**2. Human pathogenic microorganisms found in honey**

Due to characteristics cited above, only pathogenic bacteria capable of sporulation have the ability to keep in honey, but they have no reproductive capacity or vegetative cells. Fungi and yeasts are able to maintain their vegetative form [2].

Fungal growth is followed by the production of mycotoxins, which are secondary metabolites of filamentous fungi and toxic to humans and animals even in small concentrations. These are produced by fungi to reduce the incidence of competitors in environment [8]. The main producers of mycotoxins are fungi of the genus *Aspergillus*, *Alternaria*, *Fusarium*, and *Penicillium* [9]. Among which we should highlight *Aspergillus* spp. and *Penicillium* spp. because they are the most commonly found in honey. Articles about these microorganisms in honey record these genera in isolated colonies in the United Kingdom, Pakistan, Italy, and Brazil [10–13]. They are also associated with disease in honeybees.

In research performed with honey samples of different blossoming, fungi of different species were isolated, *Alternaria alternata*, *Aspergillus niger*, *Aspergillus proliferans*, *Aspergillus spelunceus*, *Chaetomium globosum*, *Cladosporium cladosporioides*, *Daldinia concentrica*, *Emericella discophora*, *Emericella qinqixianii*, *Penicillium corylophilum*, *Penicillium decumbens*, *Penicillium polonicum*, and *Penicillium echinulatum*, of which *P*. *corylophilum* and *A*. *niger* were the most frequent, but in low count, indicating that the honey is capable of containing multiplication of these fungi [13]. The presence of fungi does not imply the presence of mycotoxin; it has necessary ideal conditions such as high water activity, the presence of sugars, and the presence of organic acids capable of reducing pH. Necessary conditions for fungal growth are not always the necessary conditions for production of mycotoxins [9]. As an example, we can cite the patulin produced by species of *Penicillium*, *Aspergillus*, and *Byssochlamys* whose optimum temperature for production is 23–25°C, with minimal water activity of 0.82–0.83. Aflatoxins produced by *Aspergillus flavus* and *Aspergillus parasiticus* have ideal temperature of 30–52°C and 0.80–0.95 water activity, and ochratoxin that is produced by species of *Aspergillus* and *Penicillium* needs temperature between 30–35°C and 0.93–0.99 of water activity [9].

low redox potential due to the presence of reducing sugars; and chemical agents present as lysozyme, phenolic acids, pinocembrin, terpenes, benzyl alcohol, and volatile substances [1, 2]. High osmotic pressure results from its composition: 85–95% of sugar, of which it has 28–31% of glucose, 22–38% of fructose, 1–4% of sucrose, and 1–9% maltose [3]. Isomaltose and some oligosaccharides are also present in honey and vary according to flowering, climate, and local production [4, 5]. As honey is a product developed from changes in nectar, the bees incorporate the glucose oxidase enzyme that converts glucose into hydrogen peroxide and gluconic acid; this compound is indeed important for the taste of products as well as their bioactivity [5, 6]. The presence of acids and other chemicals varies with the composition of the transformed nectar; for this reason, some honeys have higher antimicrobial activity with respect to

About these conditions, few microorganisms have the capacity to develop or remain in honey. These microorganisms are derived from primary or secondary sources of contamination. The primary sources are related to digestive tract of honeybees, which have natural microorganisms and sources of material collection such as nectar, pollen and propolis, air, flowers, and the environment inside the beehive, while the secondary sources are incorporation of honey

Due to characteristics cited above, only pathogenic bacteria capable of sporulation have the ability to keep in honey, but they have no reproductive capacity or vegetative cells. Fungi and

Fungal growth is followed by the production of mycotoxins, which are secondary metabolites of filamentous fungi and toxic to humans and animals even in small concentrations. These are produced by fungi to reduce the incidence of competitors in environment [8]. The main producers of mycotoxins are fungi of the genus *Aspergillus*, *Alternaria*, *Fusarium*, and *Penicillium* [9]. Among which we should highlight *Aspergillus* spp. and *Penicillium* spp. because they are the most commonly found in honey. Articles about these microorganisms in honey record these genera in isolated colonies in the United Kingdom, Pakistan, Italy, and Brazil [10–13].

In research performed with honey samples of different blossoming, fungi of different species were isolated, *Alternaria alternata*, *Aspergillus niger*, *Aspergillus proliferans*, *Aspergillus spelunceus*, *Chaetomium globosum*, *Cladosporium cladosporioides*, *Daldinia concentrica*, *Emericella discophora*, *Emericella qinqixianii*, *Penicillium corylophilum*, *Penicillium decumbens*, *Penicillium polonicum*, and *Penicillium echinulatum*, of which *P*. *corylophilum* and *A*. *niger* were the most frequent, but in low count, indicating that the honey is capable of containing multiplication of these fungi [13]. The presence of fungi does not imply the presence of mycotoxin; it has necessary ideal conditions such as high water activity, the presence of sugars, and the presence of organic acids capable of reducing pH. Necessary conditions for fungal growth are not

microorganisms postharvest, processing plants, and appliances [5].

**2. Human pathogenic microorganisms found in honey**

yeasts are able to maintain their vegetative form [2].

They are also associated with disease in honeybees.

other different blossoming [7].

234 Honey Analysis

Despite of inappropriate condition found in honey for mycotoxin production, it is important to say that the presence of fungus can also cause disease in different ways, as induction of allergic responses and infections. The fungi of genus *Aspergillus* are able to causing bronchopulmonary allergies among other forms of invasive aspergillosis. They are also related in acquired disease by immunocompromised patients in hospital. *Aspergillus fumigatus* is the most pathogenic followed by *A*. *flavus*, *Aspergillus terreus*, and *A*. *niger* [14]. The allergies and asthma may be caused by inhaled or ingested spores. For example, *Aspergillus clavatus* and *A*. *fumigatus* are responsible for allergies from malt workers who inhaled large amounts of spores during the malt handling for contaminated barley [15]. Foods with acidic pH, low humidity, and high concentration of sugars, such as honey, are sources for growth of the fungi *Aspergillus glaucus* [15].

Regarding the *Penicillium*, this fungus was first associated as producer of mycotoxins. They are saprophytic fungi able to grow at water activities less than 0.9; they can invade plants and animals but not as obligate parasite. They have vegetative reproduction by spores. However, the most important aspect concerns the production of toxins as aflatoxins, patulin, and ochratoxins [16]. In humans, only a minority of fungal species has pathogenicity, i.e., *Penicillium marneffei* (Southeast Asia), which is assigned lung infections in people with HIV virus in South Asia and China, and opportunistic infections—keratitis, ear infections, and endocarditis [17].

With respect to yeasts, only *Debaryomyces hansenii*, *Zygosaccharomyces rouxii*, *Zygosaccharomyces mellis*, *Aureobasidium pullulans*, and *Cryptococcus uzbekistanensis* species were isolated from honey [13]. Among them only *Cryptococcus* species was associated with human pathogenicity, i.e., the yeast *Cryptococcus neoformans* is characterized as opportunistic human pathogen able to infect the central nervous system [18].

Among bacteria, *Bacillus* sp. and *Clostridium* sp. were described in honey. *Clostridum perfringens* is known as an enterotoxin producer that happens in final stages of sporulation; thus, in adverse conditions for their development, the toxin will be released together with spore. Vegetative cells also produce enterotoxin but at low levels. Unlike *C*. *perfringens*, the toxin produced by *Clostridium botulinum* is stronger and produced during propagation. Thus, the best condition for propagation is the same for toxin production, which is 4.5 pH, water activity of 0.93, and temperature varying with strain [19].

There are about 200 species of *Clostridium*; a lot of them has pathogenicity and produce one or more toxins, assimilated by the gut and transported by blood [20]. Only *Clostridium botulinum* was found in honey [2], but was hardly detected with conventional methods; however, with molecular techniques as PCR, the detection was more accurate. In this way, samples that seem negative showed positive with molecular test [21, 22]. This microorganism enters the beehive through the contaminated water or even by contact of product with ground. This organism does not cause damage to honeybees, but it is responsible for the development of botulism in humans, especially in children or people with weakened immune systems and can lead to death [23].

Genus *Bacillus* comprises rod-shaped Gram-positive bacteria with the ability to form spores. There are 60 species of huge genetic diversity, and most of them are nonpathogenic; the pathogenicity associated with others is in opportunistic form. These pathogens belong to group *Bacillus cereus*, a subgroup *Bacillus subtilis*; however, *Bacillus licheniformis*, *Bacillus pumilus*, and *Bacillus majavensis* can cause poisoning by food too [24]. *Bacillus cereus* is an important pathogen in honey; it is an enterotoxin producer in pH 6.0–8.0 and temperature ranging from 6°C to 21°C, but it is necessary to ingest 107 cells/mL to reach toxic effect [19].

Researchers isolated some bacteria in honey samples of different geographical and botanical origins. "They found *B*. *pumilus* (ML374), *B*. *licheniformis* (ML103A and ML104B), *B*. *amyloliquefaciens*, *B. subtilis*, *B*. *cereus*, *B. thuringiensis*, *B*. *licheniformis*, *B. megaterium*, and *B*. *pumilus* [13]." The bacteria of species *B*. *cereus* are enterotoxin producers; the others of *Bacillus* species are considered safe. Due to their ability of producing bacteriocins, they are promising in the study of new antimicrobial [25].
