Importance of Yeasts in Oral Canine Mucosa

Abstract

Dentistry science is a new specialty in veterinary medicine that has been growing in recent years, accompanied by the development of professionals who seek to improve the quality of life of pets. Cases related to problems in the oral cavity have gained significant importance in the medical clinic of professionals who treat small animals. Due to lack of professional knowledge or due to animal behavioral problems, such as aggressiveness, the anamnesis of the oral cavity is not performed most of the time, which ends up delaying the diagnosis of the pathology. In addition, an animal with a problem in the oral cavity may take years to show signs of the disease. In general, animals have an oral microbiota composed of various species of fungi, which, under specific conditions, can change from saprophytes to pathogens, compromising their health. Thus, the pre-knowledge of potentially pathogenic yeasts belonging to oral microbiota of dogs and their susceptibility profile compared to the main drugs used in antifungal therapy, is of fundamental importance as it ensures a clinical auxiliary support for the diagnosis and treatment of most diseases of the oral cavity.

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
Chapter
Importance of Yeasts in Oral
Canine Mucosa
Claudete RodriguesPaula, Bianca SilvaNavarro,
Mário MendesBonci and Diana CostaNascimento
Abstract
Dentistry science is a new specialty in veterinary medicine that has been grow-
ing in recent years, accompanied by the development of professionals who seek to
improve the quality of life of pets. Cases related to problems in the oral cavity have
gained significant importance in the medical clinic of professionals who treat small
animals. Due to lack of professional knowledge or due to animal behavioral prob-
lems, such as aggressiveness, the anamnesis of the oral cavity is not performed most
of the time, which ends up delaying the diagnosis of the pathology. In addition, an
animal with a problem in the oral cavity may take years to show signs of the disease.
In general, animals have an oral microbiota composed of various species of fungi,
which, under specific conditions, can change from saprophytes to pathogens, com-
promising their health. Thus, the pre-knowledge of potentially pathogenic yeasts
belonging to oral microbiota of dogs and their susceptibility profile compared to the
main drugs used in antifungal therapy, is of fundamental importance as it ensures
a clinical auxiliary support for the diagnosis and treatment of most diseases of the
oral cavity.
Keywords: yeasts, oral cavity, dogs, antifungal, microbial resistance, fungi
. Introduction
Fungi are eukaryotic, heterotrophic organisms, multinucleated like molds, or
only with one nucleus, like yeasts. These organisms can be unicellular, or multicel-
lular, which we call mycelium. Yeasts are unicellular and do not present, in general,
morphological differences. The cells are rounded, ovoid or elongated, but some
yeast under special conditions may have successive sprouts in a chain, which we call
pseudomycelium [1].
The classification of fungi is based on morphological, reproductive and
physiological characteristics. The taxonomy of fungi is still varied, but we
can classify them in the Kingdom Fungi in the six phylas: Basidyomycota,
Ascomycota, Glomeromycota, Chytridiomycota, Blastocladiomycota and
Neocallimastigomycota [2, 3].
Approximately 200 out of a total of 100,000 species of yeast are considered
pathogenic. Of these pathogenic species, 50 of them are regularly associated with
mycoses. Yeasts are the ones that cause the greatest number of mycoses, both in man
and in animals and we highlight the genera Candida, Cryptococcus, Malassezia and
Trichosporon [3].

Canine Medicine

These yeasts can be assexual (anascoporogenous), or sexual (ascoporogenous
or basidioporogenous). In general, they are considered opportunists “waiting” for
their “opportunity”, that is, the drop in the immunity of man and animals, thus
causing a case of ringworm.
Among domestic animals, the ones that have the closest proximity to people
are dogs. Canis lupus familiaris is believed to have emerged approximately
130,000years ago, from the domestication of the gray wolf. Crossbreeding
and selection of characteristics gave rise to different breeds, including Poodle,
Yorkshire, Terrier and Labrador Retriever, but mixed breed animals are prevalent in
homes around the world (Figure ) [4].
In addition to being mere companions in people’s homes dogs have established
themselves with essential functions such as security and hunting. These dogs have
gained these and other noble functions and thus brought them even closer to human
beings in places and situations that would otherwise be dispensed with. Today they
also act as guides for the visually impaired, accompanying people to the hospital,
monitoring blood glucose levels for diabetic people and even detecting pathogens in
hospital environments [5].
These new functions, with consequently greater proximity between dogs and
people, also result in a possible greater exchange of microorganisms between
these beings, including yeasts. Among these fungi, the most present in the oral
mucosa of dogs are the genera Malassezia and Candida and found less the genus
Cryptococcus [6].
In the field of public health, these microorganisms have in common the ability
to cause disease in both animals and people, and therefore this possible increase
in the sharing of microbiota between these beings must be monitored by health
specialists.
It is important to emphasize that the exchange of microorganisms occurs in both
directions, and that the health of the animals must also be considered in these cases.
The vigilance of the clinical mycologist must be maintained for a better
understanding of how future changes can become serious public health problems,
especially for yeasts, as we have already seen in several situations.
. Ecology and sources of yeast infection
Yeasts can be found in plants, soil, air, aquatic environment, in invertebrate and
vertebrate animals, that is, in almost all ecosystems. These microorganisms can be
in their symbiotic state, in mutualism, or in parasitism. In humans, several species
Figure 1.
Mixed breed dog.

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Importance of Yeasts in Oral Canine Mucosa
DOI: http://dx.doi.org/10.5772/intechopen.95905
can be part of their natural microbiota, in the gastrointestinal tract, in mucocutane-
ous tissues and skin. In man, a large part of yeast infections, especially of the genus
Candida, are of endogenous origin and are linked to risk factors such as old age,
prematurity, avitaminosis, antibiotic therapy, cancer, and other diseases that cause
immunodepression of the host [7].
Extrinsic factors can also be important, such as the rupture of the natural bar-
rier of the skin and mucous membranes, the use of invasive hospital material and
contact with contaminated ecological niches. Direct transmission between people
can occur in sexual relations [8].
In dogs, the main yeast found on the skin and mucocutaneous surfaces is
Malassezia pachydermatis, which easily recovers in the folds of the skin and espe-
cially in the various parts of the ear. The prevalence of some types of yeasts in the
oral mucosa of dogs is related to several habits, such as licking, sniffing and explor-
ing environments.
The licking of the paws and other areas of the body explains the considerable
presence of Malassezia pachydermatis in the oral cavity of dogs. Considered a
saprophyte in the skin of dogs, this microorganism can cause dermatitis in several
situations, and in these cases, there is also an increase in its presence in the oral
mucosa. Other relevant yeasts of these animals belong to the genera Candida,
Rhodotorula and Trichosporon, which are, in most cases, in balance with the dogs’
organism [9].
It is also reported that Cyniclomyces guttulatus, present in the stomach, intestine
and feces, which in situations of imbalance with the commensal microbiota, may be
related to clinical conditions that affect the gastrointestinal tract [10].
The habit of sniffing the soil, in parks and gardens, hunting in forests and dens,
favors the sharing of microorganisms among animals linked to these environments.
The organic matter present in these places, mainly in the feces of birds and bats,
favors colonization by fungi such as Cryptococcus spp. and Histoplasma capsulatum,
which in situations favorable to microorganisms (host immunosuppression; high
microbial inoculum load) can cause serious diseases [11].
Advances in veterinary hospital techniques, especially surgical procedures and
hospitalizations, also brings new sources of infection for dogs. The ability of micro-
organisms of the genera Candida and Malassezia to form biofilms makes equipment
such as specula, probes and other surgical materials possible sources of transmis-
sion of these microorganisms. For this reason, the correct asepsis and sterilization
for handling this equipment is extremely important to avoid mycoses and severe
cases of fungemia [12].
. Predisposing and virulence factors to yeast infections
There are several yeasts that are of interest to the veterinarian, which can
cause superficial, subcutaneous, mucosal lesions, and even granulomatous and
systemic processes, and, in most cases, suspicion about the fungal etiology of
cases is neglected, hence advanced and severe cases of mycosis in dogs are not
uncommon [13].
The transition from the yeast stage to commensal to pathogenic will depend
both on factors related to the agent’s virulence, as well as on the host’s own
susceptibility [14].
The factors that can predispose humans and animals to a yeast infection are
innumerable, resulting from alterations in the defense mechanisms or by compro-
mising the anatomical barriers of protection of the organism [15–17].

Canine Medicine

Among these factors we can mention: stress; use of broad-spectrum antibiotics
or prolonged antibiotic therapy; antineoplastic agents; neutropenia; immunosup-
pression; age (senility/puppy); inadequate environment (overcrowding); long-term
use of corticosteroids; nutritional deficiencies; diets with a high concentration
of carbohydrates; pH changes, vitamin A deficiency, trichomoniasis; presence of
autoimmune diseases; changes in anatomical barriers due to trauma (maceration);
aplastic anemia; hematological infections; periodontal diseases (Figure ) and
other concomitant diseases [17, 18].
Prolonged antibiotic therapy and a high concentration of carbohydrates in the
diet can lead to the destruction or inhibition of the competitive bacterial micro-
biota, disrupting its balance with the host organism, thus allowing the accentuated
growth of yeasts [15].
Probably due to the poor oral hygiene of dogs throughout their life and associ-
ated with the other predisposing factors already mentioned, senility is considered a
significant condition for predisposition to periodontal disease. Animals older than
4years, according to a study with stray dogs, are more likely to develop this disease,
ranging from mild gingivitis to severe periodontitis (Figure ).
Virulence factors attributed to microorganisms must also be taken into account,
such as production of hydrolytic enzymes, proteases and phospholipases, adhe-
sion, formation of germ tube and biofilms. These factors favor the invasive power
and interfere with the host’s metabolism. All these factors, from hosts and yeasts,
can lead to superficial, or systemic, conditions. It is worth mentioning that the
high concentration of viable cells of the microorganism in an ecological niche of
the host is another factor that must be considered, as they may be part of the oral
microbiota.
Figure 3.
Connection: Age x Presence of Periodontal Disease in dogs [19].
Figure 2.
Dog with periodontal disease.

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Importance of Yeasts in Oral Canine Mucosa
DOI: http://dx.doi.org/10.5772/intechopen.95905
. Importance of animals’ oral health
Disorders of the oral cavity are of great importance in veterinary medicine due
to their high prevalence in dogs and their serious consequences, which can even
affect the systemic health of the animal [20]. Abnormalities, injuries or disorders
of this organ can cause discomfort and pain, leading the animal to anorexia, due
to lack of food, and adipsia, not water intake, predisposing it to conditions of
decreased immunity and clinical complications [21].
In addition to this great discomfort and the involvement of other organs, the
inflammatory response caused by diseases in the oral cavity can lead to the gingival
tissue a progressive loss of tooth fixation to the alveolar bone and, consequently, the
loosening and loss of this tooth [22].
The dentistry specialty in veterinary medicine has been evolving in recent years,
gaining space in the curriculum of some colleges. Even though the food industry
has undergone great advances in the production of diets aimed at improving oral
health, the number of professionals who perform an adequate clinical examination
is still not significant. In addition to this important factor, the lack of adequate
provision of oral hygiene care is worrying [22, 23].
Among dogs over one year of age, 95% have some degree of the disease, and in the
clinic, it is believed that 100% of adult animals have varying degrees of periodontal
disease [23]. The most common signs associated with periodontal disease are halitosis,
dental calculus, inflammation and gingival bleeding, anorexia and the consequent
weight loss, ptyalism, difficulty in chewing and grinding food, mobility and migra-
tion of teeth, loss of alveolar bone, gingival retraction and behavioral changes [24].
Periodontal disease is, therefore, the most common disease affecting dogs of all
breeds, formed from proliferative microorganisms, defense cells (leukocytes and
macrophages), epithelial cells, bacterial polysaccharides and salivary glycoproteins,
which over time become organize, occurring mineralization and formation of
dental calculus [25]. It is believed that this clinical condition is usually caused by
the formation of bacterial plaques, but the isolation of yeasts from the oral cavity of
dogs with periodontal disease is frequent (Figure ).
The greatest risk in periodontopathic is not only the loss of teeth or the develop-
ment of local infections, but the possible systemic effects of the pathological agent
in the bloodstream.
Thus, the oral health of dogs is extremely important and still needs a greater
focus on microbiological research and awareness of those responsible, regarding
food, the importance of oral hygiene, and the attention of the tutor and the veteri-
narian regarding the etiopathogenesis of diseases, such as yeasts.
Figure 4.
Connection: Presence of Periodontal Disease x Positive isolation of yeast [19].

Canine Medicine

. Main genera and species of yeasts isolated from the oral cavity of dogs
and clinical signs
Just like in humans, dogs have a known range of yeasts in their oral mucosa that
still requires more studies regarding colonization and pathogenicity. Despite its
remarkable importance in the health of dogs, studies involving the isolation and
correct identification of yeasts began to be developed in the 20th century [26].
This microbiota is not yet fully described, due to its great complexity and
diversity. Fungal colonization of the oral cavity of dogs is associated with yeasts of
the genera Candida, Malassezia, Trichosporon and Rhodotorula. Less frequently, we
can isolate yeasts of the genus Cryptococcus [27].
In a recent study conducted with 50 mixed breed dogs, a yeast profile was
found, composed of Candida albicans (39.5%), C. parapsilosis (18.6%), C. zeyl-
anoides (13.9%), C. krusei (7%), C. tropicalis (4.7%), Trichosporon spp. (4.7%),
T. asahii (4.7%), C. guilliermondii (2.3%), T. mucoides (2.3%) and Malassezia
pachydermatis (2.3%). The genus Candida showed a high prevalence, making
up a total of 82.2% of the isolated yeast profile. It is worth mentioning here the
isolation of Candida zeylanoides, a rare species, even in humans, and thus, the oral
mucosa of dogs can harbor a new “ecological niche” of this fungus species, which
can also act as an opportunistic pathogen [9].
. Genus Candida and Candidiasis
Currently, 317 species of this genus are recognized. Several of these species,
more precisely 20, have a pathogenic potential and can thus cause infections in
several species of animals, such as dogs [17]. The relationship with the host can
be commensal, parasitic or saprophytic. It can also be found in the usual form of
a yeast, or in the form of pseudohyphae. Candida albicans is the most common
colonizer in cases of infections, with a predilection for mucous surfaces and
mucocutaneous areas. Other species, such as C. kefyr, C. lusitaniae, C. guilliermon-
dii, C. tropicalis, C. krusei, C. famata, C. parapsilosis, can be isolated from animals
(Figure ) [15].
There were only few cases found in the literature in small animals, however,
reports of candidiasis in various animal species are also increasingly common,
described in photos of pyoderma of the lip folds, disseminated and localized muco-
cutaneous dermatitis, urinary tract infections, gastrointestinal and reproductive
system, ear infections, systemic and oral infections [27].
Figure 5.
Presumptive result of Candida species isolated from the oral cavity of mixed breed dogs according to the
CHROMAGAR Candida®.

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Importance of Yeasts in Oral Canine Mucosa
DOI: http://dx.doi.org/10.5772/intechopen.95905
Candidiasis related to the digestive system of dogs, such as a clinical manifesta-
tion of glossitis, is characterized by the formation of pseudomembranous plaques,
usually whitish in color, or yellowish beige. Once these plaques were removed, we
noticed erythematous regions with the presence of ulcers (Figure ) [28].
. Genus Trichosporon and Trichosporonose
The genus Trichosporon has 37 species that inhabit different ecological niches,
such as water, soil and body and mucous surfaces of humans and animals. They can
cause superficial and deep infections, such as Trichosporon asahii, T. mucoides,
T. ovoides, T. inkin, T. asteroides and T. cutaneum [29].
No cases of Trichosporon infections have been reported in the oral mucosa of
dogs, however, several species have already been isolated as colonizers. Clinical
cases of nasal granuloma in other animals, cystitis in cats, mastitis in cows and
dermatitis in horses and monkeys have already been described (Figure ) [30].
. Genus Malassezia and Malasseziosis
The genus Malassezia has 15 species, mostly lipophilic yeasts, that can be part
of the skin and mucous membranes of humans and dogs. They are opportunistic
yeasts, and in certain circumstances, they can lead to clinical manifestations [31].
Malassezia pachydermatis, the most frequent in dogs, is not lipophilic and can grow
in a culture medium common in mycology [19] (Figure ).
Figure 6.
Mixed breed dog with oral candidiasis (glossitis). Friable white and yellowish plates covering the tongue [28].
Figure 7.
A and B –Yeasts of Genus Trichosporon showing oval and rectangular artrospores - (A) 160x and (B)
400x [15].

Canine Medicine

Several clinical symptoms can be associated with Malassezia spp. and, particu-
larly, in cases of otitis and dermatitis in dogs. Cases of otitis by Malassezia pachyder-
matis are frequency, but oral infection caused by this agent have not been described
or has not yet been well studied [19].
. Genus Cryptococcus and cryptococcosis
In the Cryptococcus genus, we found 38 species, with Cryptococcus neoformans
and C. gattii being the most prominent in medical mycology in man and animals
(Figure ). The species can be found in different places in the environment, pri-
marily in association with birds’ droppings, mainly pigeons, but have an ecological
association with trees too, such as eucalyptus [32].
In dogs, can enter the body through the lung causing pulmonary disease,
and several clinical signs can be presented, such as skin lesions, nasal mucosa
(“clown nose”), and can hit the central nervous system, for its neurotropic
nature. These lesions in the nasal mucosa can extend into the oral cavity of the
animals [28].
. Collection of clinical material and identification of yeasts
. Material collection
The collection of the oral cavity of dogs is performed with the aid of a sterile,
alginate swab, moistened with sterile saline solution. The swab is introduced,
Figure 9.
Cryptococcus spp. Encapsulated yeasts - Nigrosina, 1000x.
Figure 8.
Yeasts of the Genus Malassezia spp. - single budding on wide base - Panotic, 1000x.


Importance of Yeasts in Oral Canine Mucosa
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carefully, in the oral cavity, in circular movements, passing through the entire oral
mucosa [19] (Figure ).
After this procedure, the collected samples must be sent to the laboratory and
sown in Petri dishescontaining basic mycology medium (Sabouraud dextrose agar),
plus antibiotics (chloramphenicol - 0.05g/L concentration). Incubation at 25°C for
up to two weeks [19].
There are several procedures that can be used to identify yeasts. Direct examina-
tion (fresh), or with Gram stain is also highlighted (Figure ).
. Yeast identification
The identification of yeasts can be performed by means of macro and micro-
morphological, biochemical, proteome (MALDI-TOF) and molecular tests.
In macromorphological characterization, we studied color, texture and edges
(Figure ).
In the more specific micro morphological identification, we must observe the
characteristics of the cells (oval, round, unipolar bud, or multiple buds), pseudohy-
phae, hyphae and structures characteristic of C. albicans, such as chlamydoconidia
(Figure ).
The formation of a germ tube, another important characteristic of C. albicans,
originates from blastoconidium when the yeast is sown in fetal bovine serum
(Figure ).
Figure 10.
Collection with sterile swab of the oral mucosa of a mixed breed dog - City of Campinas, São Paulo - Brazil.
Figure 11.
Yeasts of Genus Candida in an abdominal dog fluid sample - Fuchsin, 1000x.

Canine Medicine

Tests of assimilation of sources of nitrogen and carbohydrates can be performed
(auxanogram), as well as fermentation tests (zymogram). The protocol followed
for these methods is from the manual “The Yeasts: a taxonomic study” (volumes 1,
2 and 3). The MALDI-TOF technique is a mass spectrometry, which determines the
protein profile (proteome) of the yeast under study. It is a fast technique (15–20),
simple, excellent cost–benefit, however, there are limitations to the use of the labo-
ratory routine, as the device is expensive and requires specialists to use it, as well as
a robust base of standard strains.
Figure 13.
Candida albicans in culture broth. Globose, or elongated cells, pseudohyphae, hyphae, blastoconidia and
characteristic chlamydoconidia, 1000x.
Figure 12.
Culture of yeasts on Sabouraud dextrose agar.


Importance of Yeasts in Oral Canine Mucosa
DOI: http://dx.doi.org/10.5772/intechopen.95905
For the identification of yeasts, we also count on molecular biology tech-
niques, which are sensitive and specific. For the differentiation, for example, of
C. albicans and C. dubliniensis is the most accurate technique. There are several
methods such as PCR (Polymerase chain reaction), RFLP (Restriction fragment
length polymorphism) and RAPD (Random amplified polymorphic DNA). One
of the most used is PCR, which detects minimal amounts of DNA, or RNA. But
not all laboratories can use these methods, due to the higher costs and the needs of
specialized laboratories [33].
Figure 14.
Germ tube on bovine serum – Candida albicans, 400x.
Figure 15.
Yeasts of genus Candida on CHROMAGAR Candida® - Candida albicans with green color.
Figure 16.
API20CAUX method (bioMérieux®) –profile of carbohydrates assimilation.

Canine Medicine

. Chromogenic medium: CHROMagarCandida®
Sowing in chromogenic media, such as CHROMagarCandida®, can provide
presumptive identification according to the color developed by the yeast. In this
medium, the specie Candida albicans develops a light green color; C. tropicalis it is
blue/green and C. krusei light pink, for example (Figure ).
In addition to these identification methods, there are several automated and
manual systems that facilitate the laboratory routine, such as Vitek and API20C, as
examples (Figure ).
. Epidemiological markers
As we have already pointed out, yeasts (especially those of the genus Candida)
have emerged as important pathogens in humans and animals and the interrelation
between both is of great relevance, gaining prominence today.
Several markers can be used to detect new yeast species as well as their genotype.
Based on the data, we can determine the presence of these microorganisms, the
same species/genotype, in one or more anatomical areas of the host, as well as of
different ecological niches.
Confirming the colonization/infection area has often been an arduous task.
Therefore, the use of these markers shows to be of great importance for the epide-
miological study of yeasts.
Among the phenotypic markers, we can highlight those based on colony
morphology (morphotyping), enzyme production (enzyme typing), sensitivity
to “Killer” toxins and antifungal agents. They are simple and easy to perform
techniques.
Genotypic markers are more sophisticated and safer; however, they require more
elaborate techniques. The technique is based on short sequential repetition of bases
throughout the yeast genome and its reading is performed on a specific sequencing
apparatus. The patterns of the visualized DNA bands function as true “fingerprints”
of the microorganism, leading us to the recognition of the colonization/infection
area of the host. This technique can be used both for use in clinical isolates and for
environmental samples (Figure ).
With increasingly interconnected ties between man and his dog, the use of these
markers is a valuable technique for detecting epidemiological transmission between
Figure 17.
Chromosomal bands of yeasts obtained by electrophoresis pulsed field - PFGE.


Importance of Yeasts in Oral Canine Mucosa
DOI: http://dx.doi.org/10.5772/intechopen.95905
these species and a facilitator for taking therapeutic actions based on the microbio-
logical analysis of the agent’s transmitter [33].
. Antifungals, sensitivity tests and treatment
Currently, yeast mycoses have increased substantially, and it can be considered
an important public health problem, especially in systemic clinical conditions and
hospital infections. The antifungal drugs used in human and veterinary medicine
have special characteristics regarding the chemical structure and the mechanism
of action, interfering directly or indirectly in the fungal cell, with fungistatic or
fungicidal actions [34].
Among the existing antifungal drugs, the most widely used and known are
polyenic, imidazolic, pyrimidine, sulfamide, benzofurenic and other compounds
with varying degrees of success, such as iodides, thiosulfates, sulfides and
tolnaftates. In the treatment of invasive fungal infections, classes of polyene
antifungals (amphotericin B), azoles (fluconazole, voriconazole, ketoconazole,
itraconazole, posaconazole), pyrimidines (5-fluorocytosine) and echinocandin,
caspofungin, micafungin) are mainly used [35]. The increasing incidence of
yeast infections, such as those present in the oral mucosa, has been a target of
constant concern in the search for increasingly effective treatments and safer
drugs. The use in the treatment and prophylaxis of antifungals such as fewer
toxic azoles, especially fluconazole, has given rise to cases of resistance among
susceptible yeast species.
The resistance of fungi to antifungal agents can be classified into clinical and
microbiological resistance. The concept of clinical resistance is defined when there is
a persistence or progression of a fungal infection even with the administration of the
drug chosen as appropriate. In this case, “in vitro” tests may indicate the sensitivity
of the agent to the antifungal. Usually, the occurrence of clinical resistance is associ-
ated with host, iatrogenic, pharmacological factors and factors related to the fungus
virulence [36]. Microbiological resistance is a phenomenon in which the etiologic
agent can develop in the presence of therapeutic concentrations of antifungals, a
capacity verified “in vitro”. Resistance can be intrinsic, primary or secondary, or
extrinsic. This aspect is of real importance since we are increasingly faced with resis-
tant yeasts, especially the “critical” species, highlighting C. auris and C. haemulori,
whose findings should be immediately reported to the treatment team.
Intrinsic resistance is so called when no member of a species is sensitive to the
antifungal, being primary, when in a species normally sensitive to an antifungal
we find a resistant strain (without exposure to it) or secondary or acquired, when
a previously sensitive strain develops resistance after exposure to a drug, due to
phenotypic or genotypic changes [37]. The mechanism of resistance to antifungals
by fungi, both for clinical or microbiological resistance, is involved with cellular,
biochemical and/or molecular responses.
In the cellular mechanism, strains or sensitive specimens are exchanged for
resistant endogenous ones, genetic alteration, a fact that guarantees secondary
resistance, transient genetic expression and alteration in the cell type. Regarding
the biochemical mechanism, phenotypic changes in fungi occur, allowing the
absorption of the drug to be slower, altering the target site and increasing the excre-
tion of the drug. The changes from the molecular point of view causing a genetic
amplification to occur, mutations, among other modifications in the gene involved
in the defense against the antifungal. In addition to these changes, another molecu-
lar alternative of resistance is the ability to form biofilms, an efficient physical
barrier [36].

Canine Medicine

The greater phenotypic variability of Candida species, for example, together
with the increased resistance of strains to antifungals, has assumed a prominent
role as a clinical problem [9]. The different species of this yeast vary in sensitivity to
antifungals on the market, a fact that shows the great importance of identifying and
determining the minimum inhibitory concentration (MIC) [38].
Due to this aspect, the development of standardized methods of sensitivity “in
vitro” is of vital importance and serve as guide to indicate the therapeutic choice,
monitor the effectiveness of the antifungal and decrease the formation of resistant
strains [39]. The appropriate choice of antifungal agent is, therefore, decisive in the
therapeutic response of the animal. To this end, research that aims at determining
the antifungal profile of the main yeasts isolated from dogs is of great therapeutic
value [9].
The most used parameter for determining sensitivity to antifungals is the
minimum inhibitory concentration (MIC), defined as the lowest concentration
of an antifungal agent that inhibits the growth of the fungus [40]. From the MIC
value, the yeast sample is classified according to the breakpoints established by
international committees, which allows the fungus to be characterized as sensitive,
intermediate, dose-dependent and resistant sensitivity [38]. For the detection of
sensitivity/resistance to antifungals, highlight of use in therapeutic failures, we can
count on several techniques, being “Gold standard” the method recommended by
CLSI, the microdilution test.
The determination of the sensitivity of a fungus to antifungals can also be
determined by commercial methods compatible with the tests recommended
by CLSI [17, 37]. Sensitivity tests using a solid medium, such as the commercial
method “E-test”, are of real interest in several studies and in the laboratory rou-
tine. It is an excellent technique for determining the sensitivity to antifungals “in
vitro”, simple, easy to perform, with fast results, without the need for expensive
or specialized equipment [41]. “E-test” is based on a combination of dilution and
diffusion test concepts that directly quantify antifungal sensitivity. It consists of
tapes containing pre-established concentrations of the antifungal agent, which
are placed in a solid medium and with the yeast sample. When the tape is applied
to the plate, immediate drug release occurs, thus the MIC is determined by the
intersection of the inhibitory hyperbole formed by the growth of yeast in the
plate (Figure ).
Because the MIC values of “E-test” are directly proportional to the values
referenced by the dilution CLSI, this method has a good correlation with this test.
However, it may still present differences inherent to the process.
Figure 18.
“E-test” commercial method. MIC is determined by the intersection of the inhibitory area formed by yeast
growth.


Importance of Yeasts in Oral Canine Mucosa
DOI: http://dx.doi.org/10.5772/intechopen.95905
The classification by this method determines the isolate as sensitive, dose-
dependent and resistant (Table ).
For tests to determine the antifungal profile, source control strains of the
“American Type Culture Collection” (ATCC) are always used under identification,
such as, for example, ATCC64548 (C. albicans) and ATCC777 (C. dubliniensis).
In the treatment of invasive fungal infections, classes of polyene antifungals
(amphotericin B), azoles (fluconazole, voriconazole, ketoconazole, itraconazole,
posaconazole), pyrimidines (5-fluorocytosine) and echinocandin, (caspofungin,
caspofungin, micafungin) are mainly used [35].
For the systemic treatment of yeasts, we can use Amphotericin B, in the most
varied forms (liposomal, suspension of lipid complexes). Nystatin can be used
orally or in suspension, ointments and creams (as for example, in cases of oral can-
didiasis). In animals, the use of each of these antifungals is quite varied and their
recommendation and dose will depend a lot on the etiological agent in question and
the side effects that can be generated.
When analyzing the profile of sensitivity to antifungals compared to isolates from
the oral cavity of dogs (mucosa that has greater transmissibility to humans), the
best active antifungals found in the veterinary are ketoconazole and voriconazole.
Ketoconazole is still widely used in clinics and pet shops, mainly, topically. For the
treatment of candidiasis in small animal clinics, ketoconazole is one of the most
frequently used drugs, as it has a broad spectrum of activity, encompassing several
species of Candida spp. and dermatophytes. From isolates from the oral cavity
of dogs it shows high sensitivity between yeasts and has several presentations for
veterinary use, representing an economically viable alternative, however, due to its
toxicity, the trend is disuse [9, 17].
Voriconazole has a broad spectrum of activity and a potent “in vitro” action.
Its mechanism of action is like other azole antifungals, inhibiting the enzyme 14
alpha-demethylase, dependent on cytochrome P-450, essential for the ergosterol
biosynthesis. It can be indicated as a good alternative to replace ketoconazole,
however its cost is high. This drug is also used for the treatment of systemic
mycoses, mainly in candidiasis, aspergillosis and cryptococcosis in debilitated,
immunosuppressed patients or in cases of resistance to another antifungal [43]. The
antifungals fluconazole, itraconazole and miconazole are also routinely applied in
the veterinary clinic, used indiscriminately in the treatment of mycosis suggestively
diagnosed. However, resistance to these drugs has increased, so their use should be
more cautious.
Candida zeylanoides, for example, is a relatively rare yeast in humans and
animals. In humans it has been reported from skin, nails and blood isolation,
considered an opportunistic pathogen, also involved in cases of endocarditis in an
Antifungals S (μg/mL) SDD (μg/mL) R (μg/mL)
Miconazol* ˂ 8 8–16 ≥ 16
Cetoconazol ˂ 16 —≥ 16
Fluconazol ≤ 8 16–32 ≥ 64
Itraconazol ˂ 0,25 0.25–0.5 ≥ 1
Voriconazol ≤ 1 2 ≥ 4
Caspofungina ≤ 2 — ˃ 2
S: sensitive; SDD: dose dependent sensitivity; R: resistant; NS: not sensitive [, ].
Table 1.
Interpretation of the behavior of yeast strains against the concentration of antifungals (μg/ml).

Canine Medicine

HIV-positive patient [44]. Samples of this yeast were isolated for the first time in the
oral cavity of stray dogs and demonstrated significant resistance to the antifungal
fluconazole. In addition to this species, Candida krusei also obtained partial results
of resistance to this antifungal, as well as yeasts of the genus Trichosporon spp. [9].
Itraconazole is a synthetic triazole derivative with a wide spectrum of action,
widely used in the treatment of superficial mycoses by candidiasis, malasseziosis and in
systemic mycoses. When used orally right after a meal, its bioavailability is maximum,
with biphasic elimination. This antifungal has also been used successfully in dogs with
mycotic rhinitis and in systemic mycoses, such as blastomycosis. However, its use in
dogs can lead to skin rashes and, in high dosages, it can cause anorexia and increased
plasma concentration of alkaline phosphatase and aminotransferase enzymes [43].
In addition, species isolated from the oral cavity of dogs (especially Candida albi-
cans and C. tropicalis) have shown dose-dependent sensitivity to itraconazole. Yeasts
of the genus Trichosporon also isolated from this active site, show medium resistance to
fluconazole and significant resistance to itraconazole, which reveals concern about the
use of these drugs in the treatment of candidiasis and triconosporoses in dogs [9, 19].
In the veterinary medical clinic, miconazole is commonly indicated for the
treatment of dermatophytosis, malasseziosis and candidiasis. However, yeasts of
the genus Trichosporon and Malassezia pachydermatis isolated from the oral cavity of
dogs show important resistance to this antifungal. Different for Candida yeasts, in
which the antifungal profile demonstrates sensitivity to miconazole [9, 19].
Caspofungin is an antifungal with an inhibitory action on the cell wall of the
echinocandin group, important in human medicine as an alternative for the treat-
ment of isolates resistant to fluconazole [45]. Against yeasts isolated from the
oral cavity of dogs, yeasts of the genus Trichosporon and of the genus Malassezia
demonstrate significant resistance to this antifungal, resistance also demonstrated
to a lesser extent by the species Candida parapsilosis [9, 19].
In cases of systemic infections, affecting different species of animals, the use of
amphotericin B, a drug that acts on the fungal cell membrane, has efficiency against
strains of Candida spp. However, due to the high cost and serious side effects, such
as hepatotoxicity, nephrotoxicity, myelotoxicity and cardiotoxicity, this medication
is seldom used [17].
Due to the great similarity between the fungal cell and the host cell, the action
of antifungals presents relatively high toxicity. Thus, there is a need for research for
the best choice of antifungal, based on the most appropriate therapeutic response
and on the sensitivity profile of yeast against antifungal floodgates, seeking as
well to minimize the side effects that can be generated with the use of more drugs
needed in cases of therapeutic failure [43].
When information is obtained that a street animal, which in general is a dog
that, has never received therapeutic treatment based on antifungal, presents
positive isolation for resistant yeasts, it is assumed that environmental yeasts are
undergoing an important primary resistance or that the ecological niche in which
that animal lives is contaminated by resistant microorganisms originating from
direct or indirect human contamination.
Corroborating this fact, we must consider the excessive use of pesticides in the
environment and mycoherbicides (placed in plantations, vegetable gardens, and in the
soil itself), have a chemical constitution like azoles, thus representing a strong selec-
tive pressure for the emergence of strains resistant. This question of possible envi-
ronmental contamination and fungal resistance is already discussed for other yeast
species, such as Cryptococcus spp. and medical mycology becomes an important issue.
The growing data on increased resistance of fungi against antifungal drugs have
been causing great concern for human and veterinary doctors. Although data on
resistance to antifungals from yeasts isolated from dogs are scarce, their importance


Importance of Yeasts in Oral Canine Mucosa
DOI: http://dx.doi.org/10.5772/intechopen.95905
Author details
Claudete RodriguesPaula1*, Bianca SilvaNavarro2, Mário MendesBonci1 and
Diana CostaNascimento3
1 School of Dentistry, University of Sao Paulo (USP), SaoPaulo, Brazil
2 Clinical Veterinarian Animal Facility, Zoetis Industria de Produtos Veterinários
LTDA, Campinas, Brazil
3 Pontifical Catholic University of Campinas (PUC), Campinas, Brazil
*Address all correspondence to: crpmicol@uol.com.br
is notorious, directly associated with the therapeutic success of these animals
particularly important for society and human health (physical and mental).
Therefore, the ideal therapeutic choice, for both humans and animals, should be
based on prior identification of the agent and, if possible, the use of techniques for
determining the sensitivity profile of the etiologic agent against antifungals.
. Considerations
The oral cavity is an extremely important anatomical area of dogs, considered as
one of the determining factors in the longevity of this animal’s life. To reduce thera-
peutic failures and guarantee the perfect health condition of this system, knowledge
of the existing microbiota is essential, but it is still scarce.
We can then ask ourselves: Why are recurrent fungal infections more and more
frequent in dogs? What is the relationship between the microbiota of dogs and their
respective owners? And what is the relationship of resistance to fungal infections
between these species?
Possible answer to these questions could be founded in this chapter, as well as
the beginning of the knowledge of the main yeasts found in the oral cavity of dogs,
their clinical importance and profile of resistance to the main antifungals used in
the practical routine of veterinary medicine.
Acknowledgements
The authors would like to thank Professor Renata Mangione for the translation
into English and the Brazilian Official Institutes (FAPESP, CNPQ and CAPES).
© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms
of the Creative Commons Attribution License (http://creativecommons.org/licenses/
by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly cited.


Canine Medicine
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