Maximum residual limits for antibiotics in the European Union.
Honeybees (Apis mellifera) crucially pollinate agricultural crops and endemic species, in addition to producing various apiculture products. The most economically relevant and abundant beehive product is honey, a sweet substance made from the secretions of melliferous plants. Honey is a natural food rich in nutrients, including certain bioactive compounds inherited from floral nectar and pollen. Among the most dangerous diseases for bees is American foulbrood. Spores of the causative microorganism, Paenibacillus larvae, can contaminate larvae food or the operculum wax in which larval stages of honeybees are kept. Infection is further promoted by common apiculture practices, such as reusing inert material contaminated with spores, even after months of storage. American foulbrood is untreatable, and management implicates completely incinerating the infected hive and all material that could have come into contact with pathogenic spores. The purpose of such drastic measures is to decrease propagation risk for other beehives. While evidence indicates that antibiotics could effectively control and combat this disease; antibiotic use is prohibited in most honey-producing countries due to increased risks to microbial resistance. Antibiotic residues in honey can affect consumer health, since the natural biological attributes of honey can be altered.
- American foulbrood
- Paenibacillus larvae
- beehive products
- antibiotics residues
Climate-change phenomena have strongly impacted the viability of ecosystems . Prolonged droughts and high temperatures due to intense heat waves have become, in recent years, determining factors in weakened  and decreased  beehive populations across Mediterranean climates, including western Australia, southeastern Africa, central Chile, California, and the Mediterranean basin of Europe. The combination of harsh temperatures and shortened flowering periods, as associated with insufficient water, can result in reductions to fat reserves and overall body mass in bees. This status can translate into fewer pollinating and honey-producing activities , as well as an increased incidence of specific diseases affecting honey bees weakened by nutritional deficits [8, 9].
Addressing the aforementioned threats to sustainably preserve the apiculture industry requires compliance with strict international regulations and norms to ensure the quality and safety of export products. Sufficiently monitoring residues in honey and adequately controlling diseases affecting bee health and production are constant preoccupations for apiculturists and exporters worldwide. In this chapter, there will be listed the main issues related to the uses of antibiotics as effective treatment against
2. Honey and other apiculture products
Besides fulfilling the critical role of pollinizing agricultural crops,
In addition to pollen collection, young melliferous bees secrete a liquid from wax glands that, when exposed to air, hardens and forms small flakes that collect on the underside of the bee. This economically valuable natural substance is known as beeswax and is used by bees to construct hexagonal alveoli into honeycombs. The rigid structure of honeycomb cells serves to conserve honey and pollen. Likewise, alveoli serve as a place for the queen bee to deposit eggs and for larvae or pupae to develop . Beeswax contains carbohydrates (present in pollen and nectar) that have transformed into fats due to the presence of enzymes and enzyme precursors secreted by bees. More specifically, beeswax is constituted by water and minerals (1–2%), mono-esters and hydroxyl mono-esters, complex wax esthers, hydrocarbons, and free wax acids .
Despite the importance of bee pollen and beeswax, honey is the primary apiculture product. The global honey trade is valued at 2.4 billion dollars annually and involves the movement of approximately 630 thousand tons of honey. Chile accounted for 0.6% of total exports in 2017 and is ranked 30th among export countries. In 2017, the main import markets of honey were the United States and Germany. Honey has been described as a naturally sweet mixture produced by
Status as a natural functional food means that honey is the best-characterized apiculture product. Bees selectively use floral resources available in proximity to beehives [22, 23, 24]. This is important to consider as the traits of apiculture products, including honey and bee pollen, are inherited through secondary plant metabolites transferred in nectar . Consequently, the attributes of melliferous species are directly related to the biological properties of resulting honeys . Notable among the biologically active components of honey are phenolic compounds  and flavonoids [28, 29]. Phenolic compounds and flavonoids have antioxidant capacities, acting through routes complementary to enzymatic antioxidants identified in honeys, such as glucose oxidase and catalase [30, 31, 32]. Antibiotic activity, also as related to phenolic acids and flavonoids, has been reported in some honeys globally [33, 34, 35, 36].
In addition to affecting biological properties, plant origin also directly influences the market value of honey. Quantitative and qualitative melissopalynological analyses can be used to classify honeys as monofloral, bifloral, or polyfloral. The highest demand is for monofloral honeys, which are primarily constituted (>45%) by pollen grains of the same melliferous species. Therefore, honey quality depends on the presence and concentration of specific chemical compounds and on the botanical origin of said compounds .
The elaboration of the aforementioned apiculture products can, under certain conditions, concurrently occur with the production of live material. More specifically, rearing queen bees and colonies are diversification options for national apiculturists . There is a demand for bee packages, nucleus colonies, and, particularly, queen bees in countries such as Canada, France, Mexico, and Italy. This point has driven industry growth in Chile, which, over the last 3 years, has doubled in size, going from more than 10,000 exported queen bees in 2015 to more than 20,000 in 2017 .
3. American foulbrood
There are two groups of diseases that can affect beehives—exotic and endemic diseases. Exotic diseases include parasites such as the small hive beetle (
The first report of American foulbrood in Chile was in 2001, whereas the first case of European foulbrood was in 2009. According to protocols for the management of apiculture diseases issued by the Chilean Ministry of Agriculture, both foulbrood diseases are classified as endemic and with low prevalence in the country. Nevertheless, the management of European foulbrood is less complex and involves less drastic sanitary measures than American foulbrood. Indeed, the incidence of European-foulbrood outbreaks has consistently declined since initial detection, with only one incident reported in 2016.
By contrast, American foulbrood is difficult to manage and eradicate. This pathogen has been detected in most regions of Chile, but the number of reported cases has varied since 2005. Notwithstanding, a worrying 44 outbreaks were reported in 2018, and an additional 61 outbreaks have been reported as of June 2019. Most cases have been reported in the Atacama, O’Higgins, and Maule Regions of Chile . Given that antibiotic treatment of this disease is prohibited  and that sanitary control measures include the incineration of all live material, it is believed that American foulbrood outbreaks are underreported in Chile out of fear for the total loss of infected beehives. In this way, according to the World Organization for Animal Health (OIE), there are cases reported in the first half of 2019 in Europe (with declared infection in Finland), South Africa, North America, South America and Australia. Despite those data, many countries have no information available for knowing the real state of this disease around the world as it shows Figure 1.
The infectious pathway of
Bee colonies present a coexistence mechanism with
The symptoms and effects of American foulbrood manifest slowly in beehives and occur while larvae receive contaminated food. In this stage, disease is not visible, but the first signs include the presence of dark, sunken, and greasy cappings that may be perforated by bees removing brood already in the process of putrefaction . Finally, hive death occurs due to the lack of new, live brood and the aging and death of adult bees. The weakened hive then become an easy target for pillaging by bees from stronger hives seeking food reserves. Such pillaging serves to propagate the disease in nearby beehives and, consequently, the entire apiary [43, 50].
3.1 Control strategies
3.1.1 Antibiotic treatments and the analytical methods for detecting residues in honey
The need to control American foulbrood is principally driven by damage caused by infection, which can include the loss of beehives and compromised honey and queen-bee exports. The use of tetracycline prophylactics is widespread in large animals and is allowed for bees in some honey-producing countries. In most countries, however,
Where antibiotic use is allowed, maximum residual limits range between 10 and 50 ppb. These limits are intended to minimize the presence of antibiotic compounds in end-products, such as honey . Antibiotics can, undoubtedly, affect the properties, quality, and, finally, export price of honey. Additionally, some purchasing countries regulate against the presence of antibiotics in beehives, thus impacting beekeepers that export honey [54, 55, 56]. This is a particularly relevant point for Chilean beekeepers as the primary export market is Europe, which has zero tolerance for antibiotics in imported honey (Table 1) . These strict regulations require the determination of each compound in honey through highly sensitive analytical methods.
|Antibiotic||Maximum residual limit|
Several studies have aimed to develop reliable methods for detecting and quantifying the presence of antibiotics in complex organic matrixes, such as honey. Despite the ban of antibiotics in beekeeping, these substances have been detected in various European honey samples . Liquid chromatography with UV–Vis detection resulted in the isolation of tetracycline, oxytetracycline, chlortetracycline, doxycycline, minocycline, and methacycline in different fortified honey samples cleaned by solid-phase extraction . A more recent methodology with good results is QuEChERS solid-phase extraction followed by liquid chromatography tandem mass spectrometry .
Antibiotic resistance against tetracyclines by American and European foulbrood strains has led to research of other antibiotics. Sulfonamides have been widely used, but specific methods of determining and detecting these compounds in honey are needed since toxic collateral effects in association with allergies have been observed in humans . To this end, high performance liquid chromatography paired with time-of-flight mass spectrophotometry has detected trace amounts of these compounds through direct injection .
Tylosin, a macrolide antibiotic active against many Gram-positive bacteria, has been increasingly used instead of tetracyclines and sulfonamides in beekeeping. Nevertheless, American foulbrood also presents resistance against macrolides. The best methodology for detecting macrolides in honey samples is solid-phase extraction followed by liquid chromatography tandem mass spectrophotometry . Another type of antibiotic used against American and European foulbrood is streptomycin. This aminoglycoside can potentially control foulbrood disease in beehives. Traditional methods of detection include high-performance liquid chromatography with different strategies of solid-phase extraction [63, 64]. The adverse effects to consumers of honeys contaminated by streptomycin include acute otitis and allergic dermatitis .
Finally, a number of antibiotics have been fully banned in the control of American foulbrood due to adverse effects to human health. For example, nitrofurans are associated with possible carcinogenic effects while chloramphenicol can cause aplastic anemia, in addition to evidencing possible carcinogenic risks [59, 66].
3.1.2 Nuclear irradiation
One reliable and traceable treatment for efficiently eliminating the highly resistant
3.1.3 Antimicrobial peptides
An alternative strategy for controlling and combating
American foulbrood has been present since the beginning of beekeeping and has evolved over time. Nevertheless, the apiculture industry today faces a complex situation. The effects of climate change have modified the availability of nutrients and food for bees, ultimately weakening hive health. Food availability for bees has been further decreased by the use of agrochemicals and the occurrence of extensive, devastating forest fires. These situations have provoked a resurgence of American foulbrood outbreaks, which need to be controlled to mitigate population and economic losses. Researchers specializing in apiculture should focus efforts on the search for new, environmentally friendly control strategies against this disease. Such efforts will help prevent the use of antibiotics, which in addition to inducing
Funding by CONICYT—PAI/Inserción sector productivo, 1era conv. 2019, Grant number I7819010001.
Conflict of interest
The author declares no conflict of interest.