, H. Khan1, N. Kingston1, M. Knoll1, B. Maloy-Franklin1, K. Melvin1, P. Ruiz-Pelet1, N. Ozsoy1, E. Schmitt1, L. Wheeler1, M. Potter2, M.A. Rutter3, G. Yahn4 and D.H. Parente5 1 2 3 4 5
Department of Biology, Penn State Erie, The Behrend College, Erie, PA, USA RJ Lee Group, Monroeville, PA, USA Department of Mathematics, Penn State Erie, The Behrend College, Erie, PA, USA Advanced Finishing USA, Fairview, PA, USA Sam and Irene Black School of Business, Penn State Erie, The Behrend College, Erie, PA, USA
Significance and Impact of the Study: There has been a dramatic increase in the use of silver-containing antimicrobials within the medical and consumer markets despite many concerns on how increased usage will impact the environment. To begin addressing these concerns, it is important to first determine whether the silver-containing antimicrobials are significantly affecting bacterial populations outside of the controlled laboratory setting. A significant decrease was seen in this study examining the effectiveness of Agion silver zeolite technology applied to door handles across a college campus. Results showed the recovery of a bacterial population on silver-coated door handles suggesting a bias in the effectiveness of this technology.
Keywords antibacterial effect, bacteria, culture dependent, silver ions, silver zeolite. Correspondence Beth A. Potter, Penn State Erie, The Behrend College, 4205 College Drive, Erie, PA 16563, USA. E-mail: [email protected] 2014/1332: received 30 June, 2014, revised 2 October, 2014 and accepted 24 October, 2014 doi:10.1111/lam.12356
Abstract Laboratory studies have shown that small concentrations of silver are effective at inhibiting the growth micro-organisms through the disruption of important cell structures and processes. The additional ability to incorporate silver into surfaces has increased the usage of silver in the medical field and expanded its use into the consumer market. To understand the impact of increased silvercontaining antimicrobial use, it is important to determine whether silver-based consumer goods are effective at reducing bacterial populations. Our study examined the antibacterial effectiveness of Agion silver zeolite technology applied to 25 silver- and control-coated door handles across a college campus. Door handles were sampled for 6 week periods in both the fall and spring semester, and bacteria were cultured and enumerated on tryptic soy agar (TSA), MacConkey agar (MAC) and mannitol salt agar (MSA). A significant difference was observed between the bacterial populations isolated from silverand control-coated door handles after 3 years. However, bacteria were consistently isolated from silver-coated door handles suggesting that the silver zeolite was only effective against a portion of the bacterial populations, and further studies are necessary to determine the identities of the isolated bacteria and the prevalence of silver resistance.
Introduction Silver is one of the oldest antimicrobial agents, used by the ancient Egyptians and Romans to preserve liquids and 120
foods (Franke 2007; Silvestry-Rodriguez et al. 2007). Solid silver nitrate formally entered the medical field in the Middle Ages as a treatment for chronic wounds and ulcers (Klasen 2000). By the end of the 19th century,
Letters in Applied Microbiology 60, 120--127 © 2014 The Society for Applied Microbiology
B.A. Potter et al.
solutions of silver nitrate were being used to treat burn wounds and applied to the eyes of newborns to protect against gonorrhoeal disease (Klasen 2000). Silver usage stalled in the twentieth century with the discovery of antibiotics, but its use has increased significantly due to the rise in antibiotic resistance (Silver et al. 2006; Chopra 2007). In 1968, a paper showing the effectiveness of combining silver nitrate with sulfonamide led to the marketing of silver sulfadiazine which became the gold standard in burn wound care in the 1970s (Fox 1968; Atiyeh et al. 2007). Treatment effectiveness was further enhanced when silver was incorporated into dressings/bandages. Other medical applications involving silver have included dental amalgams, catheters and other in-dwelling medical devices (Silver et al. 2006). Recently, the usage of silver has expanded into the industrial and consumer markets with its incorporation into water treatment facilities, food packaging materials, household appliances, apparel, shoes and personal hygiene products (Franke 2007; SilvestryRodriguez et al. 2007; Edwards-Jones 2009). The widespread popularity of silver stems from its effectiveness at low concentrations, its ability to be integrated within plastics, textiles and surfaces, and its broadspectrum properties. Silver has been shown to be effective against viruses, yeast, fungi and bacteria including Grampositive, Gram-negative and multidrug-resistant bacteria (Silvestry-Rodriguez et al. 2007; Marambio-Jones and Hoek 2010). Several forms of silver have been used including silver salts, compounds and zeolite, which all rely on the release of silver ions for their antimicrobial success (Atiyeh et al. 2007). Research examining the antibacterial activity of silver ions has demonstrated that they have a multifactorial approach to killing bacteria. Silver ions have been shown to affect the integrity of the cell membrane through multiple mechanisms. The membranes of cells treated with silver ions have been shown to detach from the cell wall of Gram-negative and Grampositive organisms using transmission electron microscopy (Feng et al. 2000; Jung et al. 2008). Silver ions can also interact with the thiol groups of key respiratory chain enzymes causing proton leakage, the collapse of the proton motive force and the production of reactive oxygen species (Liau et al. 1997; Dibrov et al. 2002; Inoue et al. 2002; Matsumura et al. 2003; Holt and Bard 2005; Gordon et al. 2010). In addition to binding and disrupting protein function via thiol groups, silver ions can inactivate enzymes by binding to sites usually reserved for copper ions (Ghandour et al. 1988). Silver ions alter transport across the membrane by triggering the export of intracellular stores of important metabolites such as inorganic phosphate, mannitol, succinate and glutamate (Schreurs and Rosenberg 1982). DNA replication is also inhibited due to the binding of silver ions to purine and
Effect of silver zeolite on door handle bacteria
pyrimidine bases (Jensen and Davidson 1966; Fox and Modak 1974). Though silver nanoparticles have been shown to directly interact with microbial cells with varying degrees based on particle size and shape, silver ions have also been shown to contribute to their overall effectiveness (Morones et al. 2005). The studies mentioned above have increased the interest and demand for silver, expanding their use into the commercial market to the extent that most personal items such as phones and apparel contain silver (Arvidsson et al. 2011). An increase in usage is concerning as there are still many questions regarding the use of silver including the standardization of minimum inhibitory concentrations and testing methods, the development and mechanisms of resistance, and the impact of increased usage on exposure rates to us and the environment (Chopra 2007; Arvidsson et al. 2011). For instance, studies have shown that wounds treated with silver-based dressings take longer to heal and produce more scarring than wounds treated with non-silver-based dressings due to cytotoxic effects produced in fibroblasts and keratinocytes (Innes et al. 2001; Poon and Burd 2004). The disposal of silver-containing antimicrobials leads to the introduction of silver to aquatic ecosystems and terrestrial environments via sewage sludge (Blaser et al. 2008; Colman et al. 2013). Bacteria are critical to this ecosystem, and various forms of silver have been shown to inhibit the important metabolic processes performed by nitrifying bacteria and other beneficial soil micro-organisms (Choi and Hu 2008; Gajjar et al. 2009). With respect to the aquatic ecosystem, silver can disrupt the entire food chain with toxic effects on lower organisms such as algae to higher organisms including various fish (Fabrega et al. 2011). An important question in addressing these concerns is whether the silver-containing antimicrobials being introduced to the consumer market are effective in limiting bacterial populations in the absence of controlled laboratory conditions. Thus, the goal of this study was to determine the effectiveness of Agion silver zeolite technology incorporated into door handles within a college campus. Agion silver zeolite technology relies on the entrapment of silver ions within an alumino-silicate structure. This porous structure allows for the exchange of silver ions for environmental cations, such as sodium that would be found in the moisture from our hands and allows microbes to be killed upon transfer to the surface (Agion Silver Antimicrobial – How it works 2011). Results and discussion At the beginning of this study, a powder coat-containing Agion silver zeolite was applied to 25 door handles within four buildings on the Penn State Erie, The Behrend
Letters in Applied Microbiology 60, 120--127 © 2014 The Society for Applied Microbiology
121
B.A. Potter et al.
Effect of silver zeolite on door handle bacteria
College campus. Another 25 door handles were used as controls and received the powder coat without the silver zeolite. The presence or absence of silver on the door handles was confirmed using scanning electron microscopy (Fig. 1). Particles were randomly distributed within the coating (60–70 lm thick) and were all similar in size (2–5 lm) (Fig. 1c). No particles were observed in the control-coated doors (Fig. 1a). The carbon observed in the energy-dispersive spectroscopy (EDS) spectra of both the silver- and control-coating was primarily due to the carbon-rich matrix (Fig. 1b,d). Like previous studies, aluminium, silicon, oxygen and zinc were also observed in the silver coating (Fig. 1d) (Cowan et al. 2003; KwakyeAwuah et al. 2007). For three consecutive years, door handles were sampled weekly for bacteria during a 6-week period in both the fall and spring semesters. Bacterial samples were plated
(a)
onto three different agar plates. Tryptic soy agar (TSA) is an all-purpose media that can grow a wide range of bacteria including both Gram-positive and Gram-negative micro-organisms. Mannitol salt agar (MSA) is a selective media that commonly grows Gram-positive bacteria within the Staphylococcus genus while MacConkey agar (MAC) is selective for a broad range of Gram-negative bacteria including enteric pathogens. The mean log of bacterial counts (CFU ml 1) obtained each semester from silver- and control-coated door handles in each building are shown in Fig. 2a–d, and the mean log bacterial counts obtained from the door handles in all four buildings each semester is shown in Fig. 2e. While high counts were sporadically observed, the counts were relatively low, and in some instances, no bacteria were identified on the door handles (Fig 2). These lower counts tended to occur consistently within the spring semester in all four buildings.
cps/eV
(b)
18 C 16 14 12 10 8 6 4 O
2
Si AI
0
100 μm
1
CI 2
3
4
5 keV
6
7
8
9
10
9
10
cps/eV
(c)
(d)
18 16 14
C
12 10 8
AISi
O
6 4
Zn
2 0 100 μm
Ag 1
2
3
Zn 4
5 keV
6
7
8
Figure 1 Scanning electron microscope image and energy-dispersive spectroscopy spectra of the control-(a, b) and silver-coated (c, d) door handles.
122
Letters in Applied Microbiology 60, 120--127 © 2014 The Society for Applied Microbiology
B.A. Potter et al.
Effect of silver zeolite on door handle bacteria
(a) 3·0
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Log (Counts)
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1·4 1·2 1·0 0·8 0·6 0·4 0·2 0·0
Control Silver Control Silver Control Silver Control Silver Control Silver Control Silver Fall
Spring 2011–2012
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Spring 2012–2013
Fall
Spring 2013–2014
Figure 2 The mean log of bacterial counts obtained on tryptic soy agar (black bar), mannitol salt agar (grey bar), and MacConkey agar (white bar) plates each semester in the engineering (a), gymnasium (b), student union (c), science (d) buildings and the combined values for all buildings (e).
An explanation for this result is the temperature difference between the fall and spring semesters. Average daily temperatures throughout each sampling period were col-
lected from the National Oceanic and Atmospheric Administration, and a Student’s t-test revealed a significant difference in temperature between each fall and
Letters in Applied Microbiology 60, 120--127 © 2014 The Society for Applied Microbiology
123
B.A. Potter et al.
Effect of silver zeolite on door handle bacteria
corresponding spring semester (P =
ORIGINAL ARTICLE
A long-term study examining the antibacterial effectiveness of Agion silver zeolite technology on door handles within a college campus B.A. Potter1, M. Lob1, R. Mercaldo1, A. Hetzler1, V. Kaistha1, H. Khan1, N. Kingston1, M. Knoll1, B. Maloy-Franklin1, K. Melvin1, P. Ruiz-Pelet1, N. Ozsoy1, E. Schmitt1, L. Wheeler1, M. Potter2, M.A. Rutter3, G. Yahn4 and D.H. Parente5 1 2 3 4 5
Department of Biology, Penn State Erie, The Behrend College, Erie, PA, USA RJ Lee Group, Monroeville, PA, USA Department of Mathematics, Penn State Erie, The Behrend College, Erie, PA, USA Advanced Finishing USA, Fairview, PA, USA Sam and Irene Black School of Business, Penn State Erie, The Behrend College, Erie, PA, USA
Significance and Impact of the Study: There has been a dramatic increase in the use of silver-containing antimicrobials within the medical and consumer markets despite many concerns on how increased usage will impact the environment. To begin addressing these concerns, it is important to first determine whether the silver-containing antimicrobials are significantly affecting bacterial populations outside of the controlled laboratory setting. A significant decrease was seen in this study examining the effectiveness of Agion silver zeolite technology applied to door handles across a college campus. Results showed the recovery of a bacterial population on silver-coated door handles suggesting a bias in the effectiveness of this technology.
Keywords antibacterial effect, bacteria, culture dependent, silver ions, silver zeolite. Correspondence Beth A. Potter, Penn State Erie, The Behrend College, 4205 College Drive, Erie, PA 16563, USA. E-mail: [email protected] 2014/1332: received 30 June, 2014, revised 2 October, 2014 and accepted 24 October, 2014 doi:10.1111/lam.12356
Abstract Laboratory studies have shown that small concentrations of silver are effective at inhibiting the growth micro-organisms through the disruption of important cell structures and processes. The additional ability to incorporate silver into surfaces has increased the usage of silver in the medical field and expanded its use into the consumer market. To understand the impact of increased silvercontaining antimicrobial use, it is important to determine whether silver-based consumer goods are effective at reducing bacterial populations. Our study examined the antibacterial effectiveness of Agion silver zeolite technology applied to 25 silver- and control-coated door handles across a college campus. Door handles were sampled for 6 week periods in both the fall and spring semester, and bacteria were cultured and enumerated on tryptic soy agar (TSA), MacConkey agar (MAC) and mannitol salt agar (MSA). A significant difference was observed between the bacterial populations isolated from silverand control-coated door handles after 3 years. However, bacteria were consistently isolated from silver-coated door handles suggesting that the silver zeolite was only effective against a portion of the bacterial populations, and further studies are necessary to determine the identities of the isolated bacteria and the prevalence of silver resistance.
Introduction Silver is one of the oldest antimicrobial agents, used by the ancient Egyptians and Romans to preserve liquids and 120
foods (Franke 2007; Silvestry-Rodriguez et al. 2007). Solid silver nitrate formally entered the medical field in the Middle Ages as a treatment for chronic wounds and ulcers (Klasen 2000). By the end of the 19th century,
Letters in Applied Microbiology 60, 120--127 © 2014 The Society for Applied Microbiology
B.A. Potter et al.
solutions of silver nitrate were being used to treat burn wounds and applied to the eyes of newborns to protect against gonorrhoeal disease (Klasen 2000). Silver usage stalled in the twentieth century with the discovery of antibiotics, but its use has increased significantly due to the rise in antibiotic resistance (Silver et al. 2006; Chopra 2007). In 1968, a paper showing the effectiveness of combining silver nitrate with sulfonamide led to the marketing of silver sulfadiazine which became the gold standard in burn wound care in the 1970s (Fox 1968; Atiyeh et al. 2007). Treatment effectiveness was further enhanced when silver was incorporated into dressings/bandages. Other medical applications involving silver have included dental amalgams, catheters and other in-dwelling medical devices (Silver et al. 2006). Recently, the usage of silver has expanded into the industrial and consumer markets with its incorporation into water treatment facilities, food packaging materials, household appliances, apparel, shoes and personal hygiene products (Franke 2007; SilvestryRodriguez et al. 2007; Edwards-Jones 2009). The widespread popularity of silver stems from its effectiveness at low concentrations, its ability to be integrated within plastics, textiles and surfaces, and its broadspectrum properties. Silver has been shown to be effective against viruses, yeast, fungi and bacteria including Grampositive, Gram-negative and multidrug-resistant bacteria (Silvestry-Rodriguez et al. 2007; Marambio-Jones and Hoek 2010). Several forms of silver have been used including silver salts, compounds and zeolite, which all rely on the release of silver ions for their antimicrobial success (Atiyeh et al. 2007). Research examining the antibacterial activity of silver ions has demonstrated that they have a multifactorial approach to killing bacteria. Silver ions have been shown to affect the integrity of the cell membrane through multiple mechanisms. The membranes of cells treated with silver ions have been shown to detach from the cell wall of Gram-negative and Grampositive organisms using transmission electron microscopy (Feng et al. 2000; Jung et al. 2008). Silver ions can also interact with the thiol groups of key respiratory chain enzymes causing proton leakage, the collapse of the proton motive force and the production of reactive oxygen species (Liau et al. 1997; Dibrov et al. 2002; Inoue et al. 2002; Matsumura et al. 2003; Holt and Bard 2005; Gordon et al. 2010). In addition to binding and disrupting protein function via thiol groups, silver ions can inactivate enzymes by binding to sites usually reserved for copper ions (Ghandour et al. 1988). Silver ions alter transport across the membrane by triggering the export of intracellular stores of important metabolites such as inorganic phosphate, mannitol, succinate and glutamate (Schreurs and Rosenberg 1982). DNA replication is also inhibited due to the binding of silver ions to purine and
Effect of silver zeolite on door handle bacteria
pyrimidine bases (Jensen and Davidson 1966; Fox and Modak 1974). Though silver nanoparticles have been shown to directly interact with microbial cells with varying degrees based on particle size and shape, silver ions have also been shown to contribute to their overall effectiveness (Morones et al. 2005). The studies mentioned above have increased the interest and demand for silver, expanding their use into the commercial market to the extent that most personal items such as phones and apparel contain silver (Arvidsson et al. 2011). An increase in usage is concerning as there are still many questions regarding the use of silver including the standardization of minimum inhibitory concentrations and testing methods, the development and mechanisms of resistance, and the impact of increased usage on exposure rates to us and the environment (Chopra 2007; Arvidsson et al. 2011). For instance, studies have shown that wounds treated with silver-based dressings take longer to heal and produce more scarring than wounds treated with non-silver-based dressings due to cytotoxic effects produced in fibroblasts and keratinocytes (Innes et al. 2001; Poon and Burd 2004). The disposal of silver-containing antimicrobials leads to the introduction of silver to aquatic ecosystems and terrestrial environments via sewage sludge (Blaser et al. 2008; Colman et al. 2013). Bacteria are critical to this ecosystem, and various forms of silver have been shown to inhibit the important metabolic processes performed by nitrifying bacteria and other beneficial soil micro-organisms (Choi and Hu 2008; Gajjar et al. 2009). With respect to the aquatic ecosystem, silver can disrupt the entire food chain with toxic effects on lower organisms such as algae to higher organisms including various fish (Fabrega et al. 2011). An important question in addressing these concerns is whether the silver-containing antimicrobials being introduced to the consumer market are effective in limiting bacterial populations in the absence of controlled laboratory conditions. Thus, the goal of this study was to determine the effectiveness of Agion silver zeolite technology incorporated into door handles within a college campus. Agion silver zeolite technology relies on the entrapment of silver ions within an alumino-silicate structure. This porous structure allows for the exchange of silver ions for environmental cations, such as sodium that would be found in the moisture from our hands and allows microbes to be killed upon transfer to the surface (Agion Silver Antimicrobial – How it works 2011). Results and discussion At the beginning of this study, a powder coat-containing Agion silver zeolite was applied to 25 door handles within four buildings on the Penn State Erie, The Behrend
Letters in Applied Microbiology 60, 120--127 © 2014 The Society for Applied Microbiology
121
B.A. Potter et al.
Effect of silver zeolite on door handle bacteria
College campus. Another 25 door handles were used as controls and received the powder coat without the silver zeolite. The presence or absence of silver on the door handles was confirmed using scanning electron microscopy (Fig. 1). Particles were randomly distributed within the coating (60–70 lm thick) and were all similar in size (2–5 lm) (Fig. 1c). No particles were observed in the control-coated doors (Fig. 1a). The carbon observed in the energy-dispersive spectroscopy (EDS) spectra of both the silver- and control-coating was primarily due to the carbon-rich matrix (Fig. 1b,d). Like previous studies, aluminium, silicon, oxygen and zinc were also observed in the silver coating (Fig. 1d) (Cowan et al. 2003; KwakyeAwuah et al. 2007). For three consecutive years, door handles were sampled weekly for bacteria during a 6-week period in both the fall and spring semesters. Bacterial samples were plated
(a)
onto three different agar plates. Tryptic soy agar (TSA) is an all-purpose media that can grow a wide range of bacteria including both Gram-positive and Gram-negative micro-organisms. Mannitol salt agar (MSA) is a selective media that commonly grows Gram-positive bacteria within the Staphylococcus genus while MacConkey agar (MAC) is selective for a broad range of Gram-negative bacteria including enteric pathogens. The mean log of bacterial counts (CFU ml 1) obtained each semester from silver- and control-coated door handles in each building are shown in Fig. 2a–d, and the mean log bacterial counts obtained from the door handles in all four buildings each semester is shown in Fig. 2e. While high counts were sporadically observed, the counts were relatively low, and in some instances, no bacteria were identified on the door handles (Fig 2). These lower counts tended to occur consistently within the spring semester in all four buildings.
cps/eV
(b)
18 C 16 14 12 10 8 6 4 O
2
Si AI
0
100 μm
1
CI 2
3
4
5 keV
6
7
8
9
10
9
10
cps/eV
(c)
(d)
18 16 14
C
12 10 8
AISi
O
6 4
Zn
2 0 100 μm
Ag 1
2
3
Zn 4
5 keV
6
7
8
Figure 1 Scanning electron microscope image and energy-dispersive spectroscopy spectra of the control-(a, b) and silver-coated (c, d) door handles.
122
Letters in Applied Microbiology 60, 120--127 © 2014 The Society for Applied Microbiology
B.A. Potter et al.
Effect of silver zeolite on door handle bacteria
(a) 3·0
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Control Silver Control Silver Control Silver Control Silver Control Silver Control Silver Fall
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Figure 2 The mean log of bacterial counts obtained on tryptic soy agar (black bar), mannitol salt agar (grey bar), and MacConkey agar (white bar) plates each semester in the engineering (a), gymnasium (b), student union (c), science (d) buildings and the combined values for all buildings (e).
An explanation for this result is the temperature difference between the fall and spring semesters. Average daily temperatures throughout each sampling period were col-
lected from the National Oceanic and Atmospheric Administration, and a Student’s t-test revealed a significant difference in temperature between each fall and
Letters in Applied Microbiology 60, 120--127 © 2014 The Society for Applied Microbiology
123
B.A. Potter et al.
Effect of silver zeolite on door handle bacteria
corresponding spring semester (P =
