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Materials 2022, 15, 1269. https://doi.org/10.3390/ma15031269 www.mdpi.com/journal/materials
Article
Antibacterial Properties of Biodegradable Silver Nanoparticle
Foils Based on Various Strains of Pathogenic Bacteria Isolated
from The Oral Cavity of Cats, Dogs and Horses
Miłosz Rutkowski 1, Lidia Krzemińska-Fiedorowicz 2, Gohar Khachatryan 2, Julia Kabacińska 3, Marek Tischner 4,
Aleksandra Suder 5, Klaudia Kulik 5 and Anna Lenart-Boroń 5,*
1 Scientific Circle of Biotechnologists „Helisa”, Microbiology Section, Department of Microbiology and
Biomonitoring, Faculty of Agriculture and Economics, University of Agriculture in Krakow; 30-059 Krakow,
Poland; miloszr131@gmail.com
2 Faculty of Food Technology, University of Agriculture in Krakow, 30-149 Krakow, Poland;
lidia.krzeminska@urk.edu.pl (L.K.-F.); gohar.khachatryan@urk.edu.pl (G.K.)
3 „Przychodnia Weterynaryjna Uniwersytecka” Veterinary Clinic, University Center of Veterinary Medicine,
University of Agriculture in Krakow, 30-251 Krakow, Poland; julia.kabacinska@urk.edu.pl
4 Department of Animal Reproduction, Anatomy and Genomics, Faculty of Animal Science, University of
Agriculture in Krakow, 30-059 Krakow, Poland; marek.tischner@urk.edu.pl
5 Department of Microbiology and Biomonitoring, Faculty of Agriculture and Economics, University of
Agriculture in Krakow, 30-059 Krakow, Poland; aleksandra.suder@student.urk.edu.pl (A.S.);
klaudia.kulik@student.urk.edu.pl (K.K.)
* Correspondence: anna.lenart-boron@urk.edu.pl
Abstract: Frequent occurrence of microbial resistance to biocides makes it necessary to find alterna-
tive antimicrobial substances for modern veterinary medicine. The aim of this study was to obtain
biodegradable silver nanoparticle-containing (AgNPs) foils synthesized using non-toxic chemicals
and evaluation of their activity against bacterial pathogens isolated from oral cavities of cats, dogs
and horses. Silver nanoparticle foils were synthesized using sodium alginate, and glucose, maltose
and xylose were used as reducing agents. The sizes of AgNPs differed depending on the reducing
agent used (xylose < maltose < glucose). Foil without silver nanoparticles was used as control. Bac-
terial strains were isolated from cats, dogs and horses by swabbing their oral cavities. Staphylococcus
aureus, methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli and extended-spectrum
beta-lactamase (ESBL) producing E. coli were isolated on selective chromogenic microbiological me-
dia. The bactericidal effect of AgNPs foils obtained using non-toxic chemical compounds against E.
coli, ESBL, S. aureus and MRSA isolated from oral cavities of selected animals was confirmed in this
study. No statistically significant differences were observed between the foils obtained with differ-
ent reducing agents. Therefore, all types of examined foils proved to be effective against the isolated
bacteria.
Keywords: biopolymers; animals; green chemistry; silver nanoparticles; veterinary medicine
1. Introduction
Nanotechnology is among the modern fields of science that find their wide applica-
tion in human and veterinary medicine, agriculture, food and feed production, cosmetic
industry, pharmacy, heritage preservation against microbial biodeterioration, textile in-
dustry and optics [1–4]. Scientific literature describes various forms of nanoparticle syn-
thesis including physical, chemical and biological methods. However, it is the chemical
method that involves the reduction of metal salts with the use of numerous reducing sub-
stances that allows for obtaining various shapes of nanoparticles [5–7]. Due to various
shapes of the adopted structures, silver nanoparticles exhibit numerous biological effects.
Citation: Rutkowski, M.;
Krzemińska-Fiedorowicz, L.;
Khachatryan, G.; Kabacińska, J.;
Tischner, M.; Suder, A.; Kulik, K.;
Lenart-Boroń, A. Antibacterial
Properties of Biodegradable Silver
Nanoparticle Foils Based on Various
Strains of Pathogenic Bacteria
Isolated from The Oral Cavity of
Cats, Dogs and Horses.
Materials 2022, 15, 1269.
https://doi.org/10.3390/ma15031269
Academic Editor: Jung-Kul Lee
Received: 19 January 2022
Accepted: 2 February 2022
Published: 8 February 2022
Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional
claims in published maps and institu-
tional affiliations.
Copyright: © 2022 by the authors. Li-
censee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and con-
ditions of the Creative Commons At-
tribution (CC BY) license (https://cre-
ativecommons.org/licenses/by/4.0/).
Materials 2022, 15, 1269 2 of 17
One of the most important properties of metal nanoparticles is their antimicrobial activity.
It results from a number of actions, ultimately leading to the destruction of their biological
structures and thus limiting the microbial growth. The mechanisms of antibacterial action
of silver nanoparticles (AgNPs) include: disruption of cell wall and cytoplasmic mem-
brane perforation and denaturation, denaturation of ribosomes, interruption of ATP pro-
duction, cytoplasmic membrane disruption by reactive oxygen species and interference
with DNA replication [8–12].
However, one of the widely mentioned side-effects of using nanotechnology is the
possible release of nanomaterials into the environment. Furthermore, major disad-
vantages of popular AgNP production methods include high costs, the use of hazardous
chemical materials, the demand for rigorous environmental conditions, such as pH and
temperature, or the release of toxic by-products [1]. This causes understandable concerns
and the need for developing a green-approach to the nanoparticle production, i.e., cost-
effective, rapid, eco-friendly, scalable and not generating/using toxic substances [1,3].
Polysaccharides, many of which come from natural sources, can be described by the
word “biopolymers” and are a collection of cheap and renewable raw materials used in
industry to create innovative biodegradable materials [13]. The chemical structure of
many polysaccharides allows for the modification of these substances using various ana-
lytical methods, including chemical and enzymatic ones. Due to the proven active prop-
erties of these biopolymers, polysaccharides of natural origin are used in the food industry
and in clinical practice [14–16]. The possibility of using polysaccharides in the synthesis
of metal nanoparticles has been documented in the literature [17–19]. This is because pol-
ysaccharides play an important role in the synthesis of nanometals by stabilizing the re-
sulting particles, thus influencing their shape and size [20]. An interesting example of a
biodegradable polymer is sodium alginate, which in its chemical structure is based on
interconnected units of β-d-mannuronic acid and α-1-guluronic acid [21–24]. Plastic com-
posites created with the use of sodium alginate have found application in numerous eco-
nomic fields, including production of modern food packaging, in medicine, tissue engi-
neering, in pharmacy, medicaments production and biotechnology [25–27].
Periodontal diseases are among the most significant problems that often affect com-
panion animals [28]. Inflammation of the gums in animals that occurs in the oral cavity is
a consequence of the immune system’s response to an emerging bacterial infection [29].
Another aspect to be considered in terms of health problems in humans and animals is
the possible transmission of Staphylococcus aureus between humans and companion ani-
mals, due to the nasal carriage of S. aureus [30–32]. Dogs and cats, which are the most
frequently kept pets, are suggested to play a role in household S. aureus transmission and
recurrent methicillin-resistant S. aureus (MRSA) infections in humans [30,31]. Further-
more, Moodley et al. [32] indicate that veterinary practitioners are at significantly higher
risk of MRSA carriage as a result of their professional contact with animals, e.g., horses,
dogs and cats. More importantly, it has been suggested that antibiotic resistance is more
frequent in canine isolates of S. aureus than in those of human origin [30]. Staphylococcus
aureus is an example of a microorganism that is both a human skin and mucosa commen-
sal but also a frequent cause of serious infections with high mortality and healthcare-as-
sociated costs [33].
Due to the fact that clinicians and veterinarians increasingly often encounter prob-
lems related to the antibiotic resistance of pathogenic microorganisms, finding alternative
substances with antimicrobial activity is very essential nowadays. The phenomenon of
antimicrobial resistance not only causes the problem of reducing the choice of drugs, but
also contributes to the deterioration of the health and welfare of animals. The latest guide-
lines in veterinary medicine try to prevent this. In particular, it is worth paying attention
to diseases of the oral cavity, which are one of the most common diseases that occur in
companion animals. They have health consequences not only locally, but also systemi-
Materials 2022, 15, 1269 3 of 17
cally. Unfortunately, due to significant educational deficiencies in the medical and veter-
inary studies in the field of dentistry, there has been a lot of false information and beliefs
about the treatment of oral diseases.
Therefore, the aim of this study was the synthesis of biodegradable foils containing
silver nanoparticles obtained with the use of non-toxic chemicals, together with the eval-
uation of their antibacterial activity against pathogenic bacteria isolated from the oral cav-
ity of companion animals.
2. Materials and Methods
2.1. Materials
Research-grade chemical reagents were used to prepare the nanocomposites, i.e., so-
dium alginate (Sigma-Aldrich, Poznan, Poland, molecular weight ≈1.565 × 105 Da [34]);
glycerine (Sigma-Aldrich, Poznan, Poland, 99.5%) as an excipient (plasticizer); AgNO3
(Sigma-Aldrich, Poznan, Poland, 99.99%); D-(+)-xylose, D-(+)-maltose monohydrate and
D-(+)-glucose (Sigma-Aldrich, 99%) as reducers and deionized water.
Microbiological media used for the experiments were as follows: Baird Parker agar,
Tryptone-bile-X-glucuronide agar, Chromogenic MRSA Modified Lab Agar, Chromo-
genic ESBL Lab Agar and Mueller-Hinton agar, all obtained from Biomaxima (Lublin, Po-
land).
2.2. Synthesis of Alginate Films Containing Silver Nanoparticles
Sodium alginate (16 g) was dissolved in water so that the biopolymer concentration
was 2%. The resulting suspension was gelatinized at 60 °C for 24 h. Then 200 mL of the
polysaccharide gel was dispensed into four conical flasks and 2.2 mL of Tollens solution
was added. Then, a glycerin solution in a ratio of 1:2 to sodium alginate was introduced
into the gels as a plasticizer and heated for about 0.5 h. After this time, specific reducing
agents were added to each sample. Furthermore, 8 mL of 4% xylose solution were added
to sample No. 1 (Ag-xylose), 8 mL of 4% maltose solution was added to sample No. 2 (Ag-
maltose) and 8 mL of 4% glucose solution was added to sample No. 3 (Ag-glucose). Sam-
ple No. 4 was designated as a control and left without the addition of reducing substance.
The prepared gels were heated while stirring for an hour. After this time, individual gels
were poured into dried, degreased dishes and dried in an oven for 48 h, forming nano-
particle containing foils (Figure 1).
Figure 1. Obtained foils: from the left 1. Ag-xylose; 2. Ag-maltose; 3. Ag-glucose.
2.3. Fourier Transform Infrared (FTIR) Spectroscopy
The ATR-FTIR (attenuated total reflection-Fourier transform infrared) spectra were
recorded in the range of 4000–700 cm−1 at 4 cm−1 resolution using a MATTSON 3000 FT-IR
Materials 2022, 15, 1269 4 of 17
spectrophotometer (Madison, WI, USA) equipped with a 30SPEC 30° reflective cap with
the MIRacle ATR accessory from PIKE Technologies Inc., Madison, WI, USA.
2.4. Ultraviolet-Visible (UV-VIS) Spectrometry
The UV-Vis (ultraviolet-visible spectroscopy) absorption spectra were developed us-
ing a Shimadzu 2101 scanning spectrophotometer ((Shimadzu, Kyoto, Japan) in the range
of 200–800 nm.
2.5. Scanning Electron Microscopy (SEM)
The shape, size and aggregation of nanosilver was characterized using a JEOL 7550
high-resolution scanning electron microscope (SEM) (Akishima, Tokyo, Japan) equipped
with a transmission electron detector (TED) (Akishima, Tokyo, Japan), retractable
backscattered-electron detector (RBEI) (Akishima, Tokyo, Japan) and EDS (energy disper-
sive spectra) detection system of characteristic X-ray radiation INCA PentaFetx3 EDS sys-
tem.
2.6. Isolation and Identification of Bacteria from Cats, Dogs and Horses
The presented study involves material collected from animals in the form of oral
swabs. Due to the fact that the procedure involved in obtaining bacterial strains is neither
harmful, nor causes any type of distress in animals, no bioethical commission approval
was required for this study.
A total of 114 randomly selected animals (46 cats, 26 dogs and 42 horses) were exam-
ined in this study by swabbing their oral cavities. After the collection of samples with
sterile swabs, inoculations were performed on selective media for the isolation of bacterial
pathogens and opportunistic pathogens. Baird Parker agar (Biomaxima, Lublin, Poland)
was used for the isolation and identification of Staphylococcus aureus (grey to black colonies
with clear halo after incubation for 24–48 h at 37 ± 1 °C), Tryptone-bile-X-glucuronide agar
(Biomaxima, Lublin, Poland) was used for the isolation and identification of Escherichia
coli (turquoise to blue colonies after incubation for 24 h at 44 °C), Chromogenic MRSA
Modified Lab Agar (Biomaxima, Lublin, Poland) was used for the isolation of methicillin-
resistant S. aureus (MRSA—rose to mauve colonies after incubation at 35–37 °C for 24 h)
and finally Chromogenic ESBL Lab Agar (Biomaxima, Lublin, Poland) was used for the
isolation of extended-spectrum beta lactamase-producing Enterobacterales (ESBL—Esche-
richia coli: pink to burgundy; Klebsiella, Enterobacter, Serratia and Citrobacter: green/blue to
brown-green and Proteus, Providensia and Morganella: dark to light brown after incubation
at 37 °C for 24 h). After incubation the bacterial colonies characteristic of the listed spe-
cies/groups of bacteria were subcultured by plate streaking and Gram-stained prepara-
tions thereof were observed under the light microscope (1000× magnification).
2.7. Evaluation of Antibacterial Activity of Nanosilver-Containing Foils
The test of the antimicrobial activity of silver nanoparticles in alginate films was car-
ried out using a total of 79 bacterial strains, including 74 isolates collected from animals
and 5 type strains (Table 1). Bacterial isolates were transferred to sterile saline solution to
prepare 0.5 MacFarland suspensions, which were then streaked onto Mueller–Hinton agar
(Biomaxima, Lublin, Poland). Prior to the experiment, the foils were sterilized under UV
light for 20–30 min. Then, 10 × 10 mm squares were cut with a surface sterilized scissors
and applied onto the surface of bacterial cultures. The cultures were incubated at 37 ± 1
°C for 24 h. Afterwards the results were read by measuring the diameters of bacterial
growth inhibition zones around the foil fragments. Two diameters were read and the final
result was expressed as a mean of the two reads (mm). All experiments were conducted
in three replicates.
Materials 2022, 15, 1269 5 of 17
Table 1. Characteristics of bacterial strains used in the experiment.
Bacterial Spe-
cies/Groups
Cats
Dogs
Horses
Type Species
S. aureus
21
21
1
S. aureus ATCC 29213 (sus-
ceptible)
MRSA
4
2
0
S. aureus NCTC 12493
E. coli
0
0
15
E. coli ATCC 35218
E. coli ATCC 25922
ESBL
3
1
6
E. coli ESBL (+) UTI
Total
28
24
22
5
2.8. Statistical Analysis
The normality of the results was verified using the Shapiro–Wilk test. The distribu-
tion of the results was not close to normal, therefore non-parametric tests were applied in
further analyses. The Kruskal–Wallis test was used for the following analyses:
a) the significance of differences between the antibacterial activity of various types of
foils;
b) the significance of differences between the activity of foils against microorganisms
isolated from various groups of animals;
c) the significance of differences in the activity of foils against Gram-positive and Gram-
negative bacteria;
d) the significance of differences in the activity of foils against bacteria belonging to dif-
ferent species / groups.
The significance level was set at a p value of <0.05 for all statistical tests. All analyses
were performed using Statistica ver.13.1 (2021, StatSoft, Tulsa, OK, USA).
3. Results and Discussion
3.1. Physicochemical Properties of Biodegradable Foils
In order to confirm the presence of silver nanoparticles and to determine their size,
scanning electron microscopy was performed. By using secondary electron detection (in
COMPO system), the presence of nanosilver in the whole structure of the obtained com-
posites was proved (Figure 2). The resulting silver nanostructures were characterized by
different sizes and shapes which depended on the used reducer.
Materials 2022, 15, 1269 6 of 17
Figure 2. SEM micrographs of foils taken at different magnifications: (A,B) Ag-xylose (50,000 (A)
and 100,000 (B) magnification ); (C,D) Ag-maltose 10,000 (B) and 50,000 (C) magnification); (E,F)
Ag-glucose 65,000 magnification (E,F).
Silver nanoparticles obtained using xylose as a reducing agent were characterized by
regular and spherical shapes, their sizes varied between 5 and 10 nm. When maltose was
used as a reducer, we observed an increase of the size of the nanoparticles (varying be-
tween 50 and 100 nm) and change in the shape of nanocrystals. Nanoparticles obtained
using glucose formed aggregates sized approximately 100 nm on different geometrical
shapes.
Morphology and stability of the silver nanoparticles depend on the method of their
preparation [35], applied reducer and stabilizing reagent. Microscopic studies showed
that depending on the reducer used, the obtained nanoparticles had different sizes and
shapes. The shape of the nanoparticles has a strong influence on the optical and biological
properties of the samples [36,37]. The used saccharides have different structures, which
may explain the differences in reducing and capping ability. Filippo E. and colleagues [38]
explained the influence of the structure and reducing properties of the used sugars on the
reduction reaction mechanism and on the differences in the size and shape of the synthe-
sized nanoparticles. Other scientists have shown that the reaction temperature, reaction
time, the concentration of silver source, reducing agent and the amount of capping agents
play vital roles in size and yields of silver nanoparticles [39]. They showed that by con-
trolling the concentration of glucose and silver ions and by selecting the appropriate re-
duction reaction temperature, nanosilver of various sizes can be obtained.
Figure 3 shows the UV-Vis absorption spectra of the control sample and the Ag nano-
composites, which show an absorption band at 430 nm for Ag-xylose, 460 nm for Ag-
maltose and between 375 and 520 nm for Ag-glucose. The results indicate the formation
of Ag nanoparticles [34,40]. The width of the peak band indicates that the formed nano-
particles are characterized by different sizes, which has already been confirmed by the
scanning electron microscope (SEM) images.
Materials 2022, 15, 1269 7 of 17
Figure 3. UV–Vis spectra of control (black line), Ag-xylose (green line), Ag-maltose (blue line) and
Ag-glucose (red line).
Figure 4 shows the FTIR absorption spectra of obtained bionanocomposites. We ob-
served the characteristic spectrum of the sodium alginate with a broad band centered at
approximately 3210 cm−1 (hydroxyl groups stretching), low intensity bands at about 2915
cm−1 (attributed to –CH2 groups), two peaks at 1603 cm−1 and 1408 cm−1 (asymmetric and
symmetric stretching modes, respectively, of carboxylate salt groups (–COONa)), and a
number of vibrations in the range of 1100–990 cm−1 (glycoside bonds in the polysaccharide
(C–O–C stretching)) [41]. The absence of significant changes in the shape of the obtained
spectra indicates that the synthesis of nanometals did not cause structural changes in the
alginate molecule.
Materials 2022, 15, 1269 8 of 17
Figure 4. FTIR absorption spectra of control (black line), Ag-xylose (blue line), Ag-maltose (red line)
and Ag-glucose (green line).
3.2. Isolation and Identification of Bacterial Pathogens and Opportunistic Pathogens from Ani-
mals
The examination of oral swabs collected from 114 animals (46 cats, 26 dogs and 42
horses) allowed for the isolation of a total of 74 strains of bacteria, including 28 isolates
collected from cats, 24 from dogs and 22 from horses. The results indicate that 64.91% of
examined animals were carriers of potentially pathogenic bacteria. The distribution of
bacterial groups varied among the animals. In the case of cats S. aureus was the most com-
mon (n = 21, carried by 45.65% of cats), followed by four strains of MRSA (8.69%) and
three ESBL (6.52%). Similarly, among the bacteria isolated from dogs, S. aureus was pre-
dominant (n = 21, carried by as many as 80.77% of dogs), followed by MRSA (n = 2, 7.69%)
and ESBL (n = 1, 3.85%). Reversely, out of 22 equine bacterial isolates, E. coli was the most
predominant (n = 15, carried by 35.71% of horses), followed by six strains of ESBL (14.29%)
and one S. aureus (2.38%). The observed rate of canine (92.31%), feline (60.87%) and equine
(52.38%) colonization by potential bacterial pathogens is higher than the one reported by,
e.g., Boost et al. [30], i.e., 8.8% for dogs (with rate of isolations varying from 5.7 to 14% at
various veterinary clinics) or by Bierowiec et al. [31] for cats (rate of S. aureus isolations of
19.17% for domestic cats without outdoor access and only 8.3% for feral cats). As for
horses, no MRSA was identified by Burton et al. [42] and 7.9% of horses were carriers of
methicillin-susceptible S. aureus. Gergeleit et al. [43] reports that the distribution of Enter-
obacteriaceae (including E. coli) is 17.8% in horses with healthy sinuses and 46.2% in
horses with dental sinusitis. The possibility of transmitting microbial pathogens between
animals and humans has been the subject of significant concern and has been widely de-
scribed in literature [31,44,45]. However, a number of studies report that either dog or cat
ownership is unlikely to significantly increase the risk of infection in healthy people (un-
like in immunocompromised people) [30,31]. More importantly, both Boost et al. [30] and
Bierowiec et al. [31] suggest that the bacterial transmission is more likely to occur from
owner to pet animal rather than the other way round. Regardless of the potential pet-to-
Materials 2022, 15, 1269 9 of 17
human transmission, another important factor to be considered is the fact that periodontal
diseases in carnivorous animals (such as cats and dogs) are among the most common
health issues diagnosed by veterinarians [28]. Inflammations of this region are most often
caused by bacterial colonizations, including these caused by Escherichia coli and Staphylo-
coccus aureus [46]. Having in mind the high colonization rate of the examined companion
animals (particularly cats and dogs) by potentially pathogenic and harmful bacteria, cou-
pled with their possible resistance to antimicrobial agents [30,31] and potential transmis-
sion to humans [32], it is very important to explore all options of introducing new (and
possibly environmentally friendly) materials with antibacterial properties [47].
3.3. Antibacterial Effect of Nanosilver Foils
The antibacterial effect of the AgNP foils made with three different reducing agents
(maltose, glucose and xylose) and control (sole alginate) was tested against 74 strains of
bacterial pathogens isolated from companion animals and 5 type species thereof (Table 1).
Bacteria were divided into groups of Gram-positive (S. aureus and MRSA; n = 51) and
Gram-negative (E. coli and ESBL; n = 28) strains. The results of growth inhibition zones
caused by the nanosilver containing foils against each group of bacteria are presented in
Figure 5, Table S1, Table 2 and summarized in Figures 6–8. Table S1 shows the mean val-
ues, standard deviations, coefficients of variation and minimum/maximum values of
growth inhibition zones, for individual groups of bacteria, isolated from various animals,
compared with the type strains. Table 2 shows the antibacterial efficiency of AgNP foils
expressed as the percentage of bacterial isolates within various groups, whose growth was
inhibited by the AgNPs. In general, the reaction of bacterial strains to the examined AgNP
foils differed largely, as evidenced by the standard deviation and coefficient of variation
values (Table S1). The growth inhibition zone of the S. aureus ATCC 29213 (susceptible to
all antimicrobial agents) was higher than the mean values calculated for S. aureus isolates
derived from cats and dogs. The type strain of MRSA (S. aureus NCTC 12492) showed
smaller growth inhibition zones than the mean values of MRSA isolated from cats and
dogs, except from the zone caused by AgNP foil produced with glucose. Both type strains
of E. coli (ATCC 35218 and ATCC 25922) reacted very poorly to the applied AgNP foils
and their growth inhibition was either none or much smaller than the mean value ob-
tained for the strains derived from horses.
Materials 2022, 15, 1269 10 of 17
Figure 5. Activity of Ag-glucose (G), Ag-maltose (M), Ag-xylose (X) foils compared with control foil
with sole alginate (K) against E. coli (A), ESBL-positive bacteria (B), S. aureus (C) and MRSA (D).
Table 2. Percentage (%) of bacterial isolates, whose growth was inhibited by the AgNPs.
Animals
Bacteria
AgNP Foils
Maltose
Xylose
Glucose
cats
S. aureus
57.14
66.67
71.43
MRSA
100.00
100.00
100.00
ESBL
16.67
33.33
33.33
dogs
S. aureus
85.71
61.90
80.95
MRSA
100.00
100.00
0.00
ESBL
100.00
100.00
0.00
horses
S. aureus
0.00
100.00
100.00
E. coli
73.33
86.67
73.33
ESBL
75.00
83.33
75.00
total
S. aureus
70.59
64.71
76.47
MRSA
100.00
100.00
50.00
E. coli
60.00
70.00
55.00
ESBL
70.59
64.71
76.47
Materials 2022, 15, 1269 11 of 17
Figure 6. Mean growth inhibition zones (mm) caused by the three types of AgNP-containing foils
produced using different reducing agents (maltose, xylose and glucose). The results are means of
three replicates of tests conducted for all examined bacterial isolates (n = 79). Bars represent standard
deviation. Control foils (sole alginate) caused no growth inhibition.
Figure 7. Mean growth inhibition zones (mm) caused by the three types of AgNP-containing foils
produced using different reducing agents (maltose, xylose and glucose). The results are means of
three replicates for the examined bacterial isolates (n = 79) divided into groups of Gram-positives (n
= 51) and Gram-negatives (n = 28). Bars represent standard deviation. Control foils (sole alginate)
caused no growth inhibition.
0
5
10
15
20
Maltose Xylose Glucose Control
Mean growth inhibition zone (mm)
Type of foil (reducing agent used)
Gram+
Gram–
Materials 2022, 15, 1269 12 of 17
Figure 8. Mean growth inhibition zones (mm) caused by the three types of AgNP-containing foils
produced using different reducing agents (maltose, xylose and glucose). The results are means of
three replicates for the examined bacterial isolates (n = 79) of S. aureus (n = 45), MRSA (n = 6), E. coli
(n = 17) and ESBL (n = 11). Bars represent standard deviation. Control foils (sole alginate) caused no
growth inhibition.
The comparison of the effect of AgNP foils against all bacteria isolated from the three
groups of animals (Figure 8) shows that the growth inhibition of bacterial isolates derived
from horses was the highest among all groups. As there are very few studies in which the
reaction of bacterial pathogens, isolated from various animals, was examined with AgNPs
further examinations are worth considering. However, it has been demonstrated that the
antibiotic resistance of bacteria varies between strains isolated from various groups. For
instance, Rubin et al. [48] observed significant differences in the minimum inhibitory con-
centrations of a number of antibiotics against S. aureus and Staphylococcus pseudointerme-
dius of avian, bovine, equine and porcine origin. Furthermore, Middleton et al. [45] ob-
served differences among the prevalence of methicillin resistance in S. aureus isolated
from dogs, horses, cats and dogs. What is more, Bierowiec et al. [31] presented the differ-
ences among the prevalence of antibiotic resistant S. aureus between domestic and stray
cats. This suggests that there can be a number of factors affecting the reaction and suscep-
tibility of bacteria to antimicrobial agents. Among them, previous contact with antibiotics,
horizontal gene transfer and colonization by already resistant bacterial strains or strains
containing genetic determinants of resistance are the most important ones [31]. Moreover,
there are differences in the predominant groups of bacteria isolated from various animals.
Gram-negative E. coli and ESBL-positive bacteria dominated among horses, while Gram-
positive S. aureus and MRSA dominated among cats and dogs. The differences in the cell
wall thickness between these two groups of bacteria largely affect their reaction to anti-
microbial agents [49], as discussed further in more detail. Another important aspect to
refer to is that there are concerns that similarly as in the case of antibiotics, the widespread
and uncontrolled use of silver nanoparticles may cause the resistance to this compound,
where silver-resistant bacteria can be as problematic as antibiotic-resistant ones [50]. In
this study, the efficiency of AgNP foils varied between the types of bacteria and the animal
they were isolated from (Table 3). The mean antibacterial efficiency ranged between 50
and 100% (64.70–76.47% for S. aureus; 50–100% for MRSA; 73.33–86.67% for E. coli and 55–
70% for ESBL).
Materials 2022, 15, 1269 13 of 17
Table 3. Results of Kruskal–Wallis test of significance of differences in antibacterial effects of AgNP
foils obtained with various reducing agents.
Groups of
Bacteria
Tested Foils
Type of Foil
Ag-Maltose
Ag-Xylose
Ag-Glucose
Control
S. aureus
H = 109.53
p = 0.000
Ag-maltose
-
0.89/1.00
0.32/1.00
8.21/0.000
Ag-xylose
0.89/1.00
-
1.22/1.00
7.66/0.000
Ag-glucose
0.32/1.00
1.22/1.00
-
8.52/0.000
Control
8.21/0.000
7.66/0.000
8.52/0.000
-
MRSA
H = 16.99
p = 0.0007
Ag-maltose
-
0.51/1.00
1.92/0.790
3.65/0.006
Ag-xylose
0.51/1.00
-
1.41/1.00
3.14/0.007
Ag-glucose
1.92/1.00
1.41/1.00
-
1.73/1.00
Control
3.65/0.009
3.14/0.007
1.73/1.00
-
E. coli
H = 21.69
p = 0.0001
Ag-maltose
-
1.58/0.691
0.28/1.00
4.76/0.000
Ag-xylose
1.58/0.691
-
1.30/1.00
6.34/0.000
Ag-glucose
0.28/1.00
1.30/1.00
-
5.04/0.000
Control
4.76/0.000
6.34/0.000
5.04/0.030
-
ESBL
H = 50.13
p = 0.000
Ag-maltose
-
0.59/1.00
0.59/1.00
3.36/0.005
Ag-xylose
0.59/1.00
-
1.18/1.00
3.95/0.000
Ag-glucose
0.59/1.00
1.18/1.00
-
2.77/0.034
Control
3.36/0.005
3.95/0.000
2.77/0.034
-
In terms of the efficacy of action of the applied foils, no strict pattern can be observed,
as the largest mean growth inhibition in strains isolated from cats was caused by the foils
with glucose, in strains isolated from dogs—by the foils with maltose, whereas in strains
from horses—by the foils with xylose (Figure 6).
Figure 7 shows the differences of reaction of Gram-positive (MRSA and MSSA, i.e.,
S. aureus) and Gram-negative (non-ESBL producing E. coli and ESBL+) bacteria to the
tested foils. In all cases, Gram-negative bacteria were more susceptible to the action of
AgNP foils (larger mean growth inhibition zones) than the Gram-positives. However,
only differences observed for the xylose-based foils were statistically significant (H =
22.91; p = 0.000). Again, it is not possible to designate the most effective foil, as maltose-
containing AgNP foil was the most effective against Gram-positive bacteria, while foil
containing xylose was the most effective against Gram-negatives. Even more interest-
ingly, the mean value of growth inhibition zone caused by the foil with xylose was the
highest in the case of Gram-negative bacteria and the lowest for Gram-positives.
Further dividing the bacterial groups into S. aureus, MRSA, E. coli and ESBL showed
similar results to the ones obtained for Gram-positive and Gram-negative strains grouped
together (Figure 7). Both S. aureus and MRSA reacted most strongly to the AgNP foils with
maltose, while both E. coli and ESBL strains reacted most strongly to the AgNP foils with
xylose. Maltose-containing foil was the least effective against E. coli, xylose-containing foil
was the least effective against S. aureus and glucose-containing foil was the least effective
against MRSA and ESBL. Only the differences in the reaction of E. coli and S. aureus to the
xylose-based AgNP foil were statistically significant (z = 4.84; p = 0.000046). Quite an im-
portant aspect of concern in terms of antibacterial efficiency of AgNPs is their shape and
size, which varied among the examined foils with the following pattern: Ag-xylose < Ag-
maltose < Ag-glucose. According to Wei et al. [35], the AgNPs’ sizes and shapes are among
the most important factors affecting their toxicity to living cells with a general conclusion
that the smaller the size of AgNPs, the stronger the cytotoxic effect they could have. The
pattern of size differences among the AgNPs examined in this study was directly followed
by the bacterial growth inhibition pattern only in the case of ESBL-positive bacteria. The
highest growth inhibition caused by Ag-xylose was also observed in E. coli, but then Ag-
Materials 2022, 15, 1269 14 of 17
glucose caused the second most effective inhibition. It was on E. coli that thorough re-
search was carried out concerning the mechanisms of AgNPs’ bactericidal actions [36],
reporting that AgNPs increase the outer membrane permeability to toxic substances and
cause leakage of cellular materials.
Similar differences in the reaction of Gram-negative E. coli and Gram-positive S. au-
reus to the effect of photoactivated AgNPs were observed by Al-Sharqi et al. [49], with
much stronger antibacterial effect of AgNPs against E. coli. They attributed these differ-
ences to the structure of bacterial cell walls, i.e., thicker cell wall of Gram-positive bacteria
and stronger negative charge of cell walls in Gram-negatives, which promotes adhesion
of AgNP to the bacterial cell walls and thus higher effectiveness of silver nanoparticles
against bacteria.
Notwithstanding all the above, the antibacterial effectiveness of all three types of
AgNP foils is statistically significant as compared to the control (p < 0.05, Table 3), regard-
less of the reducing agent used and the type of bacteria tested. The well-known antibacte-
rial properties of AgNPs, which result from perforation/disruption of cell membranes,
generation of reactive oxygen species responsible for cell lysis and interference with vital
biomolecules, ribosome function interference, DNA translation alteration and inhibition
of DNA replication [8], have been confirmed in the case of the biodegradable AgNP foils,
examined in this study.
4. Conclusions
The conducted studies confirmed that the oral cavities of animals, such as cats, dogs
and horses, are inhabited by bacterial pathogens, such as methicillin-resistant Staphylococ-
cus aureus (MRSA) and extended spectrum beta-lactamase producing E. coli (ESBL), as
well as by opportunistic pathogens, such as methicillin-susceptible S. aureus and non-
ESBL producing E. coli. The physicochemical analyses confirmed the successful formation
of Ag nanoparticles using all types of non-toxic, biodegradable reducing agents, such as
glucose, maltose and xylose. The sizes of AgNPs varied and increased as follows: Ag-
xylose < Ag-maltose < Ag-glucose. In our study, the Ag-xylose particle size smaller than
10 nm proved to be the most effective against Gram-negative bacteria.
All types of silver-nanoparticle-containing foils proved to cause growth inhibition of
the potential bacterial pathogens. The efficiency of growth inhibition varied between the
species of bacteria. AgNP foils produced using glucose as reducing agent were most ef-
fective against bacteria isolated from cats (71.43% efficiency), foils produced using malt-
ose as reducing agent were the most effective against bacteria isolated from dogs (85.71–
100% efficiency), whereas foils produced using xylose were the most effective against bac-
teria isolated from horses (83.3–100% efficiency). AgNP foils produced using xylose were
the most effective against E. coli and ESBL, while foils produced using maltose were the
most effective against S. aureus and MRSA. Only in the case of ESBL was the growth inhi-
bition directly proportional to the changes in AgNPs’ sizes. The obtained results suggest
that the examined non-toxic, biodegradable silver nanoparticle foils proved effective
against the potential pathogenic bacteria isolated from cats, dogs and horses.
Therefore, future studies in terms of the application of the examined AgNP foils
should be first focused on their non-toxicity to animal mucosa and gums, followed by
their applicability as modern dressings, gels, toothpaste or rinsing solution in veterinary
medicine.
Supplementary Materials: The following are available online at www.mdpi.com/arti-
cle/10.3390/ma15031269/s1, Table S1: Growth inhibition zones in bacterial pathogens and opportun-
istic pathogens caused by the silver nanoparticle foils (mm). The results are means of three repli-
cates. CV (%)—coefficient of variation; min/max—the smallest and the largest diameter of bacterial
growth inhibition (mm) caused by the AgNP foils against each group of bacteria.
Materials 2022, 15, 1269 15 of 17
Author Contributions: Conceptualization, M.R. and A.L.-B.; methodology, A.L-B., G.K., L.K.-F. and
K.K.; formal analysis, A.L.-B.; investigation, M.R., K.K. and A.S.; resources, A.L.-B., G.K., L.K.-F.,
J.K. and M.T.; writing—original draft preparation, M.R. and A.L.-B.; writing—review and editing,
G.K., L.K.-F., J.K., M.T., K.K. and A.S.; visualization, A.L.-B., G.K. and L.K.-F.; supervision, A.L.-B.;
funding acquisition, A.L.-B., G.K, J.K. and M.T. All authors have read and agreed to the published
version of the manuscript.
Funding: This research was funded by the statutory measures of the University of Agriculture in
Kraków. The APC was funded by the statutory measures of the University of Agriculture in Kra-
ków, No.: 010014-D011, 070001-D020 and 080100-DZ016.
Institutional Review Board Statement: The presented study involves material collected from ani-
mals in the form of oral swabs. Due to the fact that the procedure involved in obtaining bacterial
strains is neither harmful, nor causes any type of distress in animals, no bioethical commission ap-
proval was required for this study. The remaining conditions of keeping, handling and use of equine
saliva were according to the European Union Council Directive 2010/63/EU on the Protection of
Animals Used For Scientific Purposes.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data sharing is not applicable to this article.
Conflicts of Interest: The authors declare no conflict of interest.
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