Sandra R. Fuselli

Antimicrobial activity of some Argentinean wild plant essential oils against Paenibacillus larvae larvae, causal agent of American foulbrood (AFB)

Summary Five essential oils from Argentinan wild plants were tested in vitro for antimicrobial activity against different strains of Paenibacillus larvae larvae, causal agent of American foulbrood in honey bees, Apis mellifera L. The species selected from which to extract the essential oils were aguaribay (Schinus molle var. areira L), Andean thyme (Acantholippia seriphioides A. Gray), peperine (Mintosthachys mollis H.B.K. Gris), camomile (Tagetes minuta L.) and pennyroyal (Lippia turbinata Griseb.). Oils were extracted from different parts of the plants by steam distillation. Minimal inhibitory concentration (MIC) and minimal bactericide concentration (MBC) were evaluated on Mueller-Hinton broth and on MYPGP agar respectively, by the tube dilution method. MIC and MBC minimum and maximum values obtained were 200–250 mgL−1 and 200–300 mgL−1 for Andean thyme and 800–1000 mgL−1 and 850–1100 mgL−1 for pennyroyal, respectively. The other oils had intermediate MIC and MBC values between the formers mentioned. Highly significant differences between MIC and MBC values for the essential oils tested were found. Tukeys mean separation test indicated that Andean thyme (A. seriphioides) essential oil was different from the other four essential oils at α<0.01. Andean thyme proved to be the most effective in vitro essential oil against P. larvae larvae. Therefore, it may be a promising natural alternative to traditional antibiotics, meriting further studies on its field applicability for AFB control.

Anti-quorum sensing and antimicrobial activity of aromatic species from South America

Quorum sensing (QS) is a bacterial communication mechanism that depends on population density. The interruption of QS is one example of an antipathogenic effect. We investigated the anti-QS and antimicrobial properties of essential oils from Argentina: Salvia officinalis, Minthostachys mollis, Satureja odora, Schinus molle, Lepechinia floribunda and Artemisia annua. Anti-QS activity was determined by measuring the production of violacein in Chromobacterium violaceum through UV–visible spectrophotometry and the minimal QS inhibitory concentration (MQSIC) was calculated. The antimicrobial activity was determined using Escherichia coli, Listeria innocua and Staphylococcus aureus as indicators. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined by performing the broth microdilution assay. Minthostachys mollis showed statistically significant QS inhibition properties. This essential oil reduced pigment production by 90% when it was applied at a sublethal concentration (0.02% v/v). Conversely, the highest bacteriostatic and bactericidal activity was exhibited by S. molle oil. Minthostachys mollis essential oil is a good candidate for the development of anti-QS products with a potential application in the control of bacterial diseases mediated by QS. As this strategy interferes with the expression of pathogenic traits rather than killing the microorganism or impeding microbial growth, it avoids the problem of resistance.

In vitro antibacterial and antiparasitic effect of citrus fruit essential oils on the honey bee pathogen Paenibacillus larvae and the parasitic mite Varroa destructor.

Sandra Rosa Fuselli, Matías Maggi, Susana Beatriz García de la Rosa, Judith Principal, Martín Javier Eguaras and Rosalía Fritz Universidad Nacional de Mar del Plata (UNMdP), Facultad de Ciencias Exactas y Naturales, Mar del Plata, Argentina. Comisión de Investigaciones Científicas (CIC), La Plata, Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina. Estación de Apicultura, Decanato de Ciencias Veterinarias, Universidad Centroccidental “Lisandro Alvarado” (UCLA), Tarabana, Estado Lara, Venezuela.

Natural strategies for the control of Paenibacillus larvae, the causative agent of American foulbrood in honey bees: a review

American foulbrood (AFB) is a severe bacterial disease that affects larvae of honey bees (Apis mellifera). The causative agent of AFB is the spore-forming bacteria Paenibacillus larvae. The use of antibiotics for the control of AFB has led to the appearance of resistant bacterial strains and residues in beehive products. Nowadays, antibiotics are legally banned in several countries, and the affected colonies have to be destroyed by burning the hives. Therefore, the development of alternative methods for the control and prevention of AFB is necessary. In this context, different natural strategies based on the application of essential oils, plant extracts, propolis, royal jelly, nonconventional natural molecules, bacteria, and bacteriocines, have been studied in vitro and in vivo for the prevention and control of P. larvae. The experimental data achieved from these studies are reviewed and discussed in the present review, which intend to be a starting point for future research in the field.

Optimal Concentration of Organic Solvents to be Used in the Broth Microdilution Method to Determine the Antimicrobial Activity of Natural Products Against Paenibacillus Larvae

Abstract American Foulbrood (AFB) is a bacterial disease, caused by Paenibacillus larvae, that affects honeybees (Apis mellifera). Alternative strategies to control AFB are based on the treatment of the beehives with antimicrobial natural substances such as extracts, essential oils and/or pure compounds from plants, honey by-products, bacteria and moulds. The broth microdilution method is currently one of the most widely used methods to determine the minimum inhibitory concentration (MIC) of a substance. In this regard, the fact that most natural products, due to their lipophilic nature, must be dissolved in organic solvents or their aqueous mixtures is an issue of major concern because the organic solvent becomes part of the dilution in the incubation medium, and therefore, can interfere with bacterial viability depending on its nature and concentration. A systematic study was carried out to determine by the broth microdilution method the MIC and the maximum non inhibitory concentration (MNIC) against P. larvae of the most common organic solvents used to extract or dissolve natural products, i.e. ethanol, methanol, acetonitrile, n-butanol, dimethylsulfoxide, and acidified hydromethanolic solutions. From the MIC and MNIC for each organic solvent, recommended maximum concentrations in contact with P. larvae were established: DMSO 5% (v/v), acetonitrile 7.5% (v/v), ethanol 7.5% (v/v), methanol 12% (v/v), n-butanol 1% (v/v), and methanol-water-acetic acid (1.25:98.71:0.04, v/v/v).

Chemical Composition, Antimicrobial Activity, and Mode of Action of Essential Oils against Paenibacillus larvae, Etiological Agent of American Foulbrood on Apis mellifera

This study aimed to characterize the chemical composition of Aloysia polystachia, Acantholippia seriphioides, Schinus molle, Solidago chilensis, Lippia turbinata, Minthostachys mollis, Buddleja globosa, and Baccharis latifolia essential oils (EOs), and to evaluate their antibacterial activities and their capacity to provoke membrane disruption in Paenibacillus larvae, the bacteria that causes the American Foulbrood (AFB) disease on honey bee larvae. The relationship between the composition of the EOs and these activities on P. larvae was also analyzed. Monoterpenes were the most abundant compounds in all EOs. All EOs showed antimicrobial activity against P. larvae and disrupted the cell wall and cytoplasmic membrane of P. larvae provoking the leakage of cytoplasmic constituents (with the exception of B. latifolia EO). While, the EOs’ antimicrobial activity was correlated most strongly to the content of pulegone, carvone, (Z)‐β‐ocimene, δ‐cadinene, camphene, terpinen‐4‐ol, elemol, β‐pinene, β‐elemene, γ‐cadinene, α‐terpineol, and bornyl acetate; the volatiles that better explained the membrane disruption were carvone, limonene, cis‐carvone oxide, pentadecane, trans‐carvyl acetate, trans‐carvone oxide, trans‐limonene oxide, artemisia ketone, trans‐carveol, thymol, and γ‐terpinene (positively correlated) and biciclogermacrene, δ‐2‐carene, verbenol, α‐pinene, and α‐thujene (negatively correlated). The studied EOs are proposed as natural alternative means of control for the AFB disease.

Varroa destructor: when reversion to coumaphos resistance does not happen

The ectoparasitic mite Varroa destructor is potentially the greatest threat to honey bee colonies of Apis mellifera worldwide. Acaricide rotation is widely accepted to mitigate development of resistance to synthetic acaricides. When a population has developed resistance to a compound, cessation of its use can result in some degree of susceptibility. This study aimed to follow changes in the susceptibility of mites to coumaphos in a population not treated with the acaricide over many generations. The LC50 increase over the years from 2 to 8-fold. Considering that coumaphos had not been used for nine years in the studied apiary, the results provided here lead to reflections about the problem of the resistance phenomenon and its populations dynamics. Varroa destructor: cuando la reversión de la Resistencia a cumafós no ocurre El ácaro ectoparásito Varroa destructor constituye una de las mayores amenazas en el mundo para las colmenas de Apis mellifera. La rotación de acaricidas es una medida ampliamente aceptada para mitigar el desarrollo de resistencia de acaricidas sintéticos. Se ha observado que, cuando se deja de aplicar un acaricida a una población resistente, luego de un periodo de tiempo la población se puede volver susceptible al mismo. El objetivo del presente trabajo fue estudiar si la susceptibilidad al cumafós de una población de Varroa destructor no expuesta a dicho acaricida durante varios años, decrece a lo largo del tiempo. Los valores de CL50 se fueron incrementando a lo largo de los años, este incremento fue desde 2 a 8 veces. Considerando que el apiario estudiado no ha sido expuesto al cumafós durante nueve años, estos resultados llevan a la reflexión sobre el problema de la resistencia a acaricidas de síntesis y su dinámica en las poblaciones.