The Role of Biofilms in the Pathogenesis of Animal Bacterial Infections

Abstract

Biofilms are bacterial aggregates embedded in a self-produced, protective matrix. The biofilm lifestyle offers resilience to external threats such as the immune system, antimicrobials, and other treatments. It is therefore not surprising that biofilms have been observed to be present in a number of bacterial infections. This review describes biofilm-associated bacterial infections in most body systems of husbandry animals, including fish, as well as in sport and companion animals. The biofilms have been observed in the auditory, cardiovascular, central nervous, digestive, integumentary, reproductive, respiratory, urinary, and visual system. A number of potential roles that biofilms can play in disease pathogenesis are also described. Biofilms can induce or regulate local inflammation. For some bacterial species, biofilms appear to facilitate intracellular invasion. Biofilms can also obstruct the healing process by acting as a physical barrier. The long-term protection of bacteria in biofilms can contribute to chronic subclinical infections, Furthermore, a biofilm already present may be used by other pathogens to avoid elimination by the immune system. This review shows the importance of acknowledging the role of biofilms in animal bacterial infections, as this influences both diagnostic procedures and treatment.

Full text

Citation: Nesse, L.L.; Osland, A.M.;
Vestby, L.K. The Role of Biofilms in
the Pathogenesis of Animal Bacterial
Infections. Microorganisms 2023,11,
608. https://doi.org/10.3390/
microorganisms11030608
Academic Editors: Giovanni Di
Bonaventura and Ute Römling
Received: 10 February 2023
Revised: 21 February 2023
Accepted: 22 February 2023
Published: 28 February 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
microorganisms
Review
The Role of Biofilms in the Pathogenesis of Animal
Bacterial Infections
Live L. Nesse 1, *, Ane Mohr Osland 2and Lene K. Vestby 2
1Department of Animal Health, Welfare and Food Safety, Norwegian Veterinary Institute, 1433 Ås, Norway
2Department of Analysis and Diagnostics, Norwegian Veterinary Institute, 1433 Ås, Norway
*Correspondence: live.nesse@vetinst.no
Abstract:
Biofilms are bacterial aggregates embedded in a self-produced, protective matrix. The
biofilm lifestyle offers resilience to external threats such as the immune system, antimicrobials,
and other treatments. It is therefore not surprising that biofilms have been observed to be present
in a number of bacterial infections. This review describes biofilm-associated bacterial infections
in most body systems of husbandry animals, including fish, as well as in sport and companion
animals. The biofilms have been observed in the auditory, cardiovascular, central nervous, digestive,
integumentary, reproductive, respiratory, urinary, and visual system. A number of potential roles that
biofilms can play in disease pathogenesis are also described. Biofilms can induce or regulate local
inflammation. For some bacterial species, biofilms appear to facilitate intracellular invasion. Biofilms
can also obstruct the healing process by acting as a physical barrier. The long-term protection of
bacteria in biofilms can contribute to chronic subclinical infections, Furthermore, a biofilm already
present may be used by other pathogens to avoid elimination by the immune system. This review
shows the importance of acknowledging the role of biofilms in animal bacterial infections, as this
influences both diagnostic procedures and treatment.
Keywords:
biofilm; animals; endocarditis; meningoencephalitis; wound infections; endometritis;
mastitis; respiratory disease; subclinical infections
1. Introduction
In the 1970s, biofilms were observed in a Pseudomonas aeruginosa (P. aeruginosa) chronic
lung infection in humans and have since then been presumed to play a part in the patho-
genesis of chronic infections [
1
–
4
]. Most, if not all, bacteria may form biofilms. The clinical
significance of biofilms in human disease has been investigated in several studies in recent
decades. They have been found to affect numerous organ systems such as the auditory,
cardiovascular, digestive, integumentary, reproductive, respiratory, and urinary systems,
and this was thoroughly reviewed by Vestby et al. in 2020 [
5
]. However, not nearly the
same amount of research has been devoted to biofilm infections in animals (Figure 1). Since
the 1980s, biofilm-related infections in domestic animals have been researched, with accen-
tuation on the infections with the largest economic burdens (e.g., mastitis, endometritis,
and respiratory infections). In addition to the obvious importance for food production as
well as for the individual animal, there is also a One Health aspect as infections residing in
animals may be a potential danger, also to humans. Infections may have zoonotic potential,
with animals as infective, possibly asymptomatic, carriers. An advantage of studying
animal infections is the possibility of also including experimental infections, adding even
more valuable knowledge. Concurrently, these studies may be explicitly designed and
directed specifically toward a certain species. In human diseases, merely natural infections
may be studied. To fully comprehend the response to
in vivo
biofilms, one cannot depend
on in vitro experiments alone [6].
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Microorganisms 2023,11, 608 2 of 22
Microorganisms 2023, 11, x FOR PEER REVIEW 2 of 23
diseases, merely natural infections may be studied. To fully comprehend the response to
in vivo biofilms, one cannot depend on in vitro experiments alone [6].
Figure 1. Body systems with biofilm-associated diseases addressed in this review.
Biofilms are the most prevalent mode of life for bacterial cells and may be mono- or
polymicrobial [7,8]. In biofilms, the bacteria form aggregates and are embedded in a self-
produced protective matrix composed of extracellular elements such as eDNA, polysac-
charides (e.g., alginate), and proteins (e.g., fibrin) [9]. Here, bacteria communicate through
signaling molecules, regulated by complex quorum sensing (QS) [10], and alter their gene
expression and protein production. Horizontal gene transfer may also occur in this com-
munity [11,12]. Following infection, different scenarios may occur depending on the in-
fective agent, host immune defense, and site affected. The cells in biofilms may remain
hidden from the body’s immune system due to the protection of the matrix or due to in-
tracellular biofilm formation [13,14], which will permit chronic subclinical infections to
occur. Activation of both the innate and adaptive immune systems may also ensue, lead-
ing to a longstanding inflammation, mainly with polymorphonuclear neutrophils
(PMNs). This can cause tissue damage and necrosis [6] whereas the biofilm remains unaf-
fected. As the biofilm matures and disperses [15], there is a possibility of acute infections
developing or infections spreading elsewhere in the body. Moreover, the presence of a
biofilm has been found to ease intracellular invasion [5]. Within the biofilm, bacteria are
well protected against outside forces such as bactericides/antibiotics [12,13,16,17], and the
bacteria alter their metabolism to tolerate less nutrient availability and anoxia [18], devel-
oping a tolerant subpopulation. All bacteria may acquire genetic resistance (also while in
biofilms) to antibiotics; however, the tolerance appearing in the protection of biofilms is
particular to this phenotype. This tolerance renders bacteria in biofilms substantially more
difficult to treat than planktonic bacteria. In essence, infections involving biofilms are usu-
ally slowly developing, persistent infections where the immune system is defeated and
Figure 1. Body systems with biofilm-associated diseases addressed in this review.
Biofilms are the most prevalent mode of life for bacterial cells and may be mono-
or polymicrobial [
7
,
8
]. In biofilms, the bacteria form aggregates and are embedded in
a self-produced protective matrix composed of extracellular elements such as eDNA,
polysaccharides (e.g., alginate), and proteins (e.g., fibrin) [
9
]. Here, bacteria communicate
through signaling molecules, regulated by complex quorum sensing (QS) [
10
], and alter
their gene expression and protein production. Horizontal gene transfer may also occur
in this community [
11
,
12
]. Following infection, different scenarios may occur depending
on the infective agent, host immune defense, and site affected. The cells in biofilms may
remain hidden from the body’s immune system due to the protection of the matrix or due
to intracellular biofilm formation [
13
,
14
], which will permit chronic subclinical infections to
occur. Activation of both the innate and adaptive immune systems may also ensue, leading
to a longstanding inflammation, mainly with polymorphonuclear neutrophils (PMNs).
This can cause tissue damage and necrosis [
6
] whereas the biofilm remains unaffected. As
the biofilm matures and disperses [
15
], there is a possibility of acute infections developing
or infections spreading elsewhere in the body. Moreover, the presence of a biofilm has
been found to ease intracellular invasion [
5
]. Within the biofilm, bacteria are well protected
against outside forces such as bactericides/antibiotics [
12
,
13
,
16
,
17
], and the bacteria alter
their metabolism to tolerate less nutrient availability and anoxia [
18
], developing a tolerant
subpopulation. All bacteria may acquire genetic resistance (also while in biofilms) to
antibiotics; however, the tolerance appearing in the protection of biofilms is particular to
this phenotype. This tolerance renders bacteria in biofilms substantially more difficult to
treat than planktonic bacteria. In essence, infections involving biofilms are usually slowly
developing, persistent infections where the immune system is defeated and the response to
antimicrobial treatment is inconsistent. Accordingly, biofilms are likely to be essential in
the pathogenesis of chronic disease.
This review aims to give a detailed description of observations and indications of
biofilms in bacterial infections of diverse organ systems in animals (Figure 1) as well as
the possible role biofilms may have in the pathogenesis of these infections. Biofilms on
teeth and organ implants and the treatment of biofilm infections are out of the scope of this
review. This review will encompass husbandry animals, such as ruminants, pigs, and fish.
It will also include companion and sport animals, such as dogs, cats, and horses. Lastly, it

Microorganisms 2023,11, 608 3 of 22
will enclose topics on experimental animals, such as rats, mice, and rabbits that may be
relevant and informative to the field.
2. Auditory System
Canine Otitis Externa (OE)
OE is a frequently reported disorder in dogs, associated with infections by Staphylococ-
cus pseudintermedius (S. pseudintermedius) and P. aeruginosa in addition to yeast pathogens [
19
].
Cytological observations of microbial aggregates and filamentous veil-like materials in
clinical practice have earlier been indicative of the presence of biofilms [
20
]. In smears from
human otitis media with effusion, periodic acid-Schiff (PAS) has been used to visualize
polysaccharide biofilm matrix components.
In a study by Parnell-Turner et al. to identify biofilms in dogs with OE, several
investigators studied cytological smears from both patients and controls stained with PAS
and modified Wright’s stain in a blind test [
20
]. The term microbial aggregate was defined
as: “(i) a distinct, dense mass of apparently connected micro-organisms; and (ii) different
micro-organisms that come into focus across two or more focal planes”. In addition, to meet
the criteria for aggregate-associated infection (AAI): “the slide had to satisfy the criteria
for presence of purulence, infection and at least one microbial aggregate present within
a discrete area of stained extracellular matrix (i.e., suspected EPS)”. There was a good
agreement between the investigators on the findings in PAS-stained smears, which they
found easier to read than the smears stained with modified Wright’s. In the smears from the
patients, the investigators could observe high-density aggregates of microorganisms within
a well-defined stained matrix (Figure 2). This was not observed in any of the controls. The
findings show that biofilms are associated with OE in dogs.
Microorganisms 2023, 11, x FOR PEER REVIEW 3 of 23
the response to antimicrobial treatment is inconsistent. Accordingly, biofilms are likely to
be essential in the pathogenesis of chronic disease.
This review aims to give a detailed description of observations and indications of
biofilms in bacterial infections of diverse organ systems in animals (Figure 1) as well as
the possible role biofilms may have in the pathogenesis of these infections. Biofilms on
teeth and organ implants and the treatment of biofilm infections are out of the scope of
this review. This review will encompass husbandry animals, such as ruminants, pigs, and
fish. It will also include companion and sport animals, such as dogs, cats, and horses.
Lastly, it will enclose topics on experimental animals, such as rats, mice, and rabbits that
may be relevant and informative to the field.
2. Auditory System
Canine Otitis Externa (OE)
OE is a frequently reported disorder in dogs, associated with infections by Staphylo-
coccus pseudintermedius (S. pseudintermedius) and P. aeruginosa in addition to yeast patho-
gens [19]. Cytological observations of microbial aggregates and filamentous veil-like ma-
terials in clinical practice have earlier been indicative of the presence of biofilms [20]. In
smears from human otitis media with effusion, periodic acid-Schiff (PAS) has been used
to visualize polysaccharide biofilm matrix components.
In a study by Parnell-Turner et al. to identify biofilms in dogs with OE, several inves-
tigators studied cytological smears from both patients and controls stained with PAS and
modified Wright’s stain in a blind test [20]. The term microbial aggregate was defined as:
“(i) a distinct, dense mass of apparently connected micro-organisms; and (ii) different mi-
cro-organisms that come into focus across two or more focal planes”. In addition, to meet
the criteria for aggregate-associated infection (AAI): “the slide had to satisfy the criteria
for presence of purulence, infection and at least one microbial aggregate present within a
discrete area of stained extracellular matrix (i.e., suspected EPS)”. There was a good agree-
ment between the investigators on the findings in PAS-stained smears, which they found
easier to read than the smears stained with modified Wright’s. In the smears from the
patients, the investigators could observe high-density aggregates of microorganisms
within a well-defined stained matrix (Figure 2). This was not observed in any of the con-
trols. The findings show that biofilms are associated with OE in dogs.
Figure 2.
Comparison of bacterial aggregates across two focal planes from slides assessed to have
aggregate-associated infection. In-focus microorganisms are indicated with white arrows; out-of-focus
microorganisms indicated with the white arrowheads. Note the difference in the focus of organisms across
focal planes. Microorganisms are high-density and clustered within a well-defined stained matrix, versus
organisms on the periphery, which are not associated with the matrix. Both samples are from the same
ear. (
a
,
b
) modified Wright’s stain, (
c
,
d
) periodic acid-Schiff stain.
(a–d)
100
×
(oil) objective, bar = 10
µ
m.
Reprinted from [20], Copyright (2021), with permission from John Wiley and Sons.

Microorganisms 2023,11, 608 4 of 22
3. Cardiovascular System
Myocarditis is inflammation of the heart muscle (myocardium) [
21
]. The myocardium
is the muscular layer of the heart and consists of cardiac muscle cells. The papillary muscles
of the left ventricular myocardium are often involved in myocarditis, often visualized
by purple discoloration of the overlying endocardium [
22
]. A study has also shown that
biofilm-like aggregates of bacteria can occur in capillaries and veins in the myocardium [
23
].
The number of studies on myocarditis in animals is very limited.
Bovine Myocarditis
In bovine, Histophilus somni (H. somni) has been found to be the most common causative
agent in myocarditis infection that results in sudden death, and the importance of H. somni
in this disease has been recognized since the 1980s [
21
,
23
]. H. somni is a non-motile, Gram-
negative bacterium that is a facultative anaerobe. It belongs to the family Pasteurellacease.
H. somni biofilms have been shown to be particularly prominent in the cardiac tissue of
myocarditis cases [21].
A study by Sandal et al. [
21
] claimed to have found evidence that biofilms play a role
in the pathogenesis of both myocarditis and bovine respiratory disease complex (BRDC) in
calves (see chapter 8.1 for results on BRDC). In that study, four male calves were challenged
with H. somni, while one calf was left unchallenged as a control. Post-mortem, myocardial
necrosis was found in various degrees in the challenged calves. Histopathology showed
suppurative myocarditis. A variety of advanced microscopy techniques, in combination
with different staining techniques (transmission electron microscopy, immunoelectron
microscopy, scanning electron microscopy, and fluorescence in situ hybridization) were
used to visualize biofilm matrix components. This revealed that large amounts of biofilm
matrix were present. The authors hypothesize that it is the anaerobic environment in the
myocardium that results in the prominent biofilm formation in this area [
21
]. A study by
Elswaifi et al., focusing on respiratory tract infection, suggested that one of the H. somni
virulence factors is the presence of phosphorylcholine (ChoP) on lipooligosaccharide (LOS)
occurring in the antigenic phase. This may reduce the host inflammatory response and pro-
mote the formation of stable biofilms. LOS antigenic variation may occur through variations
in the composition or structure of glycoses or their substitutions, such as ChoP [24].
4. Central Nervous System
Knowledge is sparse regarding possible biofilm formation within the central nervous
system. However, a study on tilapia fish showed biofilm formation in the brain with the
fish pathogen Streptococcus agalactiae (S. agalactiae), i.e., group B streptococcus, GBS [
25
].
Furthermore, the presence of a biofilm was associated with the development of chronic
subclinical meningoencephalitis in the fish.
Chronic Streptococcal Meningoencephalitis in Fish
S. agalactiae (GBS) was first associated with bovine mastitis. It is also known to persist
asymptomatically in the human digestive and genitourinary tracts, as well as in the upper
airways, and it is the main cause of pneumonia, bacteremia, and meningitis in neonates [
26
].
Furthermore, it infects a number of fish species [
27
,
28
]. Piscine GBS (PiGBS) penetrates
the intestinal mucous membrane and travels via the bloodstream to the brain where it
causes chronic meningoencephalitis [
29
]. It has been suggested that nucleated erythrocytes
facilitate brain invasion leading to granulomatous inflammation.
In the study of Isiaku et al. [
25
], red hybrid tilapia fish were orally given PiGBS (ex-
posed) or phosphate-buffered saline (controls). The exposed group displayed acute clinical
signs. During the first nine days, 20% died and 17% were excluded due to severe lesions.
Thereafter, 53% of the fish progressed into a chronic, subclinical state. In these individuals,
biofilms in the form of PiGBS aggregates were observed around the meningeal surfaces
and within attached substances of an exopolymeric matrix with a considerable amount of
exopolysaccharides using PAS staining (Figure 3), fluorescence in situ hybridization and

Microorganisms 2023,11, 608 5 of 22
confocal laser scanning microscopy. These PiGBS were not eliminated by inflammatory
responses in the brain, nor by treatment with antibiotics, i.e., displaying the typical feature
of biofilm-residing bacteria.
Microorganisms 2023, 11, x FOR PEER REVIEW 5 of 23
[26]. Furthermore, it infects a number of fish species [27,28]. Piscine GBS (PiGBS) pene-
trates the intestinal mucous membrane and travels via the bloodstream to the brain where
it causes chronic meningoencephalitis [29]. It has been suggested that nucleated erythro-
cytes facilitate brain invasion leading to granulomatous inflammation.
In the study of Isiaku et al. [25], red hybrid tilapia fish were orally given PiGBS (ex-
posed) or phosphate-buffered saline (controls). The exposed group displayed acute clini-
cal signs. During the first nine days, 20% died and 17% were excluded due to severe le-
sions. Thereafter, 53% of the fish progressed into a chronic, subclinical state. In these in-
dividuals, biofilms in the form of PiGBS aggregates were observed around the meningeal
surfaces and within attached substances of an exopolymeric matrix with a considerable
amount of exopolysaccharides using PAS staining (Figure 3), fluorescence in situ hybrid-
ization and confocal laser scanning microscopy. These PiGBS were not eliminated by in-
flammatory responses in the brain, nor by treatment with antibiotics, i.e., displaying the
typical feature of biofilm-residing bacteria.
Figure 3. Representative image of exopolysaccharide (EPS) matrix on meninges of the tilapia model.
Piscine GBS on the meningeal surface secretes acidic polysaccharides—light blue (red arrows). Neu-
tral mucopolysaccharides of the brain parenchyma—magenta. Nucleus—dark blue. Myelencepha-
lon, AB/PAS stain. For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article. https://doi.org/10.1016/j.micpath.2016.10.029, accessed on
7 November 2022. Reprinted from [25], Copyright (2017), with permission from Elsevier.
5. Digestive System
The huge microbiota of the gastrointestinal tract consists of a large number of bacte-
rial species moving between different lifestyles, i.e., as planktonic, biofilm-residing and
biofilm-dispersed bacteria [30]. Natural polymicrobial biofilms are present throughout the
gastrointestinal tract, where they are attached to mucin or food particles in the lumen or
to the epithelial surface. Disease-associated biofilms may have altered bacterial and/or
matrix composition, attachment, organization, metabolite production, or other abnormal
features.
In humans, inflammatory bowel disease (ulcerative colitis and Crohn’s disease) and
colorectal cancer have been associated with biofilms dominated by Bacteroides fragilis and
Enterobacteriaceae adhering to the epithelium [5]. Gastric biofilms have been observed in
Helicobacter pylori-positive patients, as well as in an H. pylori mouse model [31]. In addi-
tion, biofilms of Salmonella enterica serovar Typhi on gallstones have been indicated to
cause the carrier state of this bacteria, often resulting in complications such as hepatitis,
chronic diarrhea, pancreatitis, and even hepatobiliary carcinomas [5]. However, little is
known about the role of biofilms in animal gastrointestinal infections.
Figure 3.
Representative image of exopolysaccharide (EPS) matrix on meninges of the tilapia model.
Piscine GBS on the meningeal surface secretes acidic polysaccharides—light blue (red arrows). Neu-
tral mucopolysaccharides of the brain parenchyma—magenta. Nucleus—dark blue. Myelencephalon,
AB/PAS stain. For interpretation of the references to color in this figure legend, the reader is re-
ferred to the web version of this article. https://doi.org/10.1016/j.micpath.2016.10.029, accessed on
7 November 2022. Reprinted from [25], Copyright (2017), with permission from Elsevier.
5. Digestive System
The huge microbiota of the gastrointestinal tract consists of a large number of bacte-
rial species moving between different lifestyles, i.e., as planktonic, biofilm-residing and
biofilm-dispersed bacteria [
30
]. Natural polymicrobial biofilms are present throughout the
gastrointestinal tract, where they are attached to mucin or food particles in the lumen or to
the epithelial surface. Disease-associated biofilms may have altered bacterial and/or matrix
composition, attachment, organization, metabolite production, or other abnormal features.
In humans, inflammatory bowel disease (ulcerative colitis and Crohn’s disease) and
colorectal cancer have been associated with biofilms dominated by Bacteroides fragilis and
Enterobacteriaceae adhering to the epithelium [
5
]. Gastric biofilms have been observed in
Helicobacter pylori-positive patients, as well as in an H. pylori mouse model [
31
]. In addition,
biofilms of Salmonella enterica serovar Typhi on gallstones have been indicated to cause
the carrier state of this bacteria, often resulting in complications such as hepatitis, chronic
diarrhea, pancreatitis, and even hepatobiliary carcinomas [
5
]. However, little is known
about the role of biofilms in animal gastrointestinal infections.
Salmonella Infections
Most serovars of non-typhoid Salmonella enterica (S. enterica) rarely give clinical symp-
toms in healthy adult animals. However, the bacteria can colonize the gut and thereby enter
the human food chain, causing human outbreaks of salmonellosis. Direct or indirect contact
with domesticated or wild animals may also be a source of infection for humans. A study
in mice has strongly indicated that orally ingested Salmonella enterica serovar Typhimurium
(STM) can produce an intestinal biofilm. It is probable that this may be the case in other
animals as well, but the significance of such a biofilm in relation to the carrier state and/or
to the development of clinical disease is still unknown.

Microorganisms 2023,11, 608 6 of 22
The study was focused on an important component of the salmonella biofilm ma-
trix [
32
]. This component was curli, i.e., an extracellular, bacterial amyloid, also called curli
fimbriae. To investigate whether STM could produce curli
in vivo
, Miller et al. infected
mice orally with STM grown so that curli were not present in the inoculum. After four to six
days, small bacterial clusters or microcolonies were observed within the cecum. Immuno-
histochemistry staining confirmed that these bacterial cells were curli-producing STMs. The
bacteria were also detected in the colon, where they were tightly packed, and had the most
intense curli staining. On the other hand, STMs in the small intestine were sporadically
spread and not curli-producing. Furthermore, SDS-PAGE investigations showed that curli
was not present in the colon of control mice, confirming that the observed curli in the
inoculated mice was not produced by other bacterial species in the microbiota. Mice were
also inoculated with an STM luciferase reporter strain for the expression of the csgD gene,
which encodes the major regulator of biofilm formation in STM. The expression of this
gene was observed in the GI tracts of all infected mice. Together, these results indicate the
production of an intestinal STM biofilm in the lower GI tract.
6. Integumentary System
Wounds occur when living tissue is damaged. Most wounds containing microorgan-
isms do heal successfully but may result in infections if microorganisms multiply and the
healing process is disrupted. This applies to both humans and animals [
33
]. Although it is
generally accepted that biofilms are involved in many wound infections, only a few studies
on naturally occurring biofilm wound infections in animals have been conducted. There is a
limited number of studies on equine [
34
–
36
] and approximately the same number of studies
on companion animals such as cats and dogs [
37
–
39
]. In addition, several experimental
studies have been performed (Figure 4).
6.1. Studies on Naturally Occurring Wound Infections
As for naturally occurring wound infections in veterinary medicine, horses are at
risk of developing chronic wounds on limbs, and those wounds have many similarities
to chronic wounds in humans [
40
]. A study by Westgate et al. claims to have found
evidence that as much as 61.5% (8 out of 13) of naturally occurring wounds from equines
are biofilm infections. In that study, anaerobic and aerobic bacteria were isolated from the
wound site, and it was found that P. aeruginosa and Staphylococcus aureus (S. aureus) were
the two most dominating bacterial species isolated from equine wounds. When testing
the biofilm-forming abilities of the isolates from the wounds versus isolates from equine
skin, it was found that isolates from equine wounds formed significantly more biofilms
(p< 0.05) in microtiter plates than isolates from equine skin [
34
]. However, the validity of
in vitro
methods as models for
in vivo
biofilms is disputed and especially
in vitro
models
based on microtiter plates or flow cells. One reason for this is suggested to be due to
differences in biofilm culture conditions
in vitro
and
in vivo
as
in vitro
conditions often
involve well-defined minimal culture media, whereas
in vivo
conditions involve blood and
other bodily fluids as the growth environment [41].
A recent review article by Jørgensen et al. concluded that P. aeruginosa and S. aureus
were the two most dominant bacterial species in biofilm wound infections [
33
]. The same
authors have, in a previous experimental study, found that biofilms were present in 100% of
surgically prepared and bandaged wounds of equine limbs. In contrast, biofilms were
not found in body wounds. The authors speculate that this is due to the diminished and
prolonged inflammatory response detected in limb wounds compared to body wounds or
the hypoxic conditions of equine limb wounds. In that study, the authors present a model
for studying wound infection in horses using an equine laboratory animal model [
42
]. Mi-
croscopical techniques using peptide nucleic acid (PNA), fluorescence in situ hybridization,
and confocal laser scanning microscopy were used in the study to categorize the wounds as
biofilm-infected wounds or not [
42
]. These techniques have been suggested to be the gold
standard for the visualization of biofilm infections in wounds in veterinary medicine [33].

Microorganisms 2023,11, 608 7 of 22
Microorganisms 2023, 11, x FOR PEER REVIEW 7 of 23
Figure 4. Studies on naturally occurring and experimental wound infections, including information
on techniques that have been used in studies on natural infections in horses and dogs. Below are
explanations of the symbols used in the figure.
6.1. Studies on Naturally Occurring Wound Infections
As for naturally occurring wound infections in veterinary medicine, horses are at risk
of developing chronic wounds on limbs, and those wounds have many similarities to
chronic wounds in humans [40]. A study by Westgate et al. claims to have found evidence
that as much as 61.5% (8 out of 13) of naturally occurring wounds from equines are biofilm
infections. In that study, anaerobic and aerobic bacteria were isolated from the wound
site, and it was found that P. aeruginosa and Staphylococcus aureus (S. aureus) were the two
most dominating bacterial species isolated from equine wounds. When testing the bio-
film-forming abilities of the isolates from the wounds versus isolates from equine skin, it
was found that isolates from equine wounds formed significantly more biofilms (p < 0.05)
in microtiter plates than isolates from equine skin [34]. However, the validity of in vitro
methods as models for in vivo biofilms is disputed and especially in vitro models based
on microtiter plates or flow cells. One reason for this is suggested to be due to differences
in biofilm culture conditions in vitro and in vivo as in vitro conditions often involve well-
defined minimal culture media, whereas in vivo conditions involve blood and other bod-
ily fluids as the growth environment [41].
A recent review article by Jørgensen et al. concluded that P. aeruginosa and S. aureus
were the two most dominant bacterial species in biofilm wound infections [33]. The same
Figure 4.
Studies on naturally occurring and experimental wound infections, including information
on techniques that have been used in studies on natural infections in horses and dogs. Below are
explanations of the symbols used in the figure.
A study by Swanson et al. is claiming to be the first study that reports a bacterial
biofilm in chronic wounds in dogs. That study uses 16S rRNA fragment sequencing and
pyrosequencing to identify microorganisms present in a pressure wound on one dog. In
addition, the biofilm was claimed to be identified histologically [
39
]. A different study
using ninety-one historical formalin-fixed and paraffin-embedded samples from dogs
(
n = 68
), cats (n = 15), and horses (n = 8) reported to have found evidence of biofilms in
only two of the samples examined. Both biofilms were found in canine tissue samples and
contained exclusively cocci, but due to the nature of the samples, complete identification
of the bacterial species was not possible [
37
]. The same authors have found in a different
study that the most dominant bacterial species from wounds of dogs are staphylococci and
streptococci [
38
]. Although the number of samples tested in the study is fairly high, the
study uses criteria to determine whether or not a biofilm is present in the sample described
in a study from 2003 [
43
], so its relevance may be disputable. None of the studies addresses
the pathogenesis.

Microorganisms 2023,11, 608 8 of 22
6.2. Laboratory Studies on Wound Infections
In contrast to the limited number of studies on naturally occurring biofilm wound
infections, there are several studies that have been conducted using laboratory animals,
mainly pigs [
44
], rabbits, and rodents [
45
,
46
], but also equines as described in the above
paragraph [
42
]. Studies using laboratory animals are mostly used as models with the
purpose of better understanding infections in humans [
47
] but may also be used as models
for a better understanding of infections in animals [
42
]. Wound healing in rodents happens
by contraction in contrast to epithelialization and granulation in humans [
45
]. Using a
rodent model to study biofilms and wounds showed that polymicrobial biofilms delay
wound closure and healing [
48
,
49
]. Using a rabbit ear model, biofilms were found to
significantly delay epithelialization and granulation tissue formation. Wounds containing
biofilms were found to significantly express lower levels of inflammatory cytokines than
infected wounds [
50
]. Another study found that biofilms were formed in ischemic wounds
but not in non-ischemic wounds where neutrophils and macrophages were present [
51
]. The
wound healing process in pigs occurs through inflammation, proliferation, re-epithelization,
and remodeling, which is analogous to humans [
45
]. Pastar et al. found that using a partial
thickness wound model in pigs, the delayed healing of wounds was found due to the
suppression of epithelialization and the expression of virulence factors [52].
7. Reproductive System
7.1. Endometritis
Successful reproduction is essential in animal husbandry, and failures cause vast
economic expenses. Research on diseases of the reproductive system in animals is therefore
of major interest. Endometritis is the inflammation of the lining of the uterus, usually
involving infection. The uterine environment has been assumed to be sterile, but recent
studies in humans [
53
] and several animal species [
54
–
57
], have revealed the presence of
a uterine microbiota. Uterine biofilms have also been observed in humans [
58
], as well
as in animals. The significance of biofilms in the pathogenesis of endometritis has been
demonstrated in relation to the pyometra form of endometritis in dogs. In other animal
species and in humans, the role of biofilms in endometritis has still not been clarified.
7.1.1. Endometritis in Dogs
Pyometra is a potentially life-threatening, suppurative bacterial endometritis with the
accumulation of inflammatory exudate [
59
]. It is one of the most common diseases of adult
nulliparous female dogs. Escherichia coli (E. coli),which is most often isolated, may produce
endotoxin resulting in sepsis, shock, and renal failure. Other species of the microbiota of
the gastrointestinal and genitourinary tract of dogs, e.g., S. intermedius and
β
-hemolytic
streptococci, have also been associated with the disease.
Fiamengo et al. found biofilms associated with the disease when examining endome-
trial biopsies from 16 bitches with pyometra positive for E. coli [
60
]. Histopathology from all
cases showed suppurative inflammation, as well as surface and luminal exudate. Chronic
inflammation was observed in 15 cases by the presence of lymphocytes and plasma cells as-
sociated with cystic hyperplasia. In 14 of the 16 cases, histopathology identified superficial
basophilic amorphous acellular material, which displayed a bright pink color with PAS
staining. Furthermore, fluorescence in situ hybridization techniques identified E. coli being
present within this material. In addition, a biofilm was indicated by scanning electron
microscopy observations of the fibrous matrix on the luminal surface of the endometrium
in all samples. Neither the bacteria nor the matrix was observed in control samples from
two healthy bitches.
7.1.2. Endometritis in Mares
Endometritis is a primary cause of infertility in mares, contributing to major economic
losses. It is generally caused by post-breeding failure to remove spermatozoa, inflamma-
tory exudate, and bacteria [
61
]. Streptococcus equi subsp. zooepidemicus, E. coli,Klebsiella

Microorganisms 2023,11, 608 9 of 22
pneumoniae, and P. aeruginosa are commonly identified. Endometritis can be difficult to
treat with antibiotics, a feature that has suggested intrauterine biofilm production to play a
role [62].
Indeed, P. aeruginosa has been shown to form biofilms on the endometrial surface
of mares. Ferris et al. [
63
] developed an
in vivo
model to monitor intrauterine biofilms
in infectious endometritis, in which a mare was inoculated with three lux-engineered P.
aeruginosa strains isolated from equine uterine infections. Five days later, an ultrasound
revealed excessive edema and inflammation. When opening the uterus, the presence of a
biofilm was indicated by observations of a luminescent, strongly adherent material, which
was positive for P. aeruginosa and was observed on the endometrial surface. The model was
further used to study the spatial intrauterine localization of metabolically active bacteria in
six mares [
64
]. The biofilm matrix component Pel was observed in endometrial samples
with tissue-adherent bacteria (Figure 5). Interestingly, reduced numbers of neutrophils,
as well as the increased gene expression of the immune-modulatory, anti-inflammatory
Interleukin 10, was observed in areas surrounding tissue-adherent bacteria. This indicates
a modulated immune response in such areas. However, tissue inflammation was the same
in areas with and without biofilm.
Microorganisms 2023, 11, x FOR PEER REVIEW 10 of 23
Figure 5. Detection of tissue-adherent P. aeruginosa in endometrium samples. H&E image of the
endometrium with tissue-adherent P. aeruginosa on the luminal surface (black arrow) (A) and deep
in the endometrial glands (black arrow) (B). (C) Differential interference contrast image of an endo-
metrial gland below the luminal surface of the uterus; this is similar to the area represented in panel
B by the black arrow. Immunofluorescent staining of tissue-adherent P. aeruginosa with an anti-
Pseudomonas antibody (Alexa Fluor 405) (D) and anti-Pel lectin (Texas red) (E) and merged image
detecting the Pel exopolysaccharide colocalized with P. aeruginosa (F). Immunofluorescent images
are projected images of Z-stacks as processed by Volocity image analysis software in which 0.5-μm
scanning increments were performed through approximately 10 μm of tissue. The scale bar is 4 μm.
Reprinted from: Ferris et al. 2017 [64].
7.2. Endometritis in Cows
Postpartum endometritis is one of the main causes of reduced fertility in dairy cattle.
Metagenomic analyses have shown that the uterine microbiota of cows with clinical en-
dometritis displays an increased abundance of Fusobacterium and a unique presence of
Trueperella and Peptoniphilus [65]. The increased prevalence of Trueperella pyogenes (T. py-
ogenes), earlier named Arcanobacterium pyogenes, in the microbiome of cows with endome-
tritis, has also been reported in other studies [66,67].
Amaci et al. [68] investigated secretion samples from 20 repeat breeder cows, i.e.,
cows who failed to conceive after three or more inseminations with fertile semen without
any anatomic or infectious abnormality. The smears were prepared from the sediment of
centrifuged uterine saline lavage. Biofilms visualized as red complexes with PAS staining
were observed in 12 animals (60%). Bacteria were isolated from eight animals, all of them
displaying biofilms in the smears. There was no correlation between the presence of a
biofilm and the results from the cytological examinations of uterine secretion. Further-
more, there was no control group in this study. Consequently, the study demonstrated
the presence of an intrauterine biofilm in repeat breeder cows, but a possible effect on
fertility or the development of endometritis is unknown.
Figure 5.
Detection of tissue-adherent P. aeruginosa in endometrium samples. H&E image of the
endometrium with tissue-adherent P. aeruginosa on the luminal surface (black arrow) (
A
) and deep
in the endometrial glands (black arrow) (
B
). (
C
) Differential interference contrast image of an en-
dometrial gland below the luminal surface of the uterus; this is similar to the area represented in
panel B by the black arrow. Immunofluorescent staining of tissue-adherent P. aeruginosa with an
anti-Pseudomonas antibody (Alexa Fluor 405) (
D
) and anti-Pel lectin (Texas red) (
E
) and merged image
detecting the Pel exopolysaccharide colocalized with P. aeruginosa (
F
). Immunofluorescent images
are projected images of Z-stacks as processed by Volocity image analysis software in which 0.5-
µ
m
scanning increments were performed through approximately 10
µ
m of tissue. The scale bar is 4
µ
m.
Reprinted from: Ferris et al. 2017 [64].

Microorganisms 2023,11, 608 10 of 22
7.2. Endometritis in Cows
Postpartum endometritis is one of the main causes of reduced fertility in dairy cat-
tle. Metagenomic analyses have shown that the uterine microbiota of cows with clinical
endometritis displays an increased abundance of Fusobacterium and a unique presence
of Trueperella and Peptoniphilus [
65
]. The increased prevalence of Trueperella pyogenes (T.
pyogenes), earlier named Arcanobacterium pyogenes, in the microbiome of cows with en-
dometritis, has also been reported in other studies [66,67].
Amaci et al. [
68
] investigated secretion samples from 20 repeat breeder cows, i.e.,
cows who failed to conceive after three or more inseminations with fertile semen without
any anatomic or infectious abnormality. The smears were prepared from the sediment of
centrifuged uterine saline lavage. Biofilms visualized as red complexes with PAS staining
were observed in 12 animals (60%). Bacteria were isolated from eight animals, all of them
displaying biofilms in the smears. There was no correlation between the presence of a
biofilm and the results from the cytological examinations of uterine secretion. Furthermore,
there was no control group in this study. Consequently, the study demonstrated the
presence of an intrauterine biofilm in repeat breeder cows, but a possible effect on fertility
or the development of endometritis is unknown.
Interestingly, the commonly found bacterium related to endometritis in cows, T.
pyogenes, has a number of adherence mechanisms, including several extracellular matrix-
binding proteins and fimbriae [
69
]. Furthermore, biofilm formation is upregulated by
the same two-component regulatory system that upregulates the expression of the major
virulence factor pyolisin. Although this may indicate that T. pyogenes is pathogenic when
residing in biofilm, it is presently not known whether biofilms are in fact a part of the
pathogenesis of endometritis.
7.3. Mastitis
Mastitis is the inflammation of the mammary gland. It is one of the most challenging
diseases in the dairy industry, causing enormous economic losses. This has prompted a
large interest in bovine and small ruminant mastitis research in general and a possible role
of biofilms in particular. However, the majority of the latter studies have been restricted
to
in vitro
investigations on the biofilm-forming abilities of clinical isolates. Relatively
few studies have focused on direct or indirect observations of biofilms’ presence in the
udders of affected animals
in vivo
. Even so, these few studies indicate both the presence of
biofilms and a possible role in the pathogenesis of mastitis.
Mastitis in Cows
Bovine mastitis occurs primarily during lactation. The main pathogen is S. aureus.
Other mastitis-associated bacteria are Streptococcus agalactiae (causing contagious mastitis),
coagulase-negative staphylococci (CoNS), E. coli,Klebsiella spp., Enterobacter spp., Citrobacter
spp., S. dysgalactiae,S. uberis,Enterococcus spp., and Pseudomonas spp.
Observations indicating presence of biofilms were made by Hensen et al. when
they experimentally infected two quarters in each of three lactating cows with low doses
of S. aureus [
70
]. The cows were slaughtered at different time points, and the udders
were subjected to macro- and microscopic examinations. During the first four days after
inoculation, all cows displayed mild clinical mastitis in the infected quarters, and two of
the cows were slaughtered. When the third cow was slaughtered after 122 days, it showed
signs of chronic subclinical mastitis with high somatic cell counts in the milk and diffuse
changes related to fibrosis in the inoculated quarters. In tissue samples from the cows in
both the clinical and chronic subclinical stages, Gram-positive cocci were mainly located
in clusters in the alveoli and lactiferous ducts in association with inflammatory cells. In
the chronic stage, the bacteria were associated with the epithelium and were also observed
within the interstitial tissue.
In another study, Schönborn and Krömker investigated swabs from slaughtered S.
aureus-infected dairy cows [
71
]. Immunofluorescence staining identified polysaccharide

Microorganisms 2023,11, 608 11 of 22
intercellular adhesion PIA in 71 of the 184 samples. As PIA is a component of the biofilm
matrix of S. aureus, such findings are indicative of S. aureus biofilms.
Vaccination studies also indicate a possible contribution by biofilms to the pathogenesis
of mastitis. Gogoi-Tiwari et al. used a mouse model to study the effect of immunization
with formalin-killed S. aureus from biofilms versus formalin-killed planktonic bacteria.
The mice immunized with biofilm-residing bacteria displayed significantly lower S. aureus
colonization, less severe clinical symptoms, and less tissue damage in their mammary
glands [
72
]. In a study on sheep by Perez et al., vaccination with crude bacterial extracts
from strong S. aureus biofilm formers displayed the highest production of antibodies and the
best protection against infection and mastitis after intra-mammary challenge, as compared
to immunization with extracts from weak biofilm formers and controls [73].
Studies on the expression of the biofilm-associated surface protein Bap in staphylo-
cocci [
74
] may provide even more information on a possible role for biofilms in mastitis
pathogenesis. S. aureus bap-positive isolates from sub-clinical mastitis were found to be
better to colonize and persist in the mammary gland
in vivo
. Furthermore, antibodies
against Bap were observed in the serum of these animals, thus confirming that Bap, and
thereby probably also a biofilm, was produced during the infection. Zuniga et al. [
75
] found
that medium somatic cell counts in the milk (i.e., markers of inflammation) were higher in
milk samples from sub-clinical mastitis with bap-positive staphylococci than in samples
with bap-negative staphylococci. These results indicate that biofilms may contribute to the
inflammatory magnitude in the udder.
Intracellular live S. aureus have been observed in alveolar cells in milk samples from
chronic bovine mastitis [
76
]. Interestingly, a three-year follow-up of a dairy herd showed
that strains of the most prevalent and persistent S. aureus genotype displayed both high
biofilm production and high cellular invasiveness [
77
]. A similar link between biofilms and
invasiveness was also observed in a study on human patients with chronic rhinosinusitis, in
which the location of intracellular S. aureus was associated with surface biofilms [
78
]. This
indicates that epithelial-associated biofilms may facilitate cellular invasion by S. aureus.
8. Respiratory System
Biofilms have been observed or indicated to play a role in the pathogenesis of a
number of bacterial infections in the human respiratory system, e.g., chronic rhinosinusitis,
pharyngitis and laryngitis, pertussis, and cystic fibrosis [
5
]. Most of the bacterial pathogens
involved also infect animals. It is therefore not surprising that experimental studies show
that the same pathogens can also produce biofilms in the respiratory systems of animals.
As an example, S. aureus, which was inoculated in sheep sinuses, produced a biofilm
identifiable by confocal laser scanning microscopy, scanning electron microscopy, and
transmission electron microscopy [
79
]. In addition, biofilms have also been observed as
being present in animal respiratory diseases.
8.1. Bovine Respiratory Disease Complex (BRDC)
Bovine respiratory disease complex (BRDC) causes substantial economic losses to the
beef industry. Polymicrobial infections are common, with H. somni, Pasteurella multocida (P.
multocida), and Mannheimia haemolytica being predominant bacterial species, in addition to
Mycoplasma bovis and several viruses. Pulmonary biofilms have been observed in calves
with BRDC after being experimentally challenged with H. somni.
Sandal et al. [
21
] exposed four calves to transtracheal challenge with an H. somni strain
after four days of immunosuppressive treatment. One calf was inoculated with saline
and kept as a control. All the exposed calves developed fever, respiratory symptoms,
and increased respiratory and heart rates. H. somni was recovered from nasal swabs and
transtracheal washes from all four challenged calves and P. multocida from three of them.
The animals were euthanized after 7 days. A post-mortem examination showed suppura-
tive bronchopneumonia. Transmission electron microscopy, immunoelectron microscopy,
scanning electron microscopy (Figure 6), and fluorescence in situ hybridization investiga-

Microorganisms 2023,11, 608 12 of 22
tions showed that an H. somni biofilm was present. Investigations of tissue from the control
calf did not display such biofilms or any of the lesions observed in the infected calves. The
results indicate that biofilm production is part of the pathogenesis of this disease.
Microorganisms 2023, 11, x FOR PEER REVIEW 13 of 23
Figure 6. Scanning electron microscopy of freeze-fractured samples of cardiac (see chapter 3) and
lung tissues. Top panel: (A) Scanning electron microscopy images of normal cardiac tissue; (B,C)
infected cardiac tissue containing coccus-shaped bacteria surrounded in a matrix network. Scale
bars: (A) 2 μm with magnification 20,000×; (B,C) 5 μm with magnification 10,000×. Bottom panel:
(D) normal lung tissue; (E,F) infected lung tissue containing few coccobacilli within an extracellular
matrix. Scale bars: (E) 1 μm with magnification 30,000×; (F) 0.5 μm with magnification 60,000×. Re-
printed from [21], Copyright (2009), with permission from Elsevier.
Interestingly, P. multocida was observed in transtracheal washes and pulmonary tis-
sue of the calves even though they had only been challenged with H. somni. Similar was
observed in another experiment with two calves challenged with H. somni [80]. In these
animals, both H. somni and P. multocida were present in the lungs. Furthermore, the calves
produced higher antibody titers against the biofilm form than the planktonic form of both
bacteria. However, only matrix components of H. somni were detected in the lungs. These
findings taken together suggest that P. multocida may have entered and used the H. somni
biofilm for persistence during chronic BRD.
8.2. Porcine Enzootic Pneumonia
Porcine enzootic pneumonia is a globally spread disease, which is caused by Myco-
plasma hyopneumoniae (M. hyopneumoniae) and exacerbated by secondary infections and
environmental stressors [81]. It usually affects growing and fattening pigs and may last
for many weeks. Although the mortality is low, the morbidity is high with the pigs dis-
playing respiratory symptoms, reduced feed efficiency, and growth retardation. This can
have devastating economic consequences. Carrier pigs are responsible for persistent in-
fections on the farm.
M. hyopneumoniae adheres to the ciliated respiratory epithelium, resulting in cilial loss
and epithelial cell death. Early observations of the bacteria on the epithelium in experi-
mentally infected pigs showed that they were enveloped by a thick, dark layer of capsular
material. Furthermore, they had many fibrils on the surface which seemed to connect the
bacteria to each other, as well to the cells [82]. Although these and other similar observa-
tions could have indicated the presence of biofilms, it took almost 40 years before biofilms
were actually demonstrated in infected pigs.
Raymond et al. [83] examined tracheal sections of pigs experimentally or naturally
infected with M. hyopneumoniae. Immunohistochemistry microscopy showed that the bac-
teria were located at the ciliary border of the epithelium, and aggregates of staining that
Figure 6.
Scanning electron microscopy of freeze-fractured samples of cardiac (see chapter 3) and lung
tissues. Top panel: (
A
) Scanning electron microscopy images of normal cardiac tissue; (
B
,
C
) infected
cardiac tissue containing coccus-shaped bacteria surrounded in a matrix network. Scale bars: (
A
) 2
µ
m
with magnification 20,000
×
; (
B
,
C
) 5
µ
m with magnification 10,000
×
. Bottom panel: (
D
) normal
lung tissue; (
E
,
F
) infected lung tissue containing few coccobacilli within an extracellular matrix.
Scale bars: (
E
) 1
µ
m with magnification 30,000
×
; (
F
) 0.5
µ
m with magnification 60,000
×
. Reprinted
from [21], Copyright (2009), with permission from Elsevier.
Interestingly, P. multocida was observed in transtracheal washes and pulmonary tissue
of the calves even though they had only been challenged with H. somni. Similar was
observed in another experiment with two calves challenged with H. somni [
80
]. In these
animals, both H. somni and P. multocida were present in the lungs. Furthermore, the calves
produced higher antibody titers against the biofilm form than the planktonic form of both
bacteria. However, only matrix components of H. somni were detected in the lungs. These
findings taken together suggest that P. multocida may have entered and used the H. somni
biofilm for persistence during chronic BRD.
8.2. Porcine Enzootic Pneumonia
Porcine enzootic pneumonia is a globally spread disease, which is caused by My-
coplasma hyopneumoniae (M. hyopneumoniae) and exacerbated by secondary infections and
environmental stressors [
81
]. It usually affects growing and fattening pigs and may last for
many weeks. Although the mortality is low, the morbidity is high with the pigs displaying
respiratory symptoms, reduced feed efficiency, and growth retardation. This can have
devastating economic consequences. Carrier pigs are responsible for persistent infections
on the farm.
M. hyopneumoniae adheres to the ciliated respiratory epithelium, resulting in cilial
loss and epithelial cell death. Early observations of the bacteria on the epithelium in
experimentally infected pigs showed that they were enveloped by a thick, dark layer of
capsular material. Furthermore, they had many fibrils on the surface which seemed to
connect the bacteria to each other, as well to the cells [
82
]. Although these and other similar

Microorganisms 2023,11, 608 13 of 22
observations could have indicated the presence of biofilms, it took almost 40 years before
biofilms were actually demonstrated in infected pigs.
Raymond et al. [
83
] examined tracheal sections of pigs experimentally or naturally
infected with M. hyopneumoniae. Immunohistochemistry microscopy showed that the
bacteria were located at the ciliary border of the epithelium, and aggregates of staining that
resembled biofilms were observed in chronically infected pigs. Using scanning electron
microscopy on samples six weeks after the experimental infection, large biofilms (100–
150
µ
m in diameter) were observed on the epithelium. The epithelium was deciliated.
Biofilms were also observed in vitro on porcine kidney epithelial-like monolayers.
8.3. Porcine Pleuropneumonia
Porcine pleuropneumonia is caused by Actinobacillus pleuropneumoniae (A. pleuropneu-
moniae). The disease, which is highly contagious and widespread, is characterized by high
morbidity as well as high mortality leading to significant economic losses.
The presence of biofilms was indicated by studies of lungs from two naturally infected
pigs displaying clinical signs consistent with acute porcine pleuropneumonia [
84
]. A
microscopic examination of the lungs confirmed the clinical diagnosis by displaying typical,
multiple foci of coagulation necrosis in the pulmonary parenchyma with microcolonies of
small Gram-negative bacilli present. The isolation and characterization of these bacteria
from the two pigs showed that they were A. pleuropneumoniae of serotype 7 and serotype
5, respectively, i.e., two of the most prevalent serotypes in North America. Through the
use of fluorescence in situ hybridization with a species-specific oligonucleotide probe,
the bacteria were observed growing as aggregates (~30–45
µ
m) in the lungs of both pigs,
i.e., in the same way as in earlier descriptions of
in vivo
biofilms in human chronic lung
infections [
6
,
41
]. The bacteria also formed similar aggregates in an
in vitro
agarose model
mimicking the porous conditions of the infected loci in the lungs. The fluorescent staining
of these aggregates showed the presence of poly-N-acetylglucosamine, which is a known
component of the A. pleuropneumoniae biofilm matrix. These findings support the notion of
the aggregates observed in vivo being biofilms.
8.4. Bordetella Bronchiseptica Infections
Bordetella bronchiseptica (B. bronchiseptica) infections have been associated with respi-
ratory disease in a number of mammals [
85
]. It is widespread in swine populations and
causes highly contagious tracheobronchitis in dogs and cats. In addition, long-term to
life-long asymptomatic carriers shed the bacteria and infect susceptible individuals. This
is most probably due to the presence of chronic, asymptomatic colonization in the form
of a biofilm in the upper respiratory tract, as demonstrated in experimental infections in
laboratory animals [85].
B. bronchiseptica biofilms
in vivo
have been observed in intranasal mouse models.
Sloan et al. observed both bacteria and the biofilm matrix component Bps polysaccharide
on the nasal septum of inoculated mice when using confocal laser scanning microscopy [
86
]
(Figure 7A). Furthermore, scanning electron microscopy showed a densely packed mul-
ticellular community of bacteria on the ciliated epithelium with architectural features
characteristic of many biofilms, i.e., mats, towers, or pillars (Figure 7B). Inoculation with a
knockout mutant lacking the essential biofilm component Bps showed that this component
was needed for the efficient long-term survival of B. bronchiseptica in the respiratory tract.
No biofilms were observed in control mice. A biofilm on the nasal septa of mice after expo-
sure to B. bronchiseptica was also observed by Conover et al. [
87
]. Confocal laser scanning
microscopy showed B. bronchiseptica being present in scattered mat-like structures on the
nasal epithelia. Scanning electron microscopy revealed that thick mats of encased bacteria
in a matrix material were covering the underlying ciliated epithelium.

Microorganisms 2023,11, 608 14 of 22
Microorganisms 2023, 11, x FOR PEER REVIEW 15 of 23
Figure 7. (A) Confocal laser scanning microscopy (CLSM) of biofilms formed within the murine
nasal cavity by B. bronchiseptica. C57BL/6 mice were inoculated with either PBS or B. bronchiseptica
RB50. Nasal septa were harvested at 15 or 38 days postinoculation, immediately fixed, and probed
with rat anti-Bordetella serum followed by a secondary anti-rat antibody conjugated to Alexa Fluor
488 (which stains bacteria green). To determine the localization of the host epithelium, specimens
were stained for F-actin using phalloidin conjugated to Alexa Fluor 633 (which stains the epithelium
red) and visualized with. Each micrograph represents a Z reconstruction. For each specimen, images
were obtained from at least five areas of the nasal septum and from at least three independent ani-
mals. (B) Scanning electron microscopy of B. bronchiseptica biofilm formation on nasal septa. Spec-
imens were collected from animals either 15 days or 38 days post-inoculation, directly fixed, and
processed for scanning electron microscopy. Scale bars = 10 μm. Reprinted from [86].
9. Skeletal System
Osteomyelitis in animals is caused by traumatic, surgical, or hematogenous infection
[88]. The latter occurs more often in juveniles than in adults. Staphylococci, streptococci,
E. coli, and other Gram-negative bacteria are often found in osteomyelitis in horses, swine,
broilers, turkeys, dogs, and cats. Erysipelothrix rhusiopathiae is in addition found in swine,
whereas T. pyogenes is more common in cattle. Osteomyelitis is difficult to treat, often re-
quiring an extended duration of antibiotic therapy and repeated surgical revisions of af-
fected tissue, i.e., hallmarks of a biofilm infection.
A number of animal models using pigs, dogs, chickens, sheep, goats, rabbits, and
mice have been designed to study osteomyelitis [89,90]. Johansen et al. [91] inoculated
three groups of pigs of three animals each with S. aureus in the right femoral artery. Group
B received a porcine strain, whereas groups C and D were inoculated with two different
strains of human origin. Two animals inoculated with saline were kept as controls (group
A). Five days after inoculation, the pigs in group B displayed lameness of the right limb,
whereas none of the others showed clinical symptoms. All animals were killed after eleven
or fifteen days. Osteomyelitic lesions were observed in the right hind leg of all three pigs
in group B and one in group C. These animals also harbored S. aureus in the femoral ab-
scesses, as identified with PNA (peptide nucleic acid) fluorescence in situ hybridization.
The bacteria were shown to reside in biofilms as aggregates of loosely-packed cocci em-
bedded in an opaque matrix.
10. Urinary System
Urinary Tract Infections
As in humans, E.coli is the pathogen that is known to most frequently colonize the
urinary tract causing urinary tract infections (UTIs) in animals, but also Enterococcus spe-
cies are frequently found. Most studies on UTIs in animals have been conducted on com-
panion (cats and dogs) and sporting animals (horses) [92–94]. Studies conducted on UTIs
Figure 7.
(
A
) Confocal laser scanning microscopy (CLSM) of biofilms formed within the murine nasal
cavity by B. bronchiseptica. C57BL/6 mice were inoculated with either PBS or B. bronchiseptica RB50.
Nasal septa were harvested at 15 or 38 days postinoculation, immediately fixed, and probed with rat
anti-Bordetella serum followed by a secondary anti-rat antibody conjugated to Alexa Fluor 488 (which
stains bacteria green). To determine the localization of the host epithelium, specimens were stained
for F-actin using phalloidin conjugated to Alexa Fluor 633 (which stains the epithelium red) and
visualized with. Each micrograph represents a Z reconstruction. For each specimen, images were
obtained from at least five areas of the nasal septum and from at least three independent animals.
(
B
) Scanning electron microscopy of B. bronchiseptica biofilm formation on nasal septa. Specimens
were collected from animals either 15 days or 38 days post-inoculation, directly fixed, and processed
for scanning electron microscopy. Scale bars = 10 µm. Reprinted from [86].
9. Skeletal System
Osteomyelitis in animals is caused by traumatic, surgical, or hematogenous infec-
tion [
88
]. The latter occurs more often in juveniles than in adults. Staphylococci, strepto-
cocci, E. coli, and other Gram-negative bacteria are often found in osteomyelitis in horses,
swine, broilers, turkeys, dogs, and cats. Erysipelothrix rhusiopathiae is in addition found
in swine, whereas T. pyogenes is more common in cattle. Osteomyelitis is difficult to treat,
often requiring an extended duration of antibiotic therapy and repeated surgical revisions
of affected tissue, i.e., hallmarks of a biofilm infection.
A number of animal models using pigs, dogs, chickens, sheep, goats, rabbits, and
mice have been designed to study osteomyelitis [
89
,
90
]. Johansen et al. [
91
] inoculated
three groups of pigs of three animals each with S. aureus in the right femoral artery. Group
B received a porcine strain, whereas groups C and D were inoculated with two different
strains of human origin. Two animals inoculated with saline were kept as controls (group
A). Five days after inoculation, the pigs in group B displayed lameness of the right limb,
whereas none of the others showed clinical symptoms. All animals were killed after eleven
or fifteen days. Osteomyelitic lesions were observed in the right hind leg of all three pigs in
group B and one in group C. These animals also harbored S. aureus in the femoral abscesses,
as identified with PNA (peptide nucleic acid) fluorescence in situ hybridization. The
bacteria were shown to reside in biofilms as aggregates of loosely-packed cocci embedded
in an opaque matrix.

Microorganisms 2023,11, 608 15 of 22
10. Urinary System
Urinary Tract Infections
As in humans, E.coli is the pathogen that is known to most frequently colonize the
urinary tract causing urinary tract infections (UTIs) in animals, but also Enterococcus
species are frequently found. Most studies on UTIs in animals have been conducted on
companion (cats and dogs) and sporting animals (horses) [
92
–
94
]. Studies conducted on
UTIs in dogs have shown that especially female dogs are at risk. Uncomplicated canine
UTIs are common and have been reported to occur in 14–17% of all dogs at one time in their
lifetime. It has also been suggested that 4.5–20% of all dogs with UTIs have a recurrence of
persistent UTIs [
95
,
96
]. Bacterial UTIs have been found to occur less frequently in cats than
in dogs with only 1–2% of cats suffering from UTIs during their lifetime [97].
The pathogenesis of UTIs is multifactorial and depends on the interplay between
bacterial virulence factors and host defense systems. A UTI can develop when the host
defense mechanism is temporarily or permanently affected. Most host defense mechanisms
against bacterial establishment include, e.g., the presence of normal resident microflora
and physical urinary tract anatomy. Bacterial virulence factors enable bacteria to colonize
and invade the urinary tract [97,98].
E. coli that cause infection outside the intestinal tract are called extraintestinal
E. coli
(ExPEC), and the ExPECs that colonize the urinary tract are commonly referred to as
uropathogenic E. coli (UPEC). Experiments using intraurethral catheterization to infect
mice with UPEC isolates have shown intracellular invasion and the development of struc-
tures with typical biofilm characteristics in the bladder epithelial cells (reviewed by [
5
]).
In addition, such intracellular bacterial communities have been observed by electron mi-
croscopy in epithelial cell shedding in urine from women with UTIs.
When studying UPEC isolated from dogs, Ballash et al. found that UTI recurrence was
associated with the ability to form biofilms. The study used 104 different UPEC isolates
originating from 94 different dogs. The isolates were tested for their biofilm-forming
capabilities
in vitro
using the microtiter plate method and crystal violet staining of the
biofilm mass. As much as approximately 80% of the isolates were able to form biofilms
in microtiter plates, where approximately 56% formed weak biofilms and 24% formed
moderate or strong biofilms. Based on retrospective medical record analysis, it was found
that isolates from dogs with a history of chronic/recurring urinary tract infections had
8.5 times the odds of forming a biofilm compared to isolates from dogs without a history
of chronic UTIs. The same study also found that isolates that could form biofilms had
a slightly higher likelihood of having more virulence genes than non-biofilm producers
(
p= 0.094
) [
96
]. There are a few other studies using similar methodologies that also confirm
that E. coli isolated from UTIs from dogs are able to form biofilms [
99
,
100
]. These results
indicate that intracellular biofilm formation in epithelial cells also may occur during UTIs
in dogs.
11. Visual System
Equine Recurrent Uveitis
Equine recurrent uveitis (ERU), also known as moon blindness, is the most common
cause of equine blindness with a prevalence of 2–25% worldwide [
101
]. ERU has been
believed to be an autoimmune syndrome initiated by an episode of acute uveitis disruption
of the blood–ocular barrier. However, Leptospira spp., which is the most common initiator,
has also been found present after several episodes. Furthermore, vitrectomy, which is a
treatment option for severe ERU, is most beneficial when leptospires is implicated. It is
therefore not surprising that a recent study identified Leptospira spp. biofilms in vitrectomy
samples from horses with ERU but not in samples from horses without this disease,
Brandes et al. [
102
] examined vitreous samples obtained by the vitrectomy of 17 horses
with ERU. High titers of antibodies against leptospires were found in 16 of the 17 samples,
and PCR was positive for these bacteria in all samples. This indicated that ERU can be
a chronic Leptospira infection and that the immune response is not sufficient to eradicate

Microorganisms 2023,11, 608 16 of 22
the bacteria. Interestingly, an unknown homogeneous granular layer surrounded the few
leptospires that were observed by using transmission electron microscopy.
Based on these and other observations, Ackermann et al. [
103
] suggested that the
Leptospira biofilm might be part of the pathogenesis of ERU. To test this hypothesis, they
investigated vitreous samples from 29 horses with clinical ECU and 3 horses without this
clinical diagnosis. Antibodies against leptospires were found in 28 of the 29 samples from
the horses with ERU. Furthermore, all 29 samples were PCR-positive for Leptospira spp. The
samples from the control horses were negative in both tests. Microscopical investigations
with Warthin-Starry Silver Stain and immunohistochemistry identified leptospires display-
ing biofilm formation in all ERU samples. Laboratory-cultured WHO Leptospira spp. strains
spread on microscope slides were used as non-biofilm controls in these investigations.
Three steps of biofilm formation were observed in the positive samples. Step 1: Individual
leptospires surrounded by a partially granulated matrix. The bacteria displayed increased
thickness as compared to the non-biofilm control leptospires. Step 2: Microcolonies consist-
ing of bacterial aggregates in a granular matrix. Step 3: Condensed round structures with a
diameter of approximately 5 to 20
µ
m. It was difficult to distinguish bacteria and matrix,
but individual leptospires protruding from the aggregates were occasionally observed. No
signs of biofilm formation could be seen in the samples from the three horses without ERU.
12. Discussion
Already 50 years ago, Nils Høiby observed a link between the presence of bacterial
aggregates and the etiology of cystic fibrosis patients [
4
]. A decade later, Bill Costerton
introduced the term biofilm into medicine. Since then, a large number of studies describing
biofilms in relation to infection and disease in humans, as well as in animals, have been
published. However, the awareness of biofilm infections among both veterinarians and
physicians, in general, has been disappointingly low. This has probably affected the
diagnostics and treatment of such infections.
In this review, we describe a number of observations and/or indications of bacterial
biofilms in most body systems of husbandry animals, including fish, as well as in sport and
companion animals (Table 1). The animals were naturally or experimentally infected. For
some infections, other model animals, e.g., mice, have been used to further elucidate the
formation and role of biofilms. The biofilms have been visualized by various microscopy
techniques in biopsies, autopsies, and exudates. This is in compliance with the guideline
from the European Society of Clinical Microbiology and Infectious Diseases (ESCMID)
on the diagnosis and treatment of biofilm infections [
104
]. This guideline states that
microscopic techniques such as confocal laser scanning microscopy and scanning electron
microscopy are the most appropriate to identify biofilms in tissue samples, but light
microscopy and routine staining methods may also be used. Furthermore, several of
the studies described in this review have included investigations on the host’s immune
response, vaccination experiments, and/or characterization of bacterial phenotypes during
infection to support the observations of biofilms being present in the affected body systems.
A large number of studies published have focused on measuring biofilm production
in vitro
by pathogens isolated from diseased animals and affected organs. Comparisons
with isolates from other sources are often included. Whereas results from such studies may
support observations from
in vivo
studies, they are themselves not sufficient to establish
a link between biofilms and disease. Consequently, few such studies are included in
this review.

Microorganisms 2023,11, 608 17 of 22
Table 1. Biofilm-associated infections of different body systems and their affected organs.
Body System Affected Organs Disease
Auditory Ear Canine otitis externa
Cardiovascular Heart Bovine myocarditis
Central nervous Brain Piscine meningoencephalitis
Digestive Intestines Salmonella infection
Integumentary Skin and underlying tissue Wound infections
Reproductive Uterus Endometritis
Mammary glands Mastitis
Respiratory
Lower airways Bovine respiratory disease
Lower airways Porcine enzootic pneumonia
Lower airways Porcine pleuropneumonia
Upper and lower airways Bordetella bronchiseptica infection
Skeletal Bones Osteomyelitis
Urinary Urinary tract Uropathogenic E. coli infection
Visual Eye Equine recurrent uveitis
Biofilm infections in animals can be studied under controlled conditions in exper-
iments using the relevant animal species. The animals can be euthanized and proper
organ samples collected for macro- and microscopic studies. This is in contrast to stud-
ies in humans, which are restricted to natural infections and available samples. Despite
these advantages in animal studies, surprisingly few of them have looked at the possible
role of biofilms in the pathogenesis of the infection studied. However, as in studies on
biofilm infections in humans [
5
], a number of potential roles in disease pathogenesis are
indicated by the observations in animal studies. Biofilms in relevant body systems of
diseased animals, but not in controls, give reason to believe that biofilms are involved in
the pathogenesis of clinical and subclinical infections. Biofilms can contribute to high local
concentrations of bacteria. The bacteria, their metabolites, or other biofilm components
may then induce the local inflammation that has been observed related to the biofilm, and
that can cause or aggravate tissue damage, e.g., the deciliation of epithelium observed in
porcine enzootic pneumonia. Interestingly, signs indicating that biofilms can modulate local
inflammatory responses have also been observed, e.g., in equine endometritis where the
increased production of anti-inflammatory Interleukin 10 was found in areas surrounding
the biofilms. Furthermore, biofilms formed by one bacterial species might be used by other
pathogens to avoid elimination by the immune system, as may be the case with P. multocida
entering and using the H. somni biofilm for persistence during chronic bovine respiratory
disease complex (BRDC). Biofilms can also act as physical barriers obstructing the healing
process, e.g., in wounds. As the biofilm lifestyle offers long-term protection against both
the immune system and antimicrobial treatment, it is not surprising that biofilms play a
role in chronic subclinical infections, as observed in mastitis in cattle and meningitis in
tilapia fish. In human studies, biofilms appear to facilitate the intracellular invasion of the
underlying tissue of some bacterial species, e.g., S. aureus. This is supported by studies on
S. aureus from mastitis in cows. Furthermore, studies on humans have also revealed the
formation of intracellular biofilms [
5
], which probably increases the intracellular survival
of the bacteria. This can be expected to occur in animals as well. Likewise, the observed
link between biofilms and certain cancer forms in humans is probably also present in some
form in animals.
Biofilm-associated infections in animals are also a cause of concern in a One Health
concept. Several of the bacteria that have been found in biofilms in animals are zoonotic and
may cause disease in humans, e.g., B. bronchiseptica,S. aureus, pathogenic E. coli,Salmonella
spp., K. pneumoniae, and P. aeruginosa. As the biofilm formation of such pathogens promotes
long-term colonization in animals, it poses an increased risk of the transfer of pathogens to
humans. This can be through direct contact with animals or indirectly through contami-
nated food products or environments. Jacques and Malouin have therefore proposed the

Microorganisms 2023,11, 608 18 of 22
One Biofilm concept within the One Health concept, “where biofilm/aggregate formation
in humans, animals and the environment are also intricately linked” [105].
In conclusion, it is important to acknowledge that biofilms are involved in a number of
bacterial infections in animals and that this influences the choice of diagnostic procedures
and treatment of these infections. Although the knowledge and awareness of the general
practitioner have improved in recent years, they still need to be higher. Additionally, further
development of diagnostic criteria as well as effective treatments for such infections are
required. Finally, knowledge of the role of biofilms in the pathogenesis of such infections
should be expanded by more studies specifically designed to address this question.
Author Contributions:
All authors have contributed to the design of the work, performed literature
studies, written parts of the manuscript, and approved the submitted version. Project administration
by L.L.N. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
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