**Table 2.**

**35**

**Table 3.**

*Prevention and Control of American Foulbrood in South America with Essential Oils: Review*

Moderately toxic (>2 μg EO/bee)

Slightly toxic (24 h-LD50 = 15.94 μg b.e./bee)

Virtually non-toxic (24 h-LD50 = 122 μg b.e./bee)

Toxic/non-toxic the nanoparticles of *M. alternifolia*

Moderately toxic (≥ 3 μg EO/bee)

Moderately toxic (>8 μg EO/bee)

Not determined 1.19, 2.37, 4.75,

Virtually non-toxic 2000, 4000,

Non-toxic 2.5, 5, 10 and

Non-toxic 2.5, 5, 10 and

**tested**

Non-toxic 25% (v/v) [27]

Non-toxic 10% (v/v) [50]

Slightly toxic [50]

Non-toxic 1.56% (v/v) [27]

1, 2, 4, 8, 16 and 32 μg EO/ bee

0.19, 0.37, 0.75, 1.50, 3.0 and 6.0 μg b.e./bee

9.50, 19.0 and 28.0 μg b.e./ bee

0.625, 1.25, 2.5, 5.0, 10.0 and 20.0 μg b.e./bee

8000 and 16,000 μg/ml

20 ml per cage of EO

3, 6, 12, 24, 48 and 96 μg EO/ bee

20 μl per cage of EO

2, 4, 8, 16, 32 and 64 μg EO/ bee

Non-toxic 5% (w/v) [48]

6.25% (w/v) [49]

Non-toxic 400 μg/ml [32]

**References**

[47]

[47]

[47]

[47]

[46]

[44]

[47]

[51]

[47]

**Essential oil Technique Toxicity Amount** 

procedure

Complete exposure

*In-vivo against larva*

procedure

administration

Systemic administration

Systemic administration

Systemic administration

Systemic administration

exposure

administration

procedure

administration

exposure

procedure

administration

*DOI: http://dx.doi.org/10.5772/intechopen.85776*

*Carapa guaianensis* Spraying

*Copaifera officinalis* Spraying

*Cymbopogon citratus* Systemic

*Carapa guaianensis* nanoemulsion

*Cymbopogon citratus* + *Thymus vulgaris* (20:80, v/v)

*Cymbopogon citratus* + *Thymus vulgaris* + *Satureja hortensis* + *Origanum vulgare* + *Ocimum basilicum* (5:11:21:26:37, v/v/v/v/v)

*Cymbopogon citratus* + *Thymus vulgaris* + *Ocimum basilicum* (10:20:70,

*Cinnamomum zeylanicum*

*Eucalyptus globulus* Complete

*Eugenia* spp. Systemic

*Melaleuca alternifolia* Spraying

*Origanum vulgare* Systemic

*Rosmarinus officinalis* Complete

*Tagetes minuta* Spraying

*Thymus vulgaris* Systemic

*Essential oils toxicity assays on Apis mellifera.*

v/v/v)

*Essential oils for the in vitro Paenibacillus larvae control.*

*Prevention and Control of American Foulbrood in South America with Essential Oils: Review DOI: http://dx.doi.org/10.5772/intechopen.85776*


#### **Table 3.**

*Essential oils toxicity assays on Apis mellifera.*

*Beekeeping - New Challenges*

[40]

[48]

[41]

[42]

**34**

**Essential oil** *Tagetes minuta*

**Technique** Agar diffusion

Agar dilution Agar dilution

Broth

Inhibitory Inhibitory

macrodilution

Broth

Inhibitory

macrodilution

Agar diffusion

Broth

Inhibitory

3200–0.78 μg/

137.0 ± 12.2 μg/ml

700–800 mg/L 700–800 mg/L

950 mg/L

[42]

[36]

850 mg/L

[42]

224.8 ± 25.6 μg/ml

[36]

ml

macrodilution

Broth

Inhibitory

microdilution

Broth

Inhibitory

microdilution

Agar diffusion

Inhibitory

10 ml

Inhibitory

10 μl

Thymol (component of *Thymus vulgaris*)

*Trachyspermum ammi* L.

*Verbena officinalis* L.

*Wedelia glauca* Ortega

*Zingiber officinale* Rosc.

*aMIC, Minimal Inhibitory Concentration.*

*bMBC, Minimal Bactericidal Concentration.*

**Table 2.**

*Essential oils for the in vitro Paenibacillus larvae control.*

Inhibitory

Inhibitory

500–650 μg/ml

700–800 μl/L 900–1000 mg/L

833 mg/L 100–133 μg/ml

133 μg/ml

[26]

[36]

Inhibitory

10 μl

**Activity**

**Amount** 

**MICa**

**MBCb**

**References**

[34]

**tested**

*Pelargonium graveolens* L., were able to inhibit the growth of *P. larvae* by the agar diffusion technique [38–45].

EOs from *Cymbopogon citratus*, *Cinnamomum aromaticum*, *Citrus reticulata* var. madurensis, *Citrus paradisi*, *Heterothalamus alienus*, *Melaleuca alternifolia*, *Mentha piperita*, *Origanum majorana*, *Origanum vulgare*, *Salvia sclarea*, *Syzygium aromaticum*, *Tagetes minuta*, *Thymus vulgaris*, as well as the mixtures of *Cymbopogon citratus* and *Thymus vulgaris* EOs (20:80, v/v), and *Cymbopogon citratus*, *Thymus vulgaris*, *Satureja hortensis*, *Origanum vulgare*, and *Ocimum basilicum* EOs (5:11:21:26:37, v/v/v/v/v) showed antibacterial activity against *P. larvae* [44, 46, 47].

EOs from *Citrus sinensis*, *Cinnamomum* spp., *Eugenia* spp., *Thymus vulgaris*, *Verberna* spp., *Acantholippia seriphioides*, *Cinnamomum zeylanicum*, *Heterothalamus alienus* Spreng., *Pimpinella anisum*, *Foeniculum vulgare*, and *Eucalyptus globulosus*, and the mixture of *Thymus vulgaris* EO, thymol and *Cinnamomum zeylanicum* EO (62.5:25:12.5, v/v/v) exhibited antibacterial activity against *P. larvae* by the broth macrodilution technique [40, 48–52].

#### *1.1.2 Toxicity assays on Apis mellifera*

*Citrus sinensis*, *Cinnamomum* spp., *Cinnamomum zeylanicum*, *Cuminum cyminum*, *Eugenia* spp., *Thymus vulgaris*, and *Verbena* spp. EOs were non-toxic for adult honey bees when they were fed with candy and the EO at different concentrations by systemic administration [40, 53]. *Cymbopogon citratus*, *Thymus vulgaris* and *Ocimum basilicum* EOs, as well as *Cymbopogon citratus* and *Thymus vulgaris* EO mixture (50:50, v/v) were moderately toxic to adult honey bees. However, the *Cymbopogon citratus*, *Thymus vulgaris* and *Coriandrum sativum* EO mixture (33.3:33.3:33.3, v/v/v) presented negative mortality curves, meaning that there was less mortality at high doses. This fact disclosed that bees did not consume candy with high quantities of *Coriandrum sativum* EO [54]. When a solution containing a certain amount of EO was sprayed over a group of honey bees, *Tagetes minuta*, *Carapa guianensis* and *Carapa officinalis* EOs resulted to be non-toxic for adult bees [27, 55]; whereas *Melaleuca alternifolia* EO caused the death of the bees after 7 days of treatment. Nevertheless, the use of nanoparticles of *Melaleuca alternifolia* EO did not produce any toxic effect on honey bees [56]. *Eucalyptus globosus* and *Rosmarinus officinalis* EOs and the nanoemulsion of *Carapa officinalis* EO were not toxic for adult worker honey bees when they were completely exposed to the EO, that is, bees were in contact with the EO and ingested the EO [50, 57, 58]. The nanoemulsion of *Carapa guianensis* EO exhibited a toxic effect for larvae and adult honey bees, whereas the nanoemulsion of *Carapa officinalis* EO, a low toxic effect on larvae [57].

#### *1.1.3 Mechanism of action of essential oils on P. larvae*

Different mechanisms of action of EOs on bacteria have been reported, among others: degradation of the cell wall, affecting the cell morphology and damaging the cytoplasmic membrane; damage of membrane protein, disruption of cell wall, leading to leakage of the cell contents, reduction of proton motive force, reduction of intracellular ATP pool, via decreasing ATP synthesis; inhibition of quorum sensing and alteration of cell division [59]. The alteration of the membrane permeability can be detected by the crystal violet assay [35] and the determination of the released UV-absorbing material assays [60]. The crystal violet assay is based on the fact that the compound enters easily when the cell membrane is defective. The released of UV-absorbing material assays is based on the fact that EOs can disrupt the cell membrane leading to a leakage of the cell content which is measured in the UV spectrum. The relationship between the chemical composition of EOs

**37**

*Prevention and Control of American Foulbrood in South America with Essential Oils: Review*

and their antimicrobial mode of action against *P. larvae* has not been systematically researched so far. EOs are complex mixtures of low molecular weight volatile constituents biosynthesized by plants, which mainly include two biosynthetically related groups, i.e., terpenes and terpenoids, and aromatic and aliphatic constituents [61]. Most antimicrobial compounds are constitutively expressed by the plants, but others are synthesized as mechanism of defense in response to pathogens [59, 62]. Pellegrini et al. [62] demonstrated that the essential oils of *Acantholippia seriphioides*, *Aloysia polystachia*, *Buddleja globosa*, *Lippia turbinata*, *Minthostachys mollis*, *Schinus molle* and *Solidago chilensis* permeabilized and altered the cell membrane and the cytoplasmic membrane of *P. larvae* causing the leakage

*1.1.4 Anti-quorum sensing and antimicrobial activity of essential oils*

Antúnez et al. (2010) [70] determined that during the division *P. larvae* produces and secretes different proteins with proteolytic activity, such as metalloproteases and enolase, these proteins are secreted and remain on the surface of the spores, producing a response in the immune system of *A. mellifera* and are probably

In recent years, the detection of quorum sensing (QS) detection signals in bacteria has added a new dimension to study the infection process. Through QS, bacteria depending on population density can activate specific genes [63–66]. The QS can regulate the expression of virulence factors, bioluminescence, sporulation, biofilm formation and conjugation [67–69]. Many bacteria coordinate the expression of multiple virulence factors, such as toxins, active redox compounds, siderophores, exoproteases, lipases and biofilm formation, thus maximizing the chances of infec-

The QS signals occur while the bacterial population grows until it reaches a threshold concentration perceived by the bacteria and results in the activation or repression of specific genes. The accumulation of a stimulant amount of such molecules can occur only when a specific number of cells, known as a quorum, is present. These self-inducing molecules have been identified as acylated homoserine lactones in gram-negative and oligopeptide bacteria, thiolactone/lactone peptide, lanthionines, isoprenyl groups [65] and even acylated homoserine lactones in grampositive bacteria [72, 73]. Similar signaling mechanisms have not yet been demonstrated in *P. larvae*. It is possible that larval infection by *P. larvae* is influenced by phenotypes regulated by QS, such as proteases exported by bacteria to their environment. The concept of QS has encouraged the development of a new nonantibiotic antibacterial therapy through the use of QS inhibitor compounds [74, 75]. The increase in resistance to multiple drugs of the bacteria against traditional medicines drastically reduces the efficacy of conventional antibiotics. This multiple resistance is now recognized as a global problem [76]. Therefore, it is necessary to develop a new therapeutic strategy to prevent this type of multidrugging. A promising mechanism is to block cell-to-cell communication, establishing a strategy called quorum extinction [77]. Although traditional antimicrobial agents cause cell death of the pathogen, the use of systems that alter the QS sensors adopts a less aggressive strategy [78]. There are several sources of QS inhibitors (quorum quenchers), but so far the most diverse and abundant are derived from natural sources such as algae and plants. There are cases of QS inhibitors in bacteria, fungi, algae, bryozoans, corals, sponges [79], plant extracts [80], essential oils [42], compounds isolated

Essential oils extracted from plants, such as *Cymbopogon citratus*, *Cymbopogon martini*, *Rosmarinus officinalis*, *Mentha piperita*, *Pelargonium odoratissimum* and

*DOI: http://dx.doi.org/10.5772/intechopen.85776*

of cytoplasmic constituents.

involved in the degradation of larval tissue.

tion and allowing better propagation [70, 71].

from bacteria [81] and furanones, among others.

*Prevention and Control of American Foulbrood in South America with Essential Oils: Review DOI: http://dx.doi.org/10.5772/intechopen.85776*

and their antimicrobial mode of action against *P. larvae* has not been systematically researched so far. EOs are complex mixtures of low molecular weight volatile constituents biosynthesized by plants, which mainly include two biosynthetically related groups, i.e., terpenes and terpenoids, and aromatic and aliphatic constituents [61]. Most antimicrobial compounds are constitutively expressed by the plants, but others are synthesized as mechanism of defense in response to pathogens [59, 62]. Pellegrini et al. [62] demonstrated that the essential oils of *Acantholippia seriphioides*, *Aloysia polystachia*, *Buddleja globosa*, *Lippia turbinata*, *Minthostachys mollis*, *Schinus molle* and *Solidago chilensis* permeabilized and altered the cell membrane and the cytoplasmic membrane of *P. larvae* causing the leakage of cytoplasmic constituents.

#### *1.1.4 Anti-quorum sensing and antimicrobial activity of essential oils*

Antúnez et al. (2010) [70] determined that during the division *P. larvae* produces and secretes different proteins with proteolytic activity, such as metalloproteases and enolase, these proteins are secreted and remain on the surface of the spores, producing a response in the immune system of *A. mellifera* and are probably involved in the degradation of larval tissue.

In recent years, the detection of quorum sensing (QS) detection signals in bacteria has added a new dimension to study the infection process. Through QS, bacteria depending on population density can activate specific genes [63–66]. The QS can regulate the expression of virulence factors, bioluminescence, sporulation, biofilm formation and conjugation [67–69]. Many bacteria coordinate the expression of multiple virulence factors, such as toxins, active redox compounds, siderophores, exoproteases, lipases and biofilm formation, thus maximizing the chances of infection and allowing better propagation [70, 71].

The QS signals occur while the bacterial population grows until it reaches a threshold concentration perceived by the bacteria and results in the activation or repression of specific genes. The accumulation of a stimulant amount of such molecules can occur only when a specific number of cells, known as a quorum, is present. These self-inducing molecules have been identified as acylated homoserine lactones in gram-negative and oligopeptide bacteria, thiolactone/lactone peptide, lanthionines, isoprenyl groups [65] and even acylated homoserine lactones in grampositive bacteria [72, 73]. Similar signaling mechanisms have not yet been demonstrated in *P. larvae*. It is possible that larval infection by *P. larvae* is influenced by phenotypes regulated by QS, such as proteases exported by bacteria to their environment. The concept of QS has encouraged the development of a new nonantibiotic antibacterial therapy through the use of QS inhibitor compounds [74, 75].

The increase in resistance to multiple drugs of the bacteria against traditional medicines drastically reduces the efficacy of conventional antibiotics. This multiple resistance is now recognized as a global problem [76]. Therefore, it is necessary to develop a new therapeutic strategy to prevent this type of multidrugging. A promising mechanism is to block cell-to-cell communication, establishing a strategy called quorum extinction [77]. Although traditional antimicrobial agents cause cell death of the pathogen, the use of systems that alter the QS sensors adopts a less aggressive strategy [78]. There are several sources of QS inhibitors (quorum quenchers), but so far the most diverse and abundant are derived from natural sources such as algae and plants. There are cases of QS inhibitors in bacteria, fungi, algae, bryozoans, corals, sponges [79], plant extracts [80], essential oils [42], compounds isolated from bacteria [81] and furanones, among others.

Essential oils extracted from plants, such as *Cymbopogon citratus*, *Cymbopogon martini*, *Rosmarinus officinalis*, *Mentha piperita*, *Pelargonium odoratissimum* and

*Beekeeping - New Challenges*

diffusion technique [38–45].

macrodilution technique [40, 48–52].

*1.1.2 Toxicity assays on Apis mellifera*

*1.1.3 Mechanism of action of essential oils on P. larvae*

*Pelargonium graveolens* L., were able to inhibit the growth of *P. larvae* by the agar

v/v/v/v/v) showed antibacterial activity against *P. larvae* [44, 46, 47].

EOs from *Cymbopogon citratus*, *Cinnamomum aromaticum*, *Citrus reticulata* var. madurensis, *Citrus paradisi*, *Heterothalamus alienus*, *Melaleuca alternifolia*, *Mentha piperita*, *Origanum majorana*, *Origanum vulgare*, *Salvia sclarea*, *Syzygium aromaticum*, *Tagetes minuta*, *Thymus vulgaris*, as well as the mixtures of *Cymbopogon citratus* and *Thymus vulgaris* EOs (20:80, v/v), and *Cymbopogon citratus*, *Thymus vulgaris*, *Satureja hortensis*, *Origanum vulgare*, and *Ocimum basilicum* EOs (5:11:21:26:37,

EOs from *Citrus sinensis*, *Cinnamomum* spp., *Eugenia* spp., *Thymus vulgaris*, *Verberna* spp., *Acantholippia seriphioides*, *Cinnamomum zeylanicum*, *Heterothalamus alienus* Spreng., *Pimpinella anisum*, *Foeniculum vulgare*, and *Eucalyptus globulosus*, and the mixture of *Thymus vulgaris* EO, thymol and *Cinnamomum zeylanicum* EO (62.5:25:12.5, v/v/v) exhibited antibacterial activity against *P. larvae* by the broth

*Citrus sinensis*, *Cinnamomum* spp., *Cinnamomum zeylanicum*, *Cuminum cyminum*, *Eugenia* spp., *Thymus vulgaris*, and *Verbena* spp. EOs were non-toxic for adult honey bees when they were fed with candy and the EO at different concentrations by systemic administration [40, 53]. *Cymbopogon citratus*, *Thymus vulgaris* and *Ocimum basilicum* EOs, as well as *Cymbopogon citratus* and *Thymus vulgaris* EO mixture (50:50, v/v) were moderately toxic to adult honey bees. However, the *Cymbopogon citratus*, *Thymus vulgaris* and *Coriandrum sativum* EO mixture (33.3:33.3:33.3, v/v/v) presented negative mortality curves, meaning that there was less mortality at high doses. This fact disclosed that bees did not consume candy with high quantities of *Coriandrum sativum* EO [54]. When a solution containing a certain amount of EO was sprayed over a group of honey bees, *Tagetes minuta*, *Carapa guianensis* and *Carapa officinalis* EOs resulted to be non-toxic for adult bees [27, 55]; whereas *Melaleuca alternifolia* EO caused the death of the bees after 7 days of treatment. Nevertheless, the use of nanoparticles of *Melaleuca alternifolia* EO did not produce any toxic effect on honey bees [56]. *Eucalyptus globosus* and *Rosmarinus officinalis* EOs and the nanoemulsion of *Carapa officinalis* EO were not toxic for adult worker honey bees when they were completely exposed to the EO, that is, bees were in contact with the EO and ingested the EO [50, 57, 58]. The nanoemulsion of *Carapa guianensis* EO exhibited a toxic effect for larvae and adult honey bees, whereas the nanoemulsion of *Carapa officinalis* EO, a low toxic effect on larvae [57].

Different mechanisms of action of EOs on bacteria have been reported, among others: degradation of the cell wall, affecting the cell morphology and damaging the cytoplasmic membrane; damage of membrane protein, disruption of cell wall, leading to leakage of the cell contents, reduction of proton motive force, reduction of intracellular ATP pool, via decreasing ATP synthesis; inhibition of quorum sensing and alteration of cell division [59]. The alteration of the membrane permeability can be detected by the crystal violet assay [35] and the determination of the released UV-absorbing material assays [60]. The crystal violet assay is based on the fact that the compound enters easily when the cell membrane is defective. The released of UV-absorbing material assays is based on the fact that EOs can disrupt the cell membrane leading to a leakage of the cell content which is measured in the UV spectrum. The relationship between the chemical composition of EOs

**36**

*Negundo vitex*, and different products, such as citral, geraniol, thymol and the linalool, have been used to evaluate its protease inhibitory activity, constituting one of the virulence factors of bacteria that can be regulated by QS [82].

Pellegrini et al. [62] propose that the EO will act by inhibiting the production of proteases, inhibiting its transportation and secretion, inhibiting the detection of quorum or avoiding the loading of proteases. All extracellular bacterial proteases are synthesized as an inactive pre-proenzyme consisting of a signal peptide, a prosequence and a maturity sequence. The peptide functions as a signal for the translocation of the pre-proenzyme to the membrane. The pre-proenzyme is processed in the proenzyme by the peptidase signal. The accusation acts as a molecular chaperone that leads to a self-cleavage of the peptide bond that links the pro and mature sequences [83]. The EOs acted at some point in this regulatory mechanism. The inhibition of larval proteases by EO could be a form of therapeutic intervention; the blocking of bacterial virulence factors does not destroy or inhibit the growth of pathogenic bacteria. It is expected that this strategy will generate little pressure on the selection of bacteria and, therefore, could diminish the appearance of bacterial resistance and avoid the interruption of the microbiota of benefits in urticaria. In future investigations, it will be interesting to isolate and characterize automatically the potential autoinductors of *P. larvae* and study their relationship with protease regulation. EOs studies are promising to use EOs in hives with symptoms of Foulbrood for the control of damage caused by *P. larvae*.
