
Review

New antimicrobial therapies used against fungi present in
subgingival sites—A brief review

Janaina Cássia Orlandi Sardi, Ana Marisa Fusco Almeida,
Maria José Soares Mendes Giannini *

Faculty of Pharmaceutical Sciences of Araraquara, Department of Clinical Analysis, Laboratory of Clinical Mycology,

Univ Estadual Paulista, UNESP, Araraquara, SP, Brazil

Contents

1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 952

2. Colonization of the oral cavity by Candida spp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 953

2.1. Candida spp. in subgingival sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 954

2.2. Resistance of yeasts to synthetic antifungals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 955

2.3. Search of new natural antifungal drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 956

3. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 957

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 957

a r c h i v e s o f o r a l b i o l o g y 5 6 ( 2 0 1 1 ) 9 5 1 – 9 5 9

a r t i c l e i n f o

Article history:

Accepted 17 March 2011

Keywords:

Candida spp

Fungi

Antifungal

Periodontal disease

New antimicrobial therapies

a b s t r a c t

Although the main reservoir of Candida spp. is believed to be the buccal mucosa, these

microorganisms can coaggregate with bacteria in subgingival biofilm and adhere to epithe-

lial cells. The treatment of periodontal disease includes scaling and root planning (SRP)

associated with proper oral hygiene. However, some patients may have negative responses

to different therapeutic procedures, with a continuous loss of insertion, so the use of

antimicrobials is needed as an adjuvant to SRP treatment. The use of a broad-spectrum

antibiotic, such as tetracycline and metronidazole, as an aid in periodontal treatment has

also been a factor for the development of superinfections by resistant bacteria and Candida

species, even in patients with HIV. In the dental practice, the most commonly used

antifungals are nystatin and fluconazole. However, the introduction of new drugs like

the next generation of azoles is essential before the onset of emergent species in periodontal

disease. Plants are good options for obtaining a wide variety of drugs. This alternative could

benefit a large population that uses plants as a first treatment option. Plants have been used

in medicine for a long time and are extensively used in folk medicine, because they

represent an economic alternative, are easily accessible and are applicable to various

diseases. Herein, we briefly review the literature pertaining the presence of Candida sp.

in periodontal pockets, the conventional antifungal resistance and new therapies that

include natural antifungal agents are reviewed.

# 2011 Elsevier Ltd. All rights reserved.

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journal homepage: http://www.elsevier.com/locate/aob
* Corresponding author. Tel.: +55 16 3301 5714; fax: +55 16 3301 6701/3
E-mail address: giannini@fcfar.unesp.br (M.J.S. Mendes Giannini).

0003–9969/$ – see front matter # 2011 Elsevier Ltd. All rights reserve
doi:10.1016/j.archoralbio.2011.03.007
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http://dx.doi.org/10.1016/j.archoralbio.2011.03.007
mailto:giannini@fcfar.unesp.br
http://dx.doi.org/10.1016/j.archoralbio.2011.03.007


a r c h i v e s o f o r a l b i o l o g y 5 6 ( 2 0 1 1 ) 9 5 1 – 9 5 9952
1. Introduction

Candida species are commensal yeasts in healthy humans and

may cause systemic infection under immunocompromised

situations due to its high adaptability to different host miches.

It was suggested that when C. albicans accessed the periodon-

tal tissues, they may be harmed by the production of

metabolites by these yeasts.1

The periodontal disease is a chronic infection that affects

the gingiva and bone that supports the teeth. This chronic

inflammatory disease results from the response to micro-

rganisms in dental biofilm and may remain confined to the

gingival tissues with minimal tissue alterations; alternatively,

this disease may progress to extreme periodontal destruction

with the loss of attachment and alveolar bone. In addition to

the presence of periodontal pathogens; such as Porphyromonas

gingivalis, Aggregatibacter actinomycetemcomitans and Tannerella

forsythia; genetic and environmental factors seem to increase

the susceptibility of some individuals in developing this severe

inflammatory disease.2 Therefore, there is general support for

this concept of periodontal disease. It is also well recognized

that the presence of just pathogenic bacteria is insufficient to

cause periodontitis. Progression of this disease occurs due to a

combination of factors, including the presence of period-

ontopathogenic microorganisms, high levels of pro-inflam-

matory cytokines, matrix metalloproteinases (MMPs),

prostaglandin E2 (PGE2), low levels of anti-inflammatory

cytokines including interleukin-10 (IL-10), transforming

growth factor (TGF-b) and tissue inhibitors of MMPs (TIMPs).3,4

However most microorganisms found in subgingival biofilm is

commensal, or also occurs in individuals with a healthy

periodontium that is in equilibrium with the host. Thus,

episodes of disease resulting from deficiencies in the ability of

the host defence to fight the bacterial biofilm, changes the

quantitative or qualitative subgingival microbiota.5,6

Periodontal diseases are classified as either gingivitis or

periodontitis. Gingivitis is an infection associated with biofilm,

characterized by an occurrence of reversible inflammatory

phenomena limited to the gingival tissues and not affecting

the supporting periodontal tissues. Clinical signs of gingivitis

range from oedema, abnormal staining of normal gums,
Table 1 – Data on resistance of Candida spp. isolated from sub

Isolates % Azoles resistance %

C. albicans 4.2 0

Non-C. albicans 6.25 6

C. albicans 3.3 0

Non-C. albicans 17 N

C. albicans 0 N

Non-C. albicans 42.8 7

C. albicans 3.6 7

Table 2 – In vitro antifungal activity of natural extracts on Can

AC MP SC 

C. albicans 15 7 1 

Non-C. albicans 15–30 1–7 1 

AC, Arrabidae chica; MP, Mentha piperita; SC, Syzygium cumini; TA, Tabebuia
unusual redness, and loss of normal contour of gingival.7

Periodontitis is defined as an inflammatory disease that

involves the tissues of dental support, leading to irreversible

alveolar bone resorption and destruction of the collagen fibres

of the periodontal ligament. The severity and progression of

this disease is influenced by local or systemic conditions or

from both a combination of both.8 Local factors are associated

with poor oral hygiene, the presence of cavities, and the

presence of dentures that can cause plaque accumulation.

Systemic factors are related to the metabolism of the host,

immunosuppressive therapy, malnutrition, diabetes mellitus

and HIV infection (Tables 1 and 2).7

A complex microbiota can be found in the periodontal

pocket affected by the disease, where approximately 500

species of bacteria can occur, amongst them are A. actinomy-

cetemcomitans, and other microorganisms such as Entamoeba

sp., virus, some Enterobacteriaceae, Pseudomonas sp., and

Candida species.9–12 This fact has led to an extensive study of

microbiological samples from periodontal lesions, especially

in cases where there is a poor response to conventional

treatment. In many of these cases, fungi have been found

colonizing the periodontal pockets. There are several reports

associating the occurrence of severe periodontitis with the

isolation of Candida species from periodontal lesions.13,14

However, the clinical significance of these observations and

the role of microorganisms in the pathogenesis of periodontal

diseases are not well understood. Many scientific investiga-

tions have been performed in order to extend the knowledge in

this area.15 However, the presence of fungi in periodontal

pockets has not yet received the necessary focus to under-

stand its role as periodontal pathogens, although they have

been recognized for their ability to adhere to the epithelium,

express virulence factors and induce inflammatory reac-

tions.16

The treatment of periodontal disease includes scaling and

root planning (SRP) associated with proper oral hygiene.

However, some patients may have negative responses to

different therapeutic procedures, with a attachment loss, so

the use of antimicrobials is needed as an adjuvant to SRP

treatment.17 The use of a broad-spectrum antibiotic, such as

tetracycline and metronidazole, as an aid in periodontal

treatment has also been a factor for the development of
gingival sites.

 Amphotericin resistance References

 Waltimo et al.64

.25 Ito et al.62

 Ito et al.62

o studied Jewtuchowicz et al.56

o studied Jewtuchowicz et al.56

1.4 Furletti et al.61

2.9 Furletti et al.61

dida species (mg/mL), according Hö fling et al.94

TA RO PG Fluconazole

3 1 3 32

1–15 1–7 1–3 32–64

 avellanedae; RO, Rosmarinus officinalis; PG, Punica granatum.


a r c h i v e s o f o r a l b i o l o g y 5 6 ( 2 0 1 1 ) 9 5 1 – 9 5 9 953
superinfections by resistant bacteria and Candida species.18,19

The use of a broad-spectrum antibiotic, such as tetracycline

and metronidazole, as an aid in periodontal treatment

associated with SRP has been recommended for the treatment

of periodontal disease.18,19

Metronidazole and amoxicillin antibiotics to be seem

indicated for the treatment of periodontal infections. Lotufo

et al.,20 performed antimicrobial susceptibility tests in vitro for

105 strains of anaerobic bacteria isolated from patients with

periodontitis. According to the results, the antimicrobial

metronidazole was more action on the organism studied.

None of the isolates showed resistance to metronidazole.

Amoxicillin also showed good results, with approximately 94%

of strains sensitive to this drug.

In the dental practice, the most commonly used anti-

fungals are nystatin and fluconazole. It is believed that the

presence of C. albicans in subgingival sites is in the form of

biofilms, which could explain the resistance to antifungal

therapy.

Plants are good options for obtaining a wide variety of

drugs.21 This alternative could benefit a large population that

uses plants as a first treatment option.22 Plants have been used

in medicine for a long time and are extensively used in folk

medicine, because they represent an economic alternative, are

easily accessible and are applicable to various diseases.23

Therefore, these constitute an excellent alternative in the

search for substances that can be used to develop new

antifungal drugs.24 It is necessary to seek new antifungal

agents that are fungicides, which cause disruption or

destruction of biofilms, which are effective in isolates that

express resistance using several molecular mechanisms and

which are not toxic.

In the present report, the literature on the presence of

Candida spp. in periodontal pockets, the conventional antifun-

gal resistance and new therapies that include natural

antifungal agents are reviewed.

2. Colonization of the oral cavity by Candida
spp.

Based on their prevalence in healthy and asymptomatic

populations, the isolation of Candida spp. from the oral cavity

does not necessarily imply an infection.25 Many studies have

shown that approximately half of the healthy adult population

carries yeasts in the oral mucosa, however, the prevalence has

been found to vary amongst different population groups.25,26

Several different groups present levels of oral colonization by

yeasts larger than the average population in general, with

these groups being known at-risk populations.27 Studies

report a higher prevalence of Candida species in patients with

Down’s syndrome, in individuals with salivary gland hypo-

function, decreased flow or salivary pH and diabetes mellitus.

These conditions seem to alter the oral environment and

promote colonization by these and other species of opportu-

nistic pathogens.28 The occurrence of these fungi has also

been reported in HIV-positive patients, with rates of infection

that are higher than in other at-risk populations.29 The

increasing proportion of these fungi suggests a deficient

immune response associated with the progression of viral
infection in HIV-infected individuals, which could be a

predictive factor for the development of candidiasis.28 The

occurrence of yeast is also common in patients with advanced

cancer with oral candidiasis a serious medical condition

amongst these patients. Epidemiological studies are therefore

needed on the distribution and virulence potential of these

yeasts in different population groups, addressing risk factors

and developing strategies for the control and prevention of

infections.27,30,31 Yeasts are found colonizing various sites in

the oral cavity (lingual, palate, tonsils, mucosa of the lips and

cheeks, caries, periodontic and endodontic lesions).32–35

Siqueira and Rô ças35 found C. albicans species associated with

bacteria in teeth with periodontal pockets around areas of root

exposure. For those authors, resistance to intra canal drugs,

and the ability of these yeasts to colonize and invade the

dentine tubules, may explain the presence of yeast in

persistent endodontic infections.

The use of a prosthesis is another factor that may favour

colonization of the oral cavity by Candida spp.36 with a report

indicating that the microbiota between a prosthesis and palate

mucosa has a composition similar to dental biofilm, except for

a greater proportion of Candida species, a fact related to the

development of candidiasis on the mucosa of the palate.

Kleinegger et al.37 concluded that a number of natural

barriers existed in the mucosal surfaces and body fluids;

preventing the colonization in healthy individuals. These

barriers are more or less effective, depending on factors

related to age, gender, smoking, diet, drugs and the host

immune status. This explains the fact that not all individuals

harbour Candida spp.

Saliva helps maintain oral health, provides a buffering

capacity and provides lubrication of the mucous membranes;

therefore, qualitative and quantitative changes in saliva

inevitably affect the physiology, defence mechanisms and

microbial ecology of the mouth.38 Lactoferrin and lysozyme

are two proteins in the innate immune response present in

saliva and exert an antifungal modulating effect on the

implantation of species of Candida in the oral cavity.39 Other

important proteins in human saliva that have a cytotoxic

action on bacteria and fungi are the histatins, estaterins,

lactoperoxidase and calprotectin.37 According to Lin et al.,40

when there is a decrease when the concentration of salivary

histatins, dysfunction of these proteins occurs and candidiasis

tends to manifest. HIV-infected individuals show a reduction

in salivary flow and an anti Candida activity of saliva and are

often suffering from oropharyngeal candidiasis. For those

authors, the saliva contained mucins and aggregated IgA,

histatin, lactoferrin and lysozyme, which remained focused

on mucosal surfaces and exerted an antimicrobial effect.41 C

albicans is able to connect to several species of streptococci (S.

oralis, S. sanguinis, S. gordonii, and Fusobacterium) through

recognition receptor polysaccharides in the bacterial cell

surface. F. nucleatum is an anaerobic gram-negative bacillus

commonly associated with periodontal lesions and isolated

from subgingival biofilm.41,42 Biofilm formation has been

considered an important strategy for microbial survival and

proliferation in the oral environment. The complex structure

of a biofilm allows microorganisms to offer protection against

the antimicrobial mechanisms of saliva and hinder the action

of antimicrobial agents.43 It is believed that most of the


a r c h i v e s o f o r a l b i o l o g y 5 6 ( 2 0 1 1 ) 9 5 1 – 9 5 9954
manifestations of candidiasis are associated with biofilm

formation, and recognition of the biofilm features may help in

developing therapeutic strategies for these infections.44 For

the current author, the biofilms formed by C. albicans and C.

dubliniensis have several features in common with bacterial

biofilms, including the structural heterogeneity and reduced

susceptibility to antimicrobial agents when mature. These

biofilms consist of a mixture of yeast and filamentous cells

embedded in a matrix of exopolymers, which serves as a

reservoir for the release of infective organisms in the oral

cavity. This can allow the survival of yeast in their ecological

niches during infectious episodes, which, according to

Ramage et al.,44 has important clinical, treatment and

prevention implications. Thus, the biofilms containing mostly

C. albicans could be implicated, not only in mucosal candidosis,

but also in the development of caries45 and in the pathogenesis

of periodontal disease.46,47

2.1. Candida spp. in subgingival sites

Candida species possess virulence factors relevant in the

pathogenesis of periodontal disease, such as the ability to

adhere to the epithelium and invade the gingival connective

tissue, the ability to inhibit the function of polymorphonuclear

neutrophils, and produce enzymes such as collagenases and

proteinases which degrade immunoglobulins.32,47–49 Accord-

ing Hägewald et al.,33 microorganisms that are capable of

degrading IgA may acquire a selective advantage in the

colonization of oral surfaces. Those authors believe that the

proteolysis of immunoglobulins facilitates the penetration

and spread of potentially toxic substances or antigens released

by the subgingival microbiota. That process could perpetuate

inflammatory changes associated with destructive periodon-

tal diseases. The periodontal alterations have been considered

a result of an exacerbated immune response against the host

tissues, with changes in cellular and humoral immune

responses that allow different species, such as Candida, to

colonize the subgingival environment.50 The detection of fungi

in the subgingival region has been suggested to contribute to

the pathogenesis of periodontal disease and to increase the

possibility of candidiasis, mainly in cases of immune depres-

sion.32,46 However, the role of yeasts, mainly Candida albicans,

in chronic periodontitis is yet unclear.51

Isolation of subgingival Candida species has been described

by many authors in different manifestations of periodontal

disease, for example, in cases of aggressive periodontitis,33

chronic periodontitis,9,49 in periodontal pockets of smokers,52

diabetics,9,53 myelosuppressed patients54 and in perimplanti-

tis. Certain Candida species are considered to be commensal

organisms within the oral cavity. Indeed, the prevalence of

oral yeast in the general population is about 34%.54 In 24

patients with acute periodontal infection and chemotherapy-

induced myelosuppression, microorganisms were detected in

high concentrations in subgingival pockets with a predomi-

nance of Staphylococcus epidermidis, C. albicans, S. aureus, and

Pseudomonas aeruginosa, with combinations of these detected

in some patients.54

Raber-Durlacher et al.,55 addressed the pathogenesis of

periodontal disease and the possibility of transmission of

systemic subgingival microorganisms in patients with cancer
treated with chemotherapy. Those authors reported that oral

infections are larger problems, mainly because there is a

higher risk of infections spread from microorganisms of the

mouth during the neutropenia occurring after chemotherapy.

Thus, the inflamed periodontal tissues may act as a focus of

infection, bringing significant morbidity and, in some cases

can become life-threatening. Still, there is evidence that

gingivitis and periodontitis are associated with fever and

sepsis in these patients, because the ulcerated epithelium of

periodontal pockets may serve as a route of entry of

microorganisms into the bloodstream, and the propagation

of systemic endotoxins and other inflammatory mediators.

Jewtuchowicz et al.56 identified different species of yeasts

using conventional mycological methods and specific poly-

merase chain reaction (PCR) assays from samples at sites of

periodontal disease isolated from immunocompromised

patients, such as those with advanced HIV infection. Amongst

76 fungal organisms isolated, C. dubliniensis comprised 10.5% of

total, which corresponded to 4.4% of patients studied. C.

albicans was the most frequently isolated species of yeast.

However, Sardi et al.9 detected some species of Candida,

using the PCR method, in higher quantities in diabetic patients

when compared with non-diabetic patients with chronic

periodontal disease. C. albicans were found in 57.3%, C.

dubliniensis in 75.6%, C. tropicalis in 15.85% and C. glabrata in

4.87% of the periodontal pockets of diabetic patients. For non-

diabetic patients, 19.17% and 13.69% of the periodontal sites

presented C. albicans and C. dubliniensis, respectively. C.

tropicalis and C. glabrata were not found in the periodontal

pocket of non-diabetic patients. Urzú a et al.57 analysed the

composition of the yeast microbiota present in the mucosal

and subgingival sites of healthy individuals and patients with

aggressive and chronic periodontitis, using phenotypic and

genotypic methods. Despite the varied profiles of the species

present in the mucosa of the three groups analysed, only C.

albicans and C. dubliniensis were capable of colonizing the

periodontal pockets in patients with chronic periodontitis,

whilst only C. albicans was identified in the subgingival sites of

healthy individuals and patients with aggressive periodontitis.

Periodontal conditions were studied in two cross-sectional

studies of adult, insulin-dependent diabetics and age- and

sex-matched controls. In one study, 154 diabetics and 77

control patients participated. In the other study, 82 diabetics

and 99 control patients took part. The number of individuals

exhibiting severe periodontal disease was superior in the

diabetic group than in the control group.58 However, a

relationship between diabetes mellitus, periodontal disease

and the presence of Candida spp was not found. Additionally,

the moderately increased glucose content of diabetic patients

did not result in higher mean numbers of C. albicans. Similar

results were obtained by Yuan et al.,53 who verified that there

were no significant differences in the prevalence of the some

microorganisms, including C. albicans, between the diabetic

and the non-diabetic groups.

Järvensivu et al.47 investigated the occurrence and extent of

penetration of C. albicans in periodontal tissues of patients

with chronic periodontitis in gingival tissue specimens

collected during periodontal surgery. These specimens were

examined by immunohistochemistry using specific antibodies

to C. albicans; the presence of hyphae penetrating the


a r c h i v e s o f o r a l b i o l o g y 5 6 ( 2 0 1 1 ) 9 5 1 – 9 5 9 955
periodontal tissue was observed. Those authors suggested

that an environmental change may have promoted the

germination of hyphae that have a greater capacity to adhere

to host tissues, and that the crevicular fluid and periodontal

pockets formed a favourable environment for germination of

these morphological structures. C. albicans could then play a

role in the infrastructure of the subgingival biofilm, and their

adherence to the periodontal tissues, since they are more

resistant to immune mechanisms that most microorganisms

present at that location.

Barros et al.49 studied Candida species in the periodontal

pockets of chronic periodontitis patients without systemic

changes; the most prevalent species was albicans with only one

isolate of C. dubliniensis.

Cuesta et al.59 analysed patients with periodontal disease

and found 25.6% of Candida species with C. albicans as the most

prevalent at 76.2%.

However, one of the factors related to a lack of response to

periodontal therapy is the failure to eliminate the reservoirs of

infectious organisms, or the appearance of superinfecting

pathogens such as Enterobacteriaceae, Pseudomonas sp., Staphy-

lococcus sp. and Candida species.60 The treatment of periodon-

tal disease includes SRP associated with proper oral hygiene. It

has been shown that these procedures are essential for

successful periodontal therapy, reducing pocket depth and

eliminating periodontal microbiota.60 However, some patients

may have negative responses to different therapeutic proce-

dures, so the use of antimicrobials is needed as an adjuvant

treatment SRP.17 The use of a broad-spectrum antibiotic, such

as tetracycline or metronidazole, as an aid in periodontal

treatment has also been a factor for the development of

superinfection by resistant bacteria and Candida ecies.18,19 The

use of an antifungal is needed but many of these Candida spp.

present in periodontal pockets are resistant to existing drugs,

necessitating the search for natural alternatives.56,61–64

2.2. Resistance of yeasts to synthetic antifungals

In the treatment of fungal infections, oral antifungal drugs are

administered. The most common antifungal medications are

the azoles. However, this treatment becomes quite limited due

to resistance problems and significant efficacy of drugs.

Currently, the therapeutic practice covers a limited number

of antifungals such as amphotericin B, fluconazole, itracona-

zole and more recently, voriconazole, although others also

show promising results, such as posaconazole, ravuconazole,

caspofungin and micafungin.65

The conventional treatment of periodontal disease is

usually effective, except in cases of proven resistance to

isolates. Some studies show the inefficiency of therapy

depending on the selection of populations of the genus

Candida. In these cases, the isolate of Candida spp. reports of

resistance or treatment failure are attributed to the difficult

access of antifungal drugs in these sites.60

In the dental practice, the most commonly used anti-

fungals are nystatin and fluconazole.63,65,66 Antifungal agents

such as amphotericin B, 5-fluorocytosine, voriconazole and

terbinafine are not usually employed in the treatment of oral

candidiasis; however, they also deserve attention. Although

these antifungals are available only for systemic use and are
recommended for the treatment of disseminated infections,

the determination of a minimum inhibitory concentration

with respect to isolates from the oral cavity of patients with

immunosuppression is important, especially in cases of

periodontitis, for obtaining epidemiological data and the

possibility of the oral cavity being the original focus of

disseminated fungal infections.62,67

Waltimo et al.,64 whilst evaluating the antifungal suscepti-

bility amongst isolates of C. albicans in periodontal pockets,

showed that 100% of these isolates were sensitive to

amphotericin B and 5-flucytosine.

However, sensitivity to azole antifungals was shown to be

variable. This fact corroborates with recent data that indicates

an increasing azole resistance amongst Candida species,

suggesting that the oral cavity, seems to be a major factor

in the increased frequency of C. albicans and other non-

albicans.68–70 Dumitru et al.71 studied isolates of C. albicans

under conditions of hypoxia and found strains resistant to

amphotericin B and four azole antifungal classes, thus

concluding that these anaerobic yeasts were more resistant

to antifungal drugs; thus explaining the resistance of biofilms

of C. albicans to several antifungal drugs. Perkholfer et al.72

evaluated anidulafungin and voriconazole alone and in

combination against conidia and hyphae under conditions

of hypoxia in isolates of Aspergillus and observed that

anidulafungin exhibited excellent activity against conidia

and hyphae of Aspergillus sp. The reading of the MIC for

anidulafungin was optimal under hypoxic conditions. Results

presented by Furletti et al.61 showed that C. albicans isolated

from periodontal pockets were resistant to amphotericin B

and sensitive to fluconazole. This result shows that there may

be difficulty in the infusion of the drug in the periodontal space

or resistant strains may be due to overexpression of efflux

genes.

Jewtuchowicz et al.56 studied isolates of C. albicans and C.

dubliniensis in patients with and without periodontitis and

consistently found that only one isolate was resistant to

fluconazole and voriconazole. C. albicans appears to be

contributing to bacterial biofilm formation on these structures

and hindering the penetration of certain antimicrobial

drugs.47 Still, for these studies, C. albicans was found typically

in the outer layers of the biofilm, and seemed to act according

as expected, as a barrier protecting the microorganisms of the

deep interior from the action of immune mechanisms, aiding

the resistance of the subgingival microbiota in the face of host

defences, and contributing to the persistence of inflammation

in adjacent tissues. Biofilms of C. albicans were highly resistant

to the clinical action of antifungal and antimicrobial agents,

including amphotericin B, chlorhexidine, nystatin and flucon-

azole.72 In that work, it was demonstrated that as the C.

albicans biofilms matured, there was concurrent acquisition

and increased resistance of yeast cells in relation to the

antimicrobials. The prophylactic use of fluconazole in low

doses has been recommended for preventing fungal infections

in immunocompromised patients. However, this has led to the

selection of yeast microbes that are resistant to this antifungal

agent, causing this resistance to appear in non-albicans

species such as C. glabrata and C. krusei, in addition to having

the sensitive C albicans be replaced by another of the same

species that is fluconazole-resistant and to become resistant


a r c h i v e s o f o r a l b i o l o g y 5 6 ( 2 0 1 1 ) 9 5 1 – 9 5 9956
to fluconazole during treatment.73,74 From a clinical stand-

point, the most important feature of biofilm growth is the

resistance to antimicrobial agents exhibited by organisms.75,76

Studies have shown that biofilms formed by Candida species

were more resistant to major antifungal agents used in the

clinic, such as amphotericin B, fluconazole, itraconazole, and

ketoconazole.77 New azoles, like voriconazole and ravucona-

zol, were also ineffective against biofilms.52,78–81 The intrinsic

resistance of Candida species biofilms to fluconazole, an agent

commonly used for antifungal treatment due to greater

resistance to amphotericin B, has been reported.81 However,

therapeutic levels of echinocandins may inhibit the metabolic

activities in C. parapsilosis biofilms,82–84 whilst lipid formula-

tions of amphotericin B also showed activity against the same

biofilms.77,81 The progression of drug resistance by Candida

biofilms has been associated with the parallel increase of the

maturation process.85 Furthermore, some researchers have

also shown that biofilms of Candida developed statically with

the presence of minimal matrix and exhibited the same level

of resistance to drugs (fluconazole and amphotericin B) as the

cells grown in a lab and exhibiting large amounts of matrix.86

Therefore, there are many controversies regarding the

mechanisms of resistance to antifungal agents. In addition

to the reduced sensitivity described by some authors in

periodontal disease, it is believed that the presence of C.

albicans in subgingival sites allows the formation of biofilms,

which could explain the resistance to antifungal therapy.

Several molecular mechanisms of resistance to antifungal

agents in C. albicans have been described, highlighting in

particular: the increased efflux of antifungal agents due to the

over expression of efflux genes, CDR1, CDR2 (the family of ABC

membrane transport proteins – ATP Binding Cassette)87 and

MDR1 (family protein Major Facilitator); the amino acid

substitutions in Erg11p enzyme (lanosterol 14-a desmetilase),

encoded by the gene ERG11. This gene in turn can be expressed

in cells with super changes in several of the biosynthetic

pathways for ergosterol, as no formation of the toxic

metabolite 14-a metilergosta-8, 24-diene-3 b, a 6-diol metabo-

lite from 14 a-metilfecosterol due to changes in the ERG3

gene.87–89 Considering it essential to understand how genes

are regulated, CDR1 and CDR2 and other genes are often co-

regulated and are over-expressed simultaneously, therefore it

is believed that there is a chance of mutations in genes

regulating this expression.90

2.3. Search of new natural antifungal drugs

A search for new antifungal agents and the characterization of

new targets which are more appropriate and efficient,

including the emergence of resistant strains, has been

proposed.91 An ideal antifungal agent should have broad-

spectrum antifungal activity and would not cause toxicity to

the host.62 Plants are good options for obtaining a wide variety

of drugs.21 Plants have been used in medicine for a long time

and are extensively used in folk medicine because they

represent an economic alternative, are easily accessible and

would be applicable to various pathologies.23 These constitute

an excellent alternative for substances that can be used in the

formulation of new antifungal agents.24 The antifungal

compounds of the plants assayed are not well known;
however, the presence of flavonoids and terpenes and a

certain degree of lipophilicity might determine toxicity by the

interactions with the membrane constituents and their

arrangement. Since plants produce a variety of compounds

with antimicrobial properties, it is expected that screening

programmes for some under-represented targets, such as

antifungal activity, may yield candidate compounds for

developing new antimicrobial drugs.92 In addition, it is

expected that plant compounds showing targets sites other

than those currently used by antibiotics will be active against

drug-resistant microbial pathogens.93 Some extracts and

essential oils of medicinal plants with antifungal activity

were investigated by various researchers. Hofling et al.94

observed activity against strains of C. albicans (CBS-562), C.

dubliniensis (CBS-7987), C. parapsilosis (CBS-604), C. tropicalis

(CBS-94), C. guilliermondii (CBS-566), C. utilis (CBS-5609), C. krusei

(CBS-573), C. lusitaniae (B-060), C. glabrata (B-07), and C. rugosa

(B-12) with the extracts of Mentha piperita, Arrabidaea chica,

Rosmarinus officinalis, Tabebuia avellanedae, Syzygium cumini and

Punica granatum.

The yeast C. albicans, frequently associated with infections

in HIV (+) patients, was the most sensitive amongst all tested

microorganisms. Lippia sidoides essential oil showed an

appreciable amount of monoterpenes, a therapeutical poten-

tial that should not be ignored, and its phenolic compounds

(thymol and carvacrol) showed activity against oral patho-

gens.92 Duarte et al.95 investigated the essential oils and

ethanol extracts obtained from 35 medicinal plants for activity

against C. albicans and found that 13 of them showed

antifungal activity. The oil of Achillea millefolium, Mikania

glomerata and Stachys byzantina all had a strong activity against

C. albicans, whilst Aloysia triphylla, Anthemis nobilis, Cymbopogon

martini, Cyperus articulates, Cyperus rotundus, Lippia alba, Mentha

arvensis and M. piperita presented moderate activity. The

essential oil obtained from the leaves of Coriandrum sativum

showed antifungal activity against established biofilm and

planktonic cells of C. albicans isolated from periodontal

pockets.96 More et al.97 isolated C. albicans from periodontal

pockets and found that six of these (Annona senegalensis,

Englerophytum magalismontanum, Dicerocarym senecioides, Euclea

divinorum, Euclea natalensis, Solanum panduriforme and Parinari

curatellifolia), had an action on these organisms. Additionally,

they also indicated that eight species of plants from South

Africa had action against bacteria periodontipathogenic and

cytotoxicity in Vero cell lines. Scorzoni et al.22 indicated that

there was contact of the crude extracts derived from EtOAc

and EtOH Kielmeyera rubriflora, in addition to the commercial

drug fluconazole, against yeast C. krusei and subsequent

protein analysis by two dimensional electrophoresis. Several

changes in protein expression were observed and both

extracts were effective in inhibiting the expression of protein

C. krusei, suggesting the existence of specific targets. In

another study of Pterogyne nitens (Fabaceae), the antifungal

activity of compounds from the plant and the substance

purified pedalitina was able to inhibit the adhesion of

Cryptococcus neoformans to lung epithelial cells with similar

efficiency to conventional drugs. Additionally, changes were

observed in protein expression after contact with this

substance, showing a considerable activity in this yeast

proteome.98 Gregio et al.99 evaluated the antimicrobial


a r c h i v e s o f o r a l b i o l o g y 5 6 ( 2 0 1 1 ) 9 5 1 – 9 5 9 957
properties of ginger extract against microorganisms found in

the oral cavity, such as Streptococcus mutans, Staphylococcus

aureus, E. coli and C. albicans, and observed action from the

extracts glycol and hidroalccolicos; however, the ethanol

extract and essential oil showed no antimicrobial activity

effective under some experimental conditions.

3. Conclusion

The interest in natural medicine has increased remarkably in

recent years as a result of side effects of conventional drugs, as

well as the emergence of antimicrobial resistance to available

drugs. Plants and their derivatives are therefore an important

alternative in the search for new drugs. For the development of

specific drugs for the treatment of periodontal disease caused

by coinfection by C. albicans, further studies should be

conducted on the proteins participating in biosynthetic

pathways of vital components for this agent.

Funding: None.

Competing interests: None declared.

Ethical approval: Not required.

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	New antimicrobial therapies used against fungi present in subgingival sites-A brief review
	Introduction
	Colonization of the oral cavity by Candida spp.
	Candida spp. in subgingival sites
	Resistance of yeasts to synthetic antifungals
	Search of new natural antifungal drugs

	Conclusion
	References