20231122AI02 - Antimicrobial Treatment Effect On Fiberglass

Journal of Applied Microbiology 2004, 97, 371–377 doi:10.1111/j.1365-2672.2004.02308.
Effects of antimicrobial treatment on fiberglass-acrylic filters
C. Cecchini1, M.C. Verdenelli1, C. Orpianesi1, G.M. Dadea2 and A. Cresci1

1
Department of Comparative Morphology and Biochemistry, University of Camerino, Camerino (MC), and 2Fabriano Filter Media S.p.A.,
Località Campoginepro, Sassoferrato (AN), Italy
2003/1160: received 17 December 2003, revised 23 February 2004 and accepted 5 April 2004
ABSTRACT
C . C E C C H I N I , M . C . V E R D E N E L L I , C . O R P I A N E S I , G . M . D A D E A A N D A . C R E S C I . 2004.
Aims: The aims of the present study were to: (i) analyse a group of antimicrobial agents and to select the most
active against test microbial strains; (ii) test the effect of the antimicrobial treatment on air filters in order to reduce
microbial colonization.
Methods and Results: Different kinds of antimicrobial agents were analysed to assess their compatibility with the
production process of air filter media. The minimal inhibitory concentration for each antimicrobial agent was
determined against a defined list of microbial strains, and an antimicrobial activity assay of filter prototypes was
developed to determine the most active agent among the compatible antimicrobials. Then, the most active was
chosen and added directly to the filter during the production process. The microbial colonization of treated and
untreated filter media was assessed at different working times for different incubation times by stereomicroscope
and scanning electron microscope analysis. Some of the antimicrobial agents analysed were more active against
microbial test strains and compatible with the production process of the filter media. Filter sections analysis of
treated filter media showed a significantly lower microbial colonization than those untreated, a reduction of species
both in density and varieties and of the presence of bacteria and fungal hyphae with reproductive structures.
Conclusions: This study demonstrated the ability of antimicrobial treatments to inhibit the growth of micro-
organisms in filter media and subsequently to increase indoor air quality (IAQ), highlighting the value of adding
antimicrobials to filter media.
Significance and Impact of the Study: To make a contribution to solving the problem of microbial
contamination of air filters, by demonstrating the efficacy of incorporating antimicrobial agents in the filter media to
improve IAQ and health.
Keywords: antimicrobial agents, indoor air quality, microbial colonization, microfibre glass-acrylic filters.
und et al. 1988; Platts-Mills 1995; Singh 1996; Jones 1999).

INTRODUCTION
Indoor air pollution in buildings is a consequence of human
During the last few years, attention addressed to air quality activity, synthetic material used in advanced construction
has increased considerably, giving rise to new research in the technology and high population density. In addition, in
field of indoor air quality (IAQ) and health (Hines 1993; urban areas, outdoor contaminant agents are to be added to
Singh 1996; Jones 1998; Samet and Spengler 2003). This these indoor pollutants (Bonadonna and Marconi 1990;
developing concern is due to the realization that in Briganti 1994; Pitzurra et al. 1997; Pasquarella et al. 2000).
developed societies many people spend most of their time Air filter systems represent the most used and efficient
indoors (Spengler and Sextod 1983; Spengler 1985; Bergl- technology for indoor pollution control and prevention.
Correspondence to: Alberto Cresci, Department of Comparative Morphology and
High-efficiency particulate air filters, made of microfibre
Biochemistry, University of Camerino, V.le E. Betti, 3, 62032 Camerino (MC), glass and acrylic resins and produced in filter panels, are
Italy (e-mail: alberto.cresci@unicam.it). usually utilized for air filtration from airborne contaminants
ª 2004 The Society for Applied Microbiology
13652672, 2004, 2, Downloaded from https://ami-journals.onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.2004.02308.x by Hong Kong University Of, Wiley Online Library on [21/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
372 C . C E C C H I N I ET AL.
such as atmospheric aerosols, which consist of mineral dust The aim of the present study is to select an antimicrobial
particles or particles originating from various combustion agent from the wide group of biocides available on the
sources and particles of biological origin like plant debris, market, according to its efficacy in reducing microbial
human or animal scales, pollens, bacteria and fungal spores colonization of the filter media, compatibility with the
(Keuhn et al. 1991; Jones 1998, 1999). Despite the fact that industrial production process and activity trend on the final
air filtration systems represent a good solution for the filtration product, thus improving IAQ and, as a conse-
improvement of IAQ, they could become a source of quence, human health.
contamination from micro-organisms harmful to human
health (Price et al. 1994; Kemp et al. 1995; Simmons and
MATERIALS AND METHODS
Crow 1995; Ahearn et al. 1997; Simmons et al. 1997).
In the light of this new knowledge, the possibility of High Efficiency Particulate Air filters (HEPA, classes H13
adding, during the production process, antimicrobial agents and H14) made of microfibre glass and acrylic resins, were
to prevent filter accumulation and dispersion of micro- supplied directly by the manufacturer (Fabriano Filter
organisms downstream of the filter, could be of great Media S.p.A., Sassoferrato (AN), Italy). Used and unused
importance and contribute to the improvement of the safety filter media were tested.
and quality of the air. The reduction of bacteria, fungi,
mould and other organic substances in filtration has been,
Acrylic compatibility assay
and continues to be an important concern for filter
manufacturers and users in many applications (Darpin The antimicrobial agents used in this study were kindly
2002; Macian et al. 2003). This is important where human provided by several chemical industries. Table 1 shows the
or animal indigestion can cause illness or where a manufac- list of antimicrobials, their physical state and their chemical
turing process is directly affected. Antimicrobial treatments composition. Antimicrobial agents are indicated with cap-
for air filtration products have recently become a topic of itals instead of commercial names.
considerable interest (Foarde et al. 2000). These antimicro- A compatibility assay was conducted to determine which
bial agents, which are intended to destroy or inhibit the antimicrobials could be added to the filter media in the
growth of micro-organisms, have been used in commercial industrial process. This test was made using a standard
and industrial surface applications (i.e. walls, carpets, etc.) acrylic mixture composed of 500 ml of tap water, 200 ml of
for several years. The evidence that micro-organisms collect acrylic substances, 100 ml of water-repellent emulsions,
in filter surfaces could affect the integrity of the filters and 10 ml of anti-foam and antimicrobial agent at 20%. The
determine the release of micro-organisms (Verdenelli et al. homogeneous mixture was mixed and left at room tempera-
2003), has led to antimicrobial treatment also being applied ture for 12 h. After that, if there were no changes in
to air filter media, to improve the air quality by destroying a viscosity or colour and no formation of coagulum, the
broad range of micro-organisms and prevent the growth of antimicrobial agent was considered compatible with the filter
microbes in the dirty filter. media ingredients and so with the industrial process. Agents
Table 1 Physical state, chemical compound

Antimicrobial Compatibility
and compatibility with the industrial pro-
agents Physical state Class of chemical compound assay cess of antimicrobial agents tested
A Solid Quaternary ammonium +
B Solid Quaternary ammonium +
C Solid Quaternary ammonium +
D Liquid Vegetal extract +
E Solid Poly(hexamethylene guanide chloride) +
F Solid Poly(hexamethylene guanide chloride) +
G Liquid Poly(hexamethylene biguanide hydrochloride) +
H Liquid Poly(hexamethylene biguanide hydrochloride) +
I Liquid viscous Quaternary ammonium +
L Liquid Quaternary ammonium )
M Liquid Chlorexidin gluconate )
N Liquid viscous Aminic hydrochoride derivative )
O Liquid Benzalkonium chloride )
+ Compatible; ) noncompatible.
ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 97, 371–377, doi:10.1111/j.1365-2672.2004.02308.x
ANTIMICROBIAL TREATMENTS 373
compatible with the industrial process of filters were process and its physical characteristics, such as quality of
subjected to minimal inhibitory concentration (MIC) deter- water, drying temperature and acrylic substances used. The
mination following the dilutions method. antimicrobial agents used were those compatible and with a
MIC £3000 ppm. A filter media prototype without antimi-
crobial agents was used as positive control.
MIC determination
The antimicrobial activity trend of these prototype filter
Test microbial strains used in this determination are listed media treated with antimicrobials A, B, C, D, E, F, G,
in Table 2; they include both micro-organisms indicated as and H was determined using the Kirby-Bauer agar
air filter contaminants by other authors (Price et al. 1994; diffusion method as reported in Verdenelli et al. (2003)
Simmons and Crow 1995; Verdenelli et al. 2003) and those using Staphylococcus aureus ATCC 25923 (1Æ5 ·
suggested to be used for MIC determination of antimicrobial 108 cells ml)1) as test organism. Sections (diameter
agents (Holt 1984). 9 mm) from new filter media were tested at 30-day
One series of 10-fold dilutions of each antimicrobial agent intervals for up to 10 months. This experiment was
for each microbial strain was prepared using Tryptone Soya conducted in triplicate.
Broth (Oxoid, Unipath Ltd, Basingstoke, UK) for bacteria
and Sabouraud Liquid Medium (Oxoid) for moulds and
Experimental design
yeasts. Each series was inoculated with 10 ll of each
microbial strain (12 · 108 cells ml)1). One negative control Antimicrobial agents. Antimicrobial agent A, utilized in
(medium plus antimicrobial agent minus inoculum) and one the filter production, was selected from the more active
positive control (medium plus inoculum minus antimicro- agents by taking into account the results of the compatibility
bial agent) were included in each series. The tubes assay, the MIC, the inhibition activity assay and, in addition,
inoculated with bacterial strains were incubated at 37C the manufacturer’s requirements. The final concentration of
while the tubes inoculated with fungal strains were incuba- antimicrobial A in the filter impregnation solution was
ted at 25C, both for 48 h. This determination was 30 000 ppm because that gave the best performance in the
conducted in triplicate. filter prototypes.
The antimicrobial agent A used in this study was a
phosphated quaternary amine complex added directly by the
Antimicrobial activity assay on filter prototypes
manufacturer during the filter production cycle. It is highly
The filter media prototypes were produced in the laboratory soluble in water and has very low solubility in nonaqueous
of Fabriano Filter Media, using a glass microfibre support solvents; the pH of the aqueous solution is of 6Æ5 to 7Æ5. The
made in the industrial process. This support was impreg- active component of the antimicrobial is cis-1-(3-chloroally)-
nated with a water solution of acrylic resins, water-repellent 3,5,7-triaza-1-azoniaadamantane chloride.
emulsions, anti-foam and antimicrobial agents at 20 000,
30 000 and 40 000 ppm concentrations, levels chosen Filter sections analysis. Sections of filter media (diameter
according to the technicalities of the filter production 48 mm), both treated with the antimicrobial A and
untreated were placed onto filter holders connected to an
Table 2 Test strains used in the study
air pump. The filter holders were put in an artificially
ventilated 36 m3 room with a controlled temperature of
Strains Source 18 ± 1C and a relative humidity (RH) of 50%. The filter
holders were placed in the middle of the room at a height of
Bacteria
Bacillus subtilis ATCC 6833 1Æ5 m with fixed orientation. Two litres (per min) of air was
Enterococcus faecalis ATCC 29212 drawn through the filter. One set of treated and untreated
Escherichia coli ATCC 11775 filter sections was aspirated for 15 days (8 h day)1). Another
Micrococcus luteus ATCC 49732 set was aspirated for 30 days. All the filter sections were
Pseudomonas aeruginosa ATCC 90207 aspirated simultaneously and collected as described above.
Staphylococcus aureus ATCC 25923 After aspiration, each section was kept in a CFT 1200 S/1B
Streptococcus bovis ATCC 48147 incubator (Piardi, Italy) at 25C for up to 14 days with an
Fungi RH of 80%. As a negative control, unused treated and
Aspergillus niger ATCC 16404 untreated filters, coming from the same stock production,
Candida albicans ATCC 10291
were incubated under the same conditions. After 7 and
Cryptococcus laurentii ATCC 18803
14 days of incubation, the filter sections were removed from
Penicillium sp. ATCC 20369
the incubator and the microbial growth on them was
ATCC, American Type Culture Collection, Manassas, VA, USA. examined using a stereomicroscope (GSZ2, Askania,
Rathenow, Germany); counts were made of the number of determined by analysis of the inhibition zone diameter during
microbial colonies on each section. a 300-day period vs the Staph. aureus ATCC 25923 strain.
The same sections were gold coated (Med 010 Balzers, The eight antimicrobial filter prototypes tested showed
Fürstentum Liechtenstein) and examined in a scanning different activities against Staph. aureus (Fig. 1). However,
electron microscope (SEM) (Cambridge Stereoscan 360, all of them showed a wider inhibition zone, according to
UK). This experiment was performed in duplicate. the increase of the antimicrobial concentrations, while the
agents showed a decrease in activity and a clear reduction
of the inhibition zone diameter during the experimental
Statistical analysis
period. No inhibition zone was produced by antimicrobial
The differences in microbial growth on filter sections, both C, but only a lack of microbial growth on the filter
treated and untreated, used and unused, were evaluated by sections at all the three antimicrobial concentrations tested
one-way analysis of variance (ANOVA). The statistical (Fig. 1a–c). Antimicrobial A was the most active among
analysis was performed using the MINITAB Statistical the agents at the three concentrations and for all the
Software Package (Minitab, Inc. State College, PA, USA). experimental time (Fig. 1a–c). Antimicrobial agent A at
30 000 ppm concentration showed an antimicrobial activity
clearly stronger than that at 20 000 ppm and an only
RESULTS slightly weaker one in comparison with the filter proto-
types at 40 000 ppm (Fig. 1a–c).
Acrylic compatibility assay and MIC determination
The results of the compatibility assays are shown in Table 1.
Filter sections analysis
Nine of 13 antimicrobial agents tested proved to be compatible
with the acrylic substances used in the filter production. Stereomicroscope and SEM analysis. The results
Table 3 shows the MIC values for each compatible obtained from the stereomicroscope analysis are shown in
antimicrobial agent determined against each microbial strain Table 4. Neither treated nor untreated, unused filter media
tested. The MIC value chosen for each antimicrobial agent (0 days of aspiration), gave evidence of micro-organism
was the one, which was efficacious against all the test micro- colonization after 7 days of incubation, while after 14 days
organisms (Table 3). of incubation, there was only a slight presence of fungi on
Of the compatible agents, only one (I) proved to have an untreated, unused filters. These results were confirmed by
MIC of 24 000 ppm; those of the other eight were between SEM analysis, as reported in Fig. 2a,b. SEM analysis of
6 and 3000 ppm (Table 3). microfibre glass-acrylic filters showed the complex structure
of HEPA filters at 1000-fold magnification (Fig. 2).
The filters used for 15 days of aspiration showed a
Antimicrobial activity assay on filter prototypes
microbial growth on the untreated filters both after 7 days
The antimicrobial activity of the eight agents with an MIC and more abundantly after 14 days of incubation, when a
between 6 and 3000 ppm supplied to the filter prototypes was variety of eukaryotic and prokaryotic cells were discovered
Table 3 Minimal inhibitory concentration (MIC) of the compatible antimicrobial agents against all the tested microbial strains
MIC values (ppm)
Microbial strains
Antimicrobial Bacillus Enterococcus Escherichia Micrococcus Pseudomonas Staphylococcus Streptococcus Aspergillus Candida Cryptococcus Penicillium
agents subtilis faecalis coli luteus aeruginosa aureus bovis niger albicans laurentii sp. MIC*
A 375 1500 £47 188 188 1500 375 3000 3000 3000 94 3000
B 375 375 1500 1500 1500 375 375 3000 £94 £94 188 3000
C 375 750 1500 £94 £94 375 375 750 3000 3000 3000 3000
D £4Æ7 150 150 £4Æ7 9Æ4 £4Æ7 9Æ4 18Æ8 £4Æ7 £4Æ7 £4Æ7 150
E 440 £110 440 £110 220 £110 220 £110 £110 £110 £110 440
F £110 220 £110 £110 440 £110 £110 440 220 220 440 440
G £3 6 6 6 £3 £3 6 6 £3 6 6 6
H £4Æ5 £4Æ5 £4Æ5 £4Æ5 £4Æ5 £4Æ5 £4Æ5 9 £4Æ5 £4Æ5 9 9
I 24 000 24 000 24 000 24 000 24 000 24 000 24 000 24 000 12 000 12 000 24 000 24 000
*MIC value efficacious against all the tested micro-organisms.
(a) 25 by SEM analysis (Fig. 2d). No colonization was observed on

treated filters by stereomicroscope (Table 4) and SEM
20
Inhibition zone
analysis (Fig. 2c).
After 30 days of aspiration, the treated filters were slightly
(mm)
15
contaminated (Table 4). Hyphal elements and mature
10 conidiophores with conidia of Penicillium sp. were observed
5 by SEM analysis (Fig. 2e).
In contrast, untreated filters displayed a more marked
0 microbial colonization both after 7 and 14 days of incuba-
0 A
Tim 90 G H B
e ( 180270 F nts tion. SEM analysis showed an evident presence of bacteria
D E l age
da C
im ic robia and fungal hyphae with reproductive structures. Bacteria
ys
) Ant
cells grown on acrylic resin substrates and single eukaryotic
cells, probably of Candida albicans, were observed (Fig. 2f).
(b) 30
Furthermore, A. niger was observed in the scanning area of
25 Inhibition zone the untreated filter media examined (data not shown).
20 (mm) The analysis of variance applied to the results obtained
15 from the stereomicroscope analysis demonstrated that the
colonization of treated filters was significantly lower than
10
that of untreated ones (P £ 0Æ05).
5
0
0 A DISCUSSION
90 H B
Tim 180 E F G g e n ts
e D l a Conventional air filters could be a source of contamination as
(da 270 C icrobia
ys Antim bacteria and fungi can build up on their surface under
)
favourable nutritional and moisture conditions (Simmons and
(c) 30 Crow 1995; Maus et al. 2001; Verdenelli et al. 2003). There is
25 considerable potential need for air filters to be protected
against attack from micro-organisms with the increasing
Inhibition zone
20
demand today for safe and healthy IAQ (Jones 1999). The
(mm)
15
design of products meeting these new requirements is a
10 challenge as the tasks are becoming more and more specific
5 (Foarde et al. 2000; Darpin 2002; Macian et al. 2003).
0 The present study addressed these concerns by introdu-
0 B A cing antimicrobial filters manufactured in microfibre glass
90
F G H nts
Tim 180 D E l age and acrylics mixed with antimicrobial agents. The results
e (d
ays 270 C im ic robia
) Ant obtained demonstrated that only some of the several
antimicrobial agents present on the market are compatible
Fig. 1 Antimicrobial activity trends of filter prototypes treated with with the acrylic substances used for filter production.
antimicrobials C, D, E, F, G, H, B, and A at concentrations of (a) Furthermore, before selecting the antimicrobial agent to
20 000, (b) 30 000 and (c) 40 000 ppm (parts per million) in the be used in the filter media, it is of great importance to
impregnation solutions vs Staphylococcus aureus ATCC 25923. The
evaluate the efficacy of the chemical compounds against a list
reported values are mean of three independent tests
of contaminant micro-organisms (Table 2). The compatible
Table 4 Number of colonies (CFU per sec-

CFU grown on filter sections
tions) grown on treated and untreated, used
and unused filter sections analysed by Aspiration time (days) 0 15 30
stereomicroscopy
Days of incubation 7 14 7 14 7 14
Treated filters* 0 0 0 0 2 ± 1 3±2

Untreated filters 0 3±1 11 ± 2 21 ± 1 35 ± 2 57 ± 3
*Values are significantly different from the untreated filters (P £ 0Æ05; analysis of variance).
Values are mean ± standard deviation of two independent tests.
Fig. 2 Scanning electron microscope (SEM) analysis of filter media: unused treated (a) and untreated (b), used for 15 days of aspiration treated (c)
and untreated (d), used for 30 days of aspiration treated (e) and untreated (f). All the sections were analysed after 14 days of incubation.
Magnification 1000·
agents showed different activity against bacteria and fungi to be the best after its incorporation in the filter. These
with an MIC varying from 6 to 24 000 ppm. results pointed out that it is important and economically
The antimicrobial activity assay on filter prototypes desirable to test the activity of the agent incorporated in a
demonstrated that the activity of the agents changed after prototype filtration product before using it in industrial
their incorporation in the filter media and that sometimes it production. The activity of antimicrobial A, at the initial
did not reflect the results of the MIC determination. In fact, concentrations of 20 000, 30 000 and 40 000 ppm, showed a
even if antimicrobial A was one of the worst agents in the reduction of 21, 15 and 14%, respectively, 300 days after
in vitro MIC determination (MIC ¼ 3000 ppm), it proved production. Thus, the evidence that the antimicrobial
activity remained high for a considerable period of time also Jones, A.P. (1998) Asthma and domestic air quality. Social Science and
supports the added value of antimicrobial filters. Medicine 46, 755–764.
The filter sections stereomicroscope analysis developed on Jones, A.P. (1999) Indoor air quality and health. Atmospheric Environ-
untreated and antimicrobial A treated filter media, demon- ment 33, 4535–4564.
Kemp, S.J., Kuehn, T.H., Pui, D.Y.H. and Vesley, D. (1995) Growth
strated the effect of the treatments in reducing microbial
of microorganisms on HVAC filters under controlled temperature
colonization significantly (P £ 0Æ05).
and humidity conditions. ASHRAE Transactions 101, 305–316.
The untreated filter sections aspirated for 30 days showed Keuhn, T.H., Pui, D.Y.H., Vesley, D., Berg, C.D. and Peloquin, M.
an evident colonization of bacteria and fungi, particularly (1991) Matching filtration with health requirements. ASHRAE
after 14 days of incubation as confirmed by SEM analysis, Transactions 97, 164–169.
demonstrating the presence of several species of bacteria and Macian, V., Compte, L., Perpina, M., Ferrando, D., Cercos, A., Lloris,
mould naturally present in the air and carried on the filter’s A. and Martinaz, M. (2003) Efficacy of an antibacterial filter for
surface during the aspiration process. Those microbial cells preventing contamination of respiratory function diagnostic equip-
can survive and grow when they find suitable conditions ment. Archivos de Bronconeumologia 39, 261–265.
(Price et al. 1994; Kemp et al. 1995; Simmons et al. 1997; Maus, R., Goppelsröder, A. and Umhauer, H. (2001) Survival of
Maus et al. 2001). bacteria and mold spores in air filter media. Atmospheric Environment
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In conclusion, this study shows interesting findings about
Pasquarella, C., Pitzurra, O. and Savino, A. (2000) The index of
the usefulness of antimicrobial agents in air filters, and microbial air contamination. Journal of Hospital Infection 46, 241–256.
makes a contribution to clarifying the effect of antimicrobial Pitzurra, M., Savino, A. and Pasquarella, C. (1997) Il Monitoraggio
agents supplied to filter media. In this way, colonization and Ambientale Microbiologico (MAM). Annali di Igiene 9, 439–454.
growth of microbes in dirty filters can be prevented and the Platts-Mills, T.A.E. (1995) Is there a dose–response relationship
number of airborne micro-organisms be reduced, with the between exposure to indoor allergens and symptoms of asthma?
aim of improving IAQ and health. Journal of Clinical Immunology 96, 435–440.
Price, D.L., Simmons, R.B., Ezeonu, I.M., Crow, S.A. and Ahearn,
D.G. (1994) Colonization of fiberglass insulation used in heating,
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Journal of Applied Microbiology 2004, 97, 371–377 doi:10.1111/j.1365-2672.2004.02308.

Effects of antimicrobial treatment on fiberglass-acrylic filters

C. Cecchini1, M.C. Verdenelli1, C. Orpianesi1, G.M. Dadea2 and A. Cresci1

und et al. 1988; Platts-Mills 1995; Singh 1996; Jones 1999).

Table 1 Physical state, chemical compound

+ Compatible; ) noncompatible.

MIC values (ppm)

*MIC value efficacious against all the tested micro-organisms.

(a) 25 by SEM analysis (Fig. 2d). No colonization was observed on

Table 4 Number of colonies (CFU per sec-

Treated filters* 0 0 0 0 2 ± 1 3±2

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