Abstract
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).
Highlights
Paenibacillus larvae is the etiological agent of American Foulbrood (AFB), a bacterial disease that affects the breeding of honeybees (Apis mellifera) during the larval or pupal stages (Genersch et al, 2006; De Graaf et al, 2013)
Bacterial growth inhibition was observed in the strain viability control (SVC) columns as well as in wells containing the organic solvent in concentrations that had been previously tested and found to be innocuous to P. larvae; the antimicrobial activity results were not being repeatable
Other extracting solvents such as ethyl acetate (Lokvam et al, 2000; Boligon et al, 2013), chloroform (Reyes et al, 2013), dichloromethane (Boligon et al, 2013; HernándezLópez et al, 2014), and hexane (Sabaté et al, 2012) have been used to extract or dissolve bioactive compounds to be tested in antimicrobial activity assays against P. larvae, but they could not be assayed in the present study because the polystyrene plates commonly used for the broth microdilution method present low resistance against these solvents
Summary
Paenibacillus larvae is the etiological agent of American Foulbrood (AFB), a bacterial disease that affects the breeding of honeybees (Apis mellifera) during the larval or pupal stages (Genersch et al, 2006; De Graaf et al, 2013). The use and abuse of antibiotics to treat AFB has led to the increase in the incidence of bacterial resistance (Eguaras et al, 2005) and the contamination of the apiary products (Mutinelli, 2003) In this context, the development of alternative and effective methods for the control and prevention of AFB disease is crucial. This paper presents a systematic study to determine the MIC and the maximum non inhibitory concentration (MNIC) against P. larvae of organic solvents and their aqueous mixtures by the broth microdilution method, in order to establish the maximum content of the organic solvent in the sample solution needed to perform this dilution method properly. The solvents most commonly used to extract bioactive natural products from plants or to dissolve pure natural compounds are studied
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