Open access peer-reviewed chapter

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

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Sandra Rosa Fuselli, Pablo Gimenez Martinez, Giselle Fuentes, Rosa María Alonso-Salces and Matías Maggi

Submitted: April 23rd, 2018 Reviewed: March 11th, 2019 Published: August 8th, 2019

DOI: 10.5772/intechopen.85776

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American foulbrood (AFB) is the most severe bacterial disease that affects honey bees, having a nearly cosmopolitan distribution. AFB’s causative agent is Paenibacillus larvae. AFB kills infected honey bee larvae; however, it eventually leads to the collapse of the entire colony when left untreated. The infection takes place by the ingestion of the spores with the food provided by adult worker bees to the larvae. In South America (SA) the disease was first described in 1989 in Argentina, constituting the first sanitary challenge for beekeepers to overcome. Prevention and control measures of AFB in SA countries generally include vigilance for early diagnosis, isolation of apiaries with cases of AFB, and multiplication of healthy colonies with hygienic queens, among others. The extensive use of tetracycline hydrochloride in Argentina has led to the development of resistant P. larvae isolates. In this context, the development of alternative and effective methods for the control and prevention of AFB disease is crucial. Currently, alternative strategies for the prevention and treatment of AFB are being studied, mainly based on essential oils.


  • Paenibacillus larvae
  • essential oils
  • quorum sensing
  • American foulbrood
  • Apis mellifera

1. Introduction

Along with wild bees, honeybees are the most important crop pollinators [1, 2]. Apis mellifera pollinates 77% of the plants responsible for producing food resources which sustain the global human population [1]. Since 1998, individual beekeepers have reported the unusual weakening and mortality of colonies, particularly in France, Belgium, Switzerland, Germany, the United Kingdom, the Netherlands, Italy, Spain, and North America [3, 4]. Most scientists agree that there is no single explanation for the extensive colony losses, but that interactions between different stressors are likely involved [5].

American foulbrood (AFB) is the most severe bacterial disease that affects honey bees, having a nearly cosmopolitan distribution (Figure 1) [6]. AFB only kills infected honey bee larvae; however, it eventually leads to the collapse of the entire colony when left untreated. AFB is considered to be very contagious; therefore, it is a notifiable disease in most countries [7]. AFB’s causative agent is Paenibacillus larvae, which is a flagellated gram-positive bacterium, whose main characteristic is the formation of highly resistant endospores. This pathogen affects the breeding during the larval or pupal stages [8]; its spores being the infectious form. Honey bee larvae are more susceptible to infection during the first 36 h after egg hatching [9], indeed only 10 spores are required to make a larva of less than 24 h old ill [10]. However, at later larval developmental stages, spore doses needed to successfully infect a larva are too high to occur under natural conditions [11]. The infection takes place by the ingestion of the spores with the food provided by adult worker bees (nurses) to the larvae [12]. The spores after germinating in the midgut of the larvae proliferate for several days. After this, P. larvae reaches the peritrophic matrix, penetrates the epidermal cells, produces septicemia causing death of the larva. Finally, dead larvae are digested by vegetative bacterial cells and converted to dry flakes containing millions of spores of P. larvae [12, 13]. The most evident symptoms of AFB are the irregular coating of the offspring, which show cells with cap and uncovered irregularly dispersed through the frames of the offspring; dark, sunken, and often perforated caps emitting a characteristic AFB odor; remnants of brown glue from the dead larvae forming a characteristic cord thread when removed with a wooden stick or an inlay; and a hard scale of larval residues at the bottom of the cell. The traditional diagnosis is made based on the observation of these clinical symptoms in the hive and in the microbial culture of material from infected colonies [14].

Figure 1.

Distribution of American foulbrood. (

AFB was first described in South America (SA) in Argentina, in 1989, constituting the first sanitary challenge for beekeepers to overcome. It was hypothesized that the entrance of P. larvae, into the country was through bees imported from the USA [15]. AFB quickly spread to most important beekeeping centers of the country [16], with incidences as high as 30% in some geographic areas [17]. At least 30–45% of the colonies were lost due to AFB during those years (Eguaras, unpublished data). AFB was extended to Chile in 2002 and was controlled. New outbreaks were detected in 2005 in different regions [18] (Table 1).

ArgentinaRestricted distribution[15, 84]
BoliviaNo information available[84]
ColombiaDisease never reported[84]
EcuadorDisease never reported[84]
French GuianaDisease never reported[84]
PeruDisease not reported[84]
VenezuelaDisease never reported[84]

Table 1.

Distribution of P. larvae in South America.

In some countries the use of antibiotics, particularly tetracycline hydrochloride (OTC) [6, 12], is the most common method for prevention and treatment of infected colonies. However, in most European countries the use of antibiotics is banned, since their use is known to generate several problems including the presence of chemical residues in the beehive products (honey, pollen and wax), which eventually may even affect consumer health. Moreover, antibiotic application can affect life of bees and can increase the risk of occurrence of resistant strains [19]. To date, the presence of OTC resistant strains has been reported in Argentina, United States, Italy, New Zealand and United Kingdom [16, 20].

Prevention and control measures of AFB in SA countries generally include vigilance for early diagnosis, isolation of apiaries with cases of AFB, and multiplication of healthy colonies with hygienic queens, among others [21]. Brazilian, Chilean, and Uruguayan authorities specifically recommend the burning of colonies containing clinical signs of the disease in order to control the outbreaks [21]. The use of antibiotics in SA is not allowed, except in Argentina [18]. The extensive use of OTC in this country has led to the development of resistant P. larvae isolates [16], which is a major concern for Argentine beekeepers. In contrast, in Uruguay and Chile, where their use is not authorized, no resistant strains have been detected [22]. The endospore resistance of P. larvae is an important problem in the control and prevention of AFB because these individuals can survive for more than 35 years in honey and/or beekeeping material and is resistant to high temperatures as well as to the most used disinfectants [10]. Most treatments are based on the use of broad spectrum antibiotics, which, in most cases, have been used continuously and excessively. In fact, different antibiotics, such as sulfathiazole and OTC, are able to inhibit the growth of P. larvae, but its use and abuse during the last years has led to the appearance of resistant strains and residues that contaminate the products of the hive. For these reasons, the use of antibiotics for the treatment and prevention of AFB is prohibited in several countries, and the affected colonies must be destroyed [23].

In this context, the development of alternative and effective methods for the control and prevention of AFB disease is crucial. These methods may consider the evidence of the bacteria-resistant phenomenon and meet the strict EU standards, as well as current trends in green consumption [24, 25]. Currently, alternative strategies for the prevention and treatment of AFB are being studied, mainly based on essential oils [25, 26, 27], probiotics and propolis [28].

1.1 Essential oils

In light of developments in the scientific field, the medicinal properties of plants have received great interest due to their low toxicity, pharmacological activities and economic viability [29]. These studies have focused on the benefits of phytochemicals extracted from plants and their effect on human health. The additives naturally obtained from plants can be individual compounds, groups of compounds or essential oils (EOs). In recent times, there has been an increase in the interest of the food industry in natural compounds, either by direct addition or by its use in synergy with other compounds. It has been reported that the direct addition of essential oils and extracts of aromatic plants to food products exerts its antioxidant or antimicrobial effect [30].

Plants and other natural sources can provide a wide variety of complex and structurally diverse compounds. Plant extracts and essential oils have antifungal, antibacterial and antiviral properties and have been evaluated worldwide as potential sources of new antimicrobial compounds, agents that promote food preservation and alternatives to treat infectious diseases [31, 32]. It has been reported that essential oils possess significant antiseptic, antibacterial, antiviral, antioxidant, antiparasitic, antifungal, and insecticidal activities [33, 34]. Therefore, essential oils can serve as powerful tools to reduce bacterial resistance [33]. Oily aromatic liquids called essential oils (also called volatile oils) are obtained from plant materials (leaves, buds, fruits, flowers, herbs, branches, bark, wood, roots and seeds).

Being natural mixtures of very complex nature, the essential oils can consist of approximately 20–60 components at quite different concentrations. Essential oils are characterized by two or three main components that are present in fairly high concentrations (20–70%) compared to other components that are present in trace amounts. The amount of different components of essential oils varies between different parts of plants and different plant species since they are derived chemically from terpenes and their oxygenated derivatives, i.e., terpenoids which are esters of aromatic and aliphatic acid, and phenolic compounds. An important characteristic of essential oils and their components is their hydrophobicity, which allows them to interact with the lipids present in the cell membrane of bacteria and mitochondria, making them more permeable by altering their cellular structures. This eventually results in the death of bacterial cells due to the leakage of critical molecules and ions from the bacterial cell. Some compounds modulate drug resistance by targeting efflux mechanisms in several species of gram-negative bacteria [35]. An important function of essential oils in nature is the protection of plants by acting as antifungal, antibacterial, antiviral and insecticidal agents and also protection against herbivores by reducing the appetite of herbivores for plants with such properties. Health Services and Human Services Public Health Services have recognized essential oils as safe substances, and some of them contain compounds that can be used as antibacterial additives [33]. The efficacy of EOs has been reported in several studies against pathogens and food contaminants [36], suggesting their applications in the food industry [34, 37]. Several EOs have been evaluated for the in vitro and in vivo control of P. larvae (Table 2), as well as their acute oral toxicity to Apis mellifera (Table 3).

Essential oilTechniqueActivityAmount testedMICaMBCbReferences
Acantholippia seriphioides A. GrayBroth macrodilutionInhibitory236 mg/L[26]
Broth macrodilutionInhibitory300 mg/L[41]
Achyrocline satureioides Lam.Agar diffusionInhibitory10 μl[33]
Artemisia absinthium L.Broth microdilutionInhibitory416 mg/L647 mg/L[42]
Artemisia annua L.Broth microdilutionInhibitory402 mg/L624 mg/L[42]
Aloysia polystachia Griseb.Broth microdilutionInhibitory700–800 mg/L900 mg/L[42]
Carapa guianensis Aubl.Broth microdilutionInhibitory25% (v/v)[27]
Carum carvi L.Agar diffusionInhibitory5, 10 μl[35]
Chamomilla recutita L.Agar diffusionNon-inhibitory5, 10 μl[35]
Cinnamomum aromaticum L.Agar diffusionInhibitory10 μl[34]
Agar diffusionInhibitory0.015% (v/v) (strong activity)[34]
Cinnamomum camphora (L) J. Presl.Agar diffusionInhibitory10 μl[36]
Broth microdilutionInhibitory3200–0.78286.2 ± 27.9 μg/ml375.0 ± 34.8[36]
Cinnamomum glandulifera Nees.Agar dilutionInhibitory700 μg/ml[40]
Cinnamomum zeylanicum L.Agar diffusionInhibitory2 mg/ml[36, 59]
Broth macrodilutionInhibitory58–83 μg/ml108–112 μg/ml[42]
Inhibitory25–100 mg/L25–100 mg/L[45]
Inhibitory38–50 μg/ml[46]
Inhibitory25–67 μg/ml[6]
Cinnamomum zeylanicum + Thymus vulgaris L.Broth macrodilutionInhibitory66.6 μg/ml95.83 μg/ml[42]
Citrus limon L.Broth microdilutionInhibitory764 mg/L2293 mg/L[26]
Cinnamomum zeylanicum + Thymus vulgaris L.Broth macrodilutionInhibitory66.6 μg/ml95.83 μg/ml[42]
Citrus limon L.Broth microdilutionInhibitory764 mg/L2293 mg/L[42]
Citrus nobilis LourBroth microdilutionInhibitory815 mg/L2447 mg/L[42]
Citrus reticulata var. madurensis BlancoAgar diffusionInhibitory10 μl[34]
Agar dilutionInhibitory0.12–1.0% (v/v)[34]
Copaifera officinalis L.Broth microdilutionInhibitory1.56% (v/v)[27]
Copaifera officinalis L. nanoemulsionInhibitory0.39% (v/v)[50]
Cymbopogon citratus + Thymus vulgaris L.Agar dilutionInhibitory25–100 μg/ml[40]
C. citratus + T. vulgaris + Satureja hortensis L. + Origanum vulgare L. + Ocimum basilicum L.Agar dilutionInhibitory25–175 μg/ml[40]
C. citratus + T. vulgaris + O. basilicumAgar dilutionInhibitory50–350 μg/ml[40]
Cymbopogon martini Stapf.Broth microdilutionInhibitory1195 mg/L1208 mg/L[42]
Cymbopogon nardus L.Broth microdilutionInhibitory319 mg/L595 mg/L[42]
Daucus carota L.Agar diffusionInhibitory10 μl[36]
Broth microdilutionInhibitory3200–0.78 μg/ml412.8 ± 26.0 μg/ml589.6 ± 48.2 μg/ml[36]
Eucalyptus cinerea F. MuellAgar diffusionInhibitory10 μl[33]
Eugenia spp.Agar diffusionInhibitory[32]
Illicium verum Hook.f.Agar diffusionInhibitory10 μl[36]
Illicium verum Hook.f.Broth microdilutionInhibitory3200–0.78278.6 ± 21.2 μg/ml365.0 ± 32.1[36]
Lavandula officinalis L.Broth macrodilutionInhibitory350–400 μg/ml[45]
Laurus nobilis L.Broth microdilutionInhibitory1000 μg/ml[39]
Broth microdilutionInhibitory12,879 μg/ml[36]
Lepechinia floribunda Benth.Broth microdilutionInhibitory394 mg/L518 mg/L[26]
Lippia turbinata GrisebBroth macrodilutionInhibitory866 mg/L[26]
Litsea cubeba Pers.Agar diffusionInhibitory10 μl[36]
Litsea cubeba Pers.Broth microdilutionInhibitory3200–0.78 μg/ml85.0 ± 7.9 μg/ml186.0 ± 21.2 μg/ml[36]
Melaleuca alternifolia Maiden & BetcheAgar diffusionInhibitory10 μl[34]
Agar dilutionInhibitory0.015–0.12% (v/v) (strong activity)[34]
Broth microdilutionInhibitory1095 mg/L1187 mg/L[42]
Inhibitory0.18–1.5% (v/v)[27]
Inhibitory331 mg/L585 mg/L[42]
Broth microdilutionInhibitory1000–1800 μg/ml1600–2000[81]
Mentha arvensis L.Broth microdilutionInhibitory144.7 ± 17.2 μg/ml248.0 ± 23.4 μg/ml[36]
Mentha (hybrid)Broth microdilutionInhibitory600–700 μg/ml1000–1200 μg/ml[81]
Mentha rotundifolia L.Broth microdilutionInhibitory600–1000 μg/ml1600 ≥ 2000 μg/ml[81]
Mentha spicata L.Agar diffusionInhibitory10 μl[36]
Broth microdilutionInhibitory3200 to 0.78 μg/ml145.6 ± 15.4 μg/ml256.0 ± 26.5 μg/ml[36]
Minthostachys mollis Kunth.Broth macrodilutionInhibitory775 mg/L[42]
Minthostachys verticillata GrisebAgar diffusionInhibitory10 μl[33]
Myristica fragrans Gronov.Agar diffusionInhibitory10 μl[36]
Myristica fragrans Gronov.Broth microdilutionInhibitory3200–0.78285.8 ± 29.2 μg/ml371.3 ± 29.0[36]
Ocimum basilicum L.Agar dilutionInhibitory350–450 μg/ml[40]
Inhibitory0.06–0.12% (v/v)[34]
Ocimum tenuiflorum L.Agar diffusionInhibitory10 μl[36]
Broth microdilutionInhibitory3200–0.78412.8 ± 26.0 μg/ml589.6 ± 48.2[36]
Pimenta dioica (L.) Merr.Agar diffusionInhibitory10 μl[36]
Broth microdilutionInhibitory3200–0.78 μg/ml78.0 ± 8.2 μg/ml162.0 ± 18.2 μg/ml[36]
Pimpinella anisum L.Agar diffusionInhibitory5 μl[35]
Inhibitory10 μl[35]
Broth macrodilutionInhibitory300 μg/ml[46]
Salvia officinalis L.Agar diffusionInhibitory5 μl[35]
Inhibitory10 μl[35]
Salvia sclarea L.Agar diffusionInhibitory10 μl[34]
Agar dilutionInhibitory0.06% (v/v) (strong activity)[34]
Satureja odora Griseb.Broth microdilutionInhibitory700–800 mg/L900 mg/L[42]
Schinus molle L.Broth macrodilutionInhibitory666 mg/L[42]
Syzygium aromaticum L.Agar diffusionInhibitory10 μl[34]
Syzygium aromaticum L.Agar diffusionInhibitory5 μl[35]
Syzygium aromaticum L.Agar diffusionInhibitory10 μl[35]
Agar dilutionInhibitory0.015% (v/v) (strong activity)[34]
Tagetes minutaAgar diffusionInhibitory10 μl[34]
Agar dilutionInhibitory500–650 μg/ml[40]
Agar dilutionInhibitory700–800 μl/L[48]
Broth macrodilutionInhibitory900–1000 mg/L[41]
Inhibitory833 mg/L[42]
Thymol (component of Thymus vulgaris)Broth macrodilutionInhibitory100–133 μg/ml133 μg/ml[26]
Trachyspermum ammi L.Agar diffusionInhibitory10 μl[36]
Broth macrodilutionInhibitory3200–0.78 μg/ml137.0 ± 12.2 μg/ml224.8 ± 25.6 μg/ml[36]
Verbena officinalis L.Broth microdilutionInhibitory700–800 mg/L850 mg/L[42]
Wedelia glauca OrtegaBroth microdilutionInhibitory700–800 mg/L950 mg/L[42]
Zingiber officinale Rosc.Agar diffusionInhibitory10 ml[36]

Table 2.

Essential oils for the in vitro Paenibacillus larvae control.

MIC, Minimal Inhibitory Concentration.

MBC, Minimal Bactericidal Concentration.

Essential oilTechniqueToxicityAmount testedReferences
Carapa guaianensisSpraying procedureNon-toxic25% (v/v)[27]
Carapa guaianensis nanoemulsionComplete exposureNon-toxic10% (v/v)[50]
In-vivo against larvaSlightly toxic[50]
Copaifera officinalisSpraying procedureNon-toxic1.56% (v/v)[27]
Cymbopogon citratusSystemic administrationModerately toxic (>2 μg EO/bee)1, 2, 4, 8, 16 and 32 μg EO/bee[47]
Cymbopogon citratus + Thymus vulgaris (20:80, v/v)Systemic administrationSlightly toxic (24 h-LD50 = 15.94 μg b.e./bee)0.19, 0.37, 0.75, 1.50, 3.0 and 6.0 μg b.e./bee[47]
Cymbopogon citratus + Thymus vulgaris + Satureja hortensis + Origanum vulgare + Ocimum basilicum (5:11:21:26:37, v/v/v/v/v)Systemic administrationNot determined1.19, 2.37, 4.75, 9.50, 19.0 and 28.0 μg b.e./bee[47]
Cymbopogon citratus + Thymus vulgaris + Ocimum basilicum (10:20:70, v/v/v)Systemic administrationVirtually non-toxic (24 h-LD50 = 122 μg b.e./bee)0.625, 1.25, 2.5, 5.0, 10.0 and 20.0 μg b.e./bee[47]
Cinnamomum zeylanicumSystemic administrationVirtually non-toxic2000, 4000, 8000 and 16,000 μg/ml[46]
Eucalyptus globulusComplete exposureNon-toxic2.5, 5, 10 and 20 ml per cage of EO[44]
Eugenia spp.Systemic administrationNon-toxic400 μg/ml[32]
Melaleuca alternifoliaSpraying procedureToxic/non-toxic the nanoparticles of M. alternifolia6.25% (w/v)[49]
Origanum vulgareSystemic administrationModerately toxic (≥ 3 μg EO/bee)3, 6, 12, 24, 48 and 96 μg EO/bee[47]
Rosmarinus officinalisComplete exposureNon-toxic2.5, 5, 10 and 20 μl per cage of EO[51]
Tagetes minutaSpraying procedureNon-toxic5% (w/v)[48]
Thymus vulgarisSystemic administrationModerately toxic (>8 μg EO/bee)2, 4, 8, 16, 32 and 64 μg EO/bee[47]

Table 3.

Essential oils toxicity assays on Apis mellifera.

1.1.1 In vitro assays to control P. larvae

EOs from Achyrocline satureioides, Carum carvi, Cinnamomum spp., Cinnamomum zeylanicum, Citrus paradise, Cuminum cyminum, Cymbopogon citratus, Eucalyptus cinerea, Melaleuca alternifolia, Mentha piperita, Minthostachys verticillata, Origanum majorana, Origanum vulgare, Polygonum bistorta, Salvia officinalis, Salvia sclarea, Syzygium aromaticum, Tagetes minuta, Thymus vulgaris, Verbena, Pimenta dioica (L.) Merr., Litsea cubeba Pers., Trachyspermum ammi L., Mentha arvensis L., Mentha spicata L., Illicium verum Hook.f, Myristica fragrans Gronov., Cinnamomum camphora (L.) J. Presl., Ocimum tenuiflorum L., Daucus carota L., Zingiber officinale Rosc., and Pelargonium graveolens L., were able to inhibit the growth of P. larvae by the agar diffusion technique [38, 39, 40, 41, 42, 43, 44, 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, 49, 50, 51, 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 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, 64, 65, 66]. The QS can regulate the expression of virulence factors, bioluminescence, sporulation, biofilm formation and conjugation [67, 68, 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 gram-positive 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 non-antibiotic 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 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.


2. Conclusion

The research carried out to study the in vitro and in vivo antimicrobial activity of essential oils against P. larvae, their toxicity in adult honey bees, as well as their mode of action (degradation of the cell wall, affecting cell morphology and damaging the cytoplasm membrane, coagulation of the cytoplasm, etc.) and anti-QS activity (inhibiting the production of proteases, inhibiting transportation and secretion of proteases, inhibiting the detection of quorum, etc.), has been thoroughly reviewed throughout this chapter. As far as honeybee larvae are the target of AFB disease, future research should focus on studying the effect of essential oils that are effective in vitro and non-toxic for adult honey bees on honeybee larvae. In addition, more studies are still needed on the distribution and effects of these natural products in hives, adult honey bees, larvae, honey, royal jelly and other bee products to understand the pharmacokinetics and pharmacodynamics within the hive. As well, research on the effectiveness of these natural antimicrobials in field conditions is imperative. Moreover, further studies should be conducted on the sporicidal properties of these natural substances to destroy spores of P. larvae for the prevention of AFB disease. And last but not least, the development of adequate delivery modes of the essential oils within the hives for in vivo treatment and prevention of the disease is another important issue that requires further research, to put these natural strategies into practice under true hive conditions.


  1. 1. Buchmann S, Mirocha P. The Forgotten Pollinators. Island Press; 1996
  2. 2. Klein AM et al. Wild pollination services to California almond rely on semi-natural habitat. Journal of Applied Ecology. 2012;49(3):723-732
  3. 3. Le Conte Y, Ellis M, Ritter W. Varroa mites and honey bee health: Can Varroa explain part of the colony losses? Apidologie. 2010;41(3):353-363
  4. 4. Neumann P, Carreck NL. Honey bee colony losses. Journal of Apicultural Research. 2010;49(1):1-6
  5. 5. Potts SG et al. Global pollinator declines: Trends, impacts and drivers. Trends in Ecology & Evolution. 2010;25(6):345-353
  6. 6. Genersch E. American foulbrood in honeybees and its causative agent, Paenibacillus larvae. Journal of Invertebrate Pathology. 2010;103(Suppl. 1):S10-S19
  7. 7. Djukic M et al. How to kill the honey bee larva: Genomic potential and virulence mechanisms of Paenibacillus larvae. PLoS ONE. 2014;9(3):e90914
  8. 8. Genersch E et al. Reclassification of Paenibacillus larvae subsp. pulvifaciens and Paenibacillus larvae subsp. larvae as Paenibacillus larvae without subspecies differentiation. International Journal of Systematic and Evolutionary Microbiology. 2006;56(3):501-511
  9. 9. Ashiralieva A, Genersch E. Reclassification, genotypes and virulence of Paenibacillus larvae, the etiological agent of American foulbrood in honeybees—A review. Apidologie. 2006;37(4):411-420
  10. 10. Bamrick JF. Resistance to American foulbrood in honey bees: VI. Spore germination in larvae of different ages. Journal of Invertebrate Pathology. 1967;9(1):30-34
  11. 11. Genersch E, Ashiralieva A, Fries I. Strain- and genotype-specific differences in virulence of Paenibacillus larvae subsp. larvae, a bacterial pathogen causing American foulbrood disease in honeybees. Applied and Environmental Microbiology. 2005;71(11):7551-7555
  12. 12. Hansen H, Brødsgaard CJ. American foulbrood: A review of its biology, diagnosis and control. Bee World. 1999;80(1):5-23
  13. 13. Cornman RS, Lopez D, Evans JD. Transcriptional response of honey bee larvae infected with the bacterial pathogen Paenibacillus larvae. PLoS ONE. 2013;8(6):e65424
  14. 14. De Graaf D et al. Diagnosis of American foulbrood in honey bees: A synthesis and proposed analytical protocols. Letters in Applied Microbiology. 2006;43(6):583-590
  15. 15. Alippi A. Characterization of Bacillus larvae White, the causative agent of American foulbrood of honey-bees. First record of its occurrence in Argentina. Revista Argentina de Microbiología. 1992;24:67-72
  16. 16. Alippi A. Characterization of isolates of Paenibacillus larvae with biochemical type and oxytetracycline resistance. Revista Argentina de Microbiología. 1996;28:197-203
  17. 17. Marcangeli J. Pograma sanitario apicola del Partido de Mar Chiquita. Relevamiento de las enfermedades de abejas. In: XVII Congreso Latinoamericano de Parasitologia; Mar del Plata; 2005
  18. 18. Servicio Agrícola Ganadero (SAG). Informe final del brote de Loque Americana en Chile. 2006. Available from: s/INFORME_FINAL_BROTE_OCTUBRE_2005.PDF
  19. 19. Martel AC et al. Tetracycline residues in honey after hive treatment. Food Additives and Contaminants. 2006;23(3):265-273
  20. 20. Miyagi T et al. Verification of oxytetracycline-resistant American foulbrood pathogen Paenibacillus larvae in the United States. Journal of Invertebrate Pathology. 2000;75(1):95-96
  21. 21. Harriet J, Campa J, Mendoza Y. Loque Americana: Cartilla No 22; 2013. Available from:
  22. 22. Piccini C, Zunino P. American foulbrood in Uruguay: Isolation of Paenibacillus larvae larvae from larvae with clinical symptoms and adult honeybees and susceptibility to oxytetracycline. Journal of Invertebrate Pathology. 2001;78:176-177
  23. 23. Mutinelli F. European legislation governing the authorization of veterinary medicinal products with particular reference to the use of drugs for the control of honey bee diseases. Apiacta. 2003;38:156-168
  24. 24. Lewis K, Ausubel FM. Prospects for plant-derived antibacterials. Nature Biotechnology. 2006;24(12):1504
  25. 25. Damiani N et al. Laurel leaf extracts for honeybee pest and disease management: Antimicrobial, microsporicidal, and acaricidal activity. Parasitology Research. 2013;113:701-709
  26. 26. Fuselli S et al. Inhibition of Paenibacillus larvae employing a mixture of essential oils and thymol. Revista Argentina de Microbiología. 2006;38(2):89-92
  27. 27. Santos RCV et al. Antimicrobial activity of Amazonian oils against Paenibacillus species. Journal of Invertebrate Pathology. 2012;109(3):265-268
  28. 28. Bastos EMA et al. In vitro study of the antimicrobial activity of Brazilian propolis against Paenibacillus larvae. Journal of Invertebrate Pathology. 2008;97(3):273-281
  29. 29. Auddy B et al. Screening of antioxidant activity of three Indian medicinal plants, traditionally used for the management of neurodegenerative diseases. Journal of Ethnopharmacology. 2003;84(2-3):131-138
  30. 30. Costa DC et al. Advances in phenolic compounds analysis of aromatic plants and their potential applications. Trends in Food Science & Technology. 2015;45(2):336-354
  31. 31. Astani A, Reichling J, Schnitzler P. Comparative study on the antiviral activity of selected monoterpenes derived from essential oils. Phytotherapy Research: An International Journal Devoted to Pharmacological and Toxicological Evaluation of Natural Product Derivatives. 2010;24(5):673-679
  32. 32. Safaei-Ghomi J, Ahd AA. Antimicrobial and antifungal properties of the essential oil and methanol extracts of Eucalyptus largiflorens and Eucalyptus intertexta. Pharmacognosy Magazine. 2010;6(23):172
  33. 33. Burt S. Essential oils: Their antibacterial properties and potential applications in foods—A review. International Journal of Food Microbiology. 2004;94(3):223-253
  34. 34. Kaloustian J et al. Étude de six huiles essentielles: Composition chimique et activité antibactérienne. Phytothérapie. 2008;6(3):160-164
  35. 35. Devi KP et al. Eugenol (an essential oil of clove) acts as an antibacterial agent against Salmonella typhi by disrupting the cellular membrane. Journal of Ethnopharmacology. 2010;130(1):107-115
  36. 36. Djenane D et al. Perspectives on the use of essential oils as antimicrobials against Campylobacter jejuni CECT 7572 in retail chicken meats packaged in microaerobic atmosphere. Journal of Food Safety. 2012;32(1):37-47
  37. 37. Benjilali B et al. Méthode d’étude des propriétés antiseptiques des huiles essentielles par contact direct en milieu gélosé. Plantes Médicinales et Phytothérapie. 1986;20(2):155-167
  38. 38. Ansari MJ et al. In vitro evaluation of the effects of some plant essential oils on Paenibacillus larvae, the causative agent of American foulbrood. Biotechnology & Biotechnological Equipment. 2016;30(1):49-55
  39. 39. Fernández NJ et al. Laurus nobilis L. extracts against Paenibacillus larvae: Antimicrobial activity, antioxidant capacity, hygienic behavior and colony strength. Saudi Journal of Biological Sciences. 2018;26:906-912
  40. 40. Floris I, Carta C, Moretti M. Activity of various essential oils against Bacillus larvae white in vitro and in apiary trials [antimicrobial actions, American foulbrood control]. Apidologie (France). 1996;27:111-119
  41. 41. Gonzalez M, Marioli J. Antibacterial activity of water extracts and essential oils of various aromatic plants against Paenibacillus larvae, the causative agent of American foulbrood. Journal of Invertebrate Pathology. 2010;104(3):209-213
  42. 42. Khan SU et al. Antimicrobial potentials of medicinal plant’s extract and their derived silver nanoparticles: A focus on honey bee pathogen. Saudi Journal of Biological Sciences. 2018. In press
  43. 43. Kuzyšinová K et al. Testing of inhibition activity of essential oils against Paenibacillus larvae—The causative agent of American foulbrood. Acta Veterinaria Brno. 2014;83(1):9-12
  44. 44. Roussenova N. Antibacterial activity of essential oils against the etiological agent of American foulbrood disease (Paenibacillus larvae). Bulgarian Journal of Veterinary Medicine. 2011;14(1):17-24
  45. 45. Tutun H, Koç N, Kart A. Plant essential oils used against some bee diseases. Turkish Journal of Agriculture-Food Science and Technology. 2018;6(1):34-45
  46. 46. Alippi A et al. Actividad antimicrobiana in vitro de algunos aceites esenciales y mezclas de esencias sobre Paenibacillus larvae subsp. larvae. Vida Apícola. 2001;106:41-44
  47. 47. Alippi AM et al. Antimicrobial activity of some essential oils against Paenibacillus larvae, the causal agent of American foulbrood disease. Journal of Herbs Spices & Medicinal Plants. 1996;4(2):9-16
  48. 48. Fuselli S et al. Inhibition of Paenibacillus larvae subsp. larvae by the essential oils of two wild plants and their emulsifying agents. Spanish Journal of Agricultural Research. 2005;3(2):220-224
  49. 49. Fuselli SR et al. Antimicrobial activity of some Argentinean wild plant essential oils against Paenibacillus larvae larvae, causal agent of American foulbrood (AFB). Journal of Apicultural Research. 2006;45(1):2-7
  50. 50. Gende LB et al. Susceptibility of Paenibacillus larvae isolates to a tetracycline hydrochloride and cinnamon (Cinnamomum zeylanicum) essential oil mixture. Bulletin of Insectology. 2010;63(2):247-250
  51. 51. Gende LB et al. Antimicrobial activity of cinnamon (Cinnamomum zeylanicum) essential oil and its main components against Paenibacillus larvae from Argentine. Bulletin of insectology. 2008;61(1):1
  52. 52. Ruffinengo SR et al. Laboratory evaluation of Heterothalamus alienus essential oil against different pests of Apis mellifera. Journal of Essential Oil Research. 2006;18(6):704-707
  53. 53. Gende LB et al. Antimicrobial activity of Pimpinella anisum and Foeniculum vulgare essential oils against Paenibacillus larvae. Journal of Essential Oil Research. 2009;21(1):91-93
  54. 54. Albo G et al. Toxicidad de aceites esenciales con efecto fungistático sobre Ascosphaera apis en larvas y adultos de. Apis mellifera. 2008;1(7):1-7
  55. 55. Eguaras MJ et al. An in vitro evaluation of Tagetes minuta essential oil for the control of the honeybee pathogens Paenibacillus larvae and Ascosphaera apis, and the parasitic mite Varroa destructor. Journal of Essential Oil Research. 2005;17(3):336-340
  56. 56. Santos RCV et al. Antimicrobial activity of tea tree oil nanoparticles against American and European foulbrood diseases agents. Journal of Asia-Pacific Entomology. 2014;17(3):343-347
  57. 57. de Almeida Vaucher R et al. Antimicrobial activity of nanostructured Amazonian oils against Paenibacillus species and their toxicity on larvae and adult worker bees. Journal of Asia-Pacific Entomology. 2015;18(2):205-210
  58. 58. Maggi M et al. Bioactivity of Rosmarinus officinalis essential oils against Apis mellifera, Varroa destructor and Paenibacillus larvae related to the drying treatment of the plant material. Natural Product Research. 2011;25(4):397-406
  59. 59. Nazzaro F, Fratianni F, Coppola R. Quorum sensing and phytochemicals. International Journal of Molecular Sciences. 2013;14(6):12607-12619
  60. 60. Zhou K et al. Mode of action of pentocin 31-1: An antilisteria bacteriocin produced by Lactobacillus pentosus from Chinese traditional ham. Food Control. 2008;19(8):817-822
  61. 61. Bassolé IHN, Juliani HR. Essential oils in combination and their antimicrobial properties. Molecules. 2012;17(4):3989-4006
  62. 62. Pellegrini MC et al. Inhibitory action of essential oils against proteases activity of Paenibacillus larvae, the etiological agent of American foulbrood disease. Spanish Journal of Agricultural Research. 2018;15(4):0504
  63. 63. Henke JM, Bassler BL. Bacterial social engagements. Trends in Cell Biology. 2004;14(11):648-656
  64. 64. Kleerebezem M et al. Quorum sensing by peptide pheromones and two-component signal-transduction systems in gram-positive bacteria. Molecular Microbiology. 1997;24(5):895-904
  65. 65. Waters CM, Bassler BL. Quorum sensing: Cell-to-cell communication in bacteria. Annual Review of Cell and Developmental Biology. 2005;21:319-346
  66. 66. Fuqua C, Parsek MR, Greenberg EP. Regulation of gene expression by cell to cell communication: Acyl homoserine lactone quorum sensing. Annual Review of Genetics. 2001;35(1):439-468
  67. 67. Boyer M, Wisniewski-Dyé F. Cell-cell signalling in bacteria: Not simply a matter of quorum. FEMS Microbiology Ecology. 2009;70(1):1-19
  68. 68. Cámara M, Williams P, Hardman A. Controlling infection by tuning in and turning down the volume of bacterial small-talk. The Lancet Infectious Diseases. 2002;2(11):667-676
  69. 69. Williams P. Quorum sensing, communication and cross-kingdom signalling in the bacterial world. Microbiology. 2007;153(12):3923-3938
  70. 70. Antúnez K et al. Secreted and immunogenic proteins produced by the honeybee bacterial pathogen, Paenibacillus larvae. Veterinary Microbiology. 2010;141(3-4):385-389
  71. 71. Castillo-Juárez I et al. Role of quorum sensing in bacterial infections. World Journal of Clinical Cases: WJCC. 2015;3(7):575
  72. 72. Biswa P, Doble M. Production of acylated homoserine lactone by Gram-positive bacteria isolated from marine water. FEMS Microbiology Letters. 2013;343(1):34-41
  73. 73. Monnet V, Gardan R. Quorum-sensing regulators in Gram-positive bacteria: ‘cherchez le peptide’. Molecular Microbiology. 2015;97(2):181-184
  74. 74. Hentzer M et al. Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors. The EMBO Journal. 2003;22(15):3803-3815
  75. 75. Rasmussen TB, Givskov M. Quorum-sensing inhibitors as anti-pathogenic drugs. International Journal of Medical Microbiology. 2006;296(2-3):149-161
  76. 76. Olivero-Verbel J et al. Composition, anti-quorum sensing and antimicrobial activity of essential oils from Lippia alba. Brazilian Journal of Microbiology. 2014;45(3):759-767
  77. 77. Zhang L-H. Quorum quenching and proactive host defense. Trends in Plant Science. 2003;8(5):238-244
  78. 78. Otero Casal A et al. Quorum Sensing: El Lenguaje de las Bacterias. Zaragoza: Acribia, SA; 2005
  79. 79. Tang K, Zhang X-H. Quorum quenching agents: Resources for antivirulence therapy. Marine Drugs. 2014;12(6):3245-3282
  80. 80. Gala V, Desai K. Plant based quorum sensing inhibitors of Pseudomonas aeruginosa. International Journal of Pharmacy and Pharmaceutical Sciences. 2014;6(8):20-25
  81. 81. Chu Y-Y et al. A new class of quorum quenching molecules from Staphylococcus species affects communication and growth of gram-negative bacteria. PLoS Pathogens. 2013;9(9):e1003654
  82. 82. Sivamani P et al. Comparative molecular docking analysis of essential oil constituents as elastase inhibitors. Bioinformation. 2012;8(10):457
  83. 83. Inouye K et al. Engineering, expression, purification, and production of recombinant thermolysin. Biotechnology Annual Review. 2007;13:43-64
  84. 84. OIE. World Animal Health Information Database - Version: 1.4. World Animal Health Information Database. Paris, France: World Organisation for Animal Health; 2009. Available from:

Written By

Sandra Rosa Fuselli, Pablo Gimenez Martinez, Giselle Fuentes, Rosa María Alonso-Salces and Matías Maggi

Submitted: April 23rd, 2018 Reviewed: March 11th, 2019 Published: August 8th, 2019