**7. Microorganism detection methodologies in honey and honeybee products**

#### **7.1. Microbial diversity**

Much has been discussed about the succession of gut microbiota among queens, workers, and larvae and the role of the diversity on the quality of honey, safety, and health of the colony [11, 53, 114–117]. New methodologies have made it possible to access information about the differences in the profile of this microbiota in different apiculture sources [118–121], species [53, 122] and genetic diversity [116] of honeybees, development stages [53, 68, 117, 122–126], nutrition [116, 127], location inside the gut [49, 53, 68] and digestive system [120], ontogenetic stage and geographic location [118, 122, 125], environmental conditions [128], health control [129], and individual [116, 125].

with a reported impact on the intestinal barrier mechanism [94], it is not surprising that lactic acid bacteria, especially fructophilic lactic acid bacteria, may be involved with honeybee health. Symbiosis is common in nature, in which symbionts as commensals or mutualists evolved to benefit each other. Culture-independent studies of the human microbiota identified recently a complex symbiotic environment with more than 1000 bacterial phylotypes representing more than 7000 strains [95]. The composition of this microbiota has been suggested to be a result of a highly coevolved symbiosis and commensalism influenced by nutrition, physiology, and immunological factors. It varied with the sources of nectar and the presence of other bacterial

Products of stingless bees are consumed since before the discovery of the Americas to the present day. Honey of these bees has activities against microorganisms, having importance in the colony maintenance as a microbiologically stable environment [96]. Stingless bee honey has characteristics that confer antimicrobial character, i.e., activity against Gram-negative and Gram-positive bacteria such as *Enterococcus*, *Staphylococcus faecalis*, *Staphylococcus aureus*, *E*. *coli*, *Pseudomonas aeruginosa*, *Bacillus cereus*, and *Candida albicans* [1, 97, 98], which justifies its

However, *Meliponini* also feature mutualistic interaction with microorganisms, i.e., lactic acid bacteria are found in Australian species as *Tetragonula carbonaria*, *T*. *hockingsi*, and *Austroplebeia australis* [102]. Yeasts such as *Starmerella meliponinorum*, *Starmerella neotropicalis*, *Candida apicola*, and *Zygosaccharomyces* spp. are commonly found in the Neotropical species of stingless bees such as *Tetragonisca angustula*, *Frieseomelitta varia*, *Melipona quinquefasciata*, and *Melipona quadrifasciata* [103–105] and provide sensory and conservation to food characteristics [106–109]. About fungi, the interesting fact is that bees cultivate them as food [110] and protection against other pathogenic microorganisms [111], i.e., *Scaptotrigona aff. depilis* young larvae, needs to be fed from the mycelium of *Monascus* genus (*Ascomycotina*) to complete their development [112], which reinforces the intrinsic evolutionary relationship between microorganisms and these bees. Little is known about pathogens in stingless bees; however, there are no pathogen transfer record from *A*. *mellifera* [113], which shows the lack of information about microorganisms in *Meliponini*.

**7. Microorganism detection methodologies in honey and honeybee products**

Much has been discussed about the succession of gut microbiota among queens, workers, and larvae and the role of the diversity on the quality of honey, safety, and health of the colony [11, 53, 114–117]. New methodologies have made it possible to access information about the

genera within the honeybee and ended up eventually in the honey (**Figure 1**).

**6. Microorganisms in stingless bee honey**

use in popular medicine [6, 41, 99–101].

242 Honey Analysis

**7.1. Microbial diversity**

This access has been carried out mainly by sequencing the coding region of the 16S subunit of the bacterial ribosome [53, 121, 130], both from genomic DNA from microorganisms growing on selective media as Man-Rogosa-Sharpe agar, Sabouraud dextrose agar, and *Candida* agar [117, 120, 131, 132], such as process-independent culture as specific PCR [68], denaturing gradient gel electrophoresis [124, 125], mixed and deep 16S sequencing [49, 128], pyrosequencing [53, 116, 121], and clone library [115, 118, 120, 122]. While culturedependent methods are ideal for quantification of microorganisms and phenotypic testing, culture-independent methods generally have greater coverage in relation to the amount of different species accessed and are ideal for fingerprinting studies, and the identification of these species may be performed by real-time PCR analysis [49, 68, 125, 128]. These methods, although they have different principles, were able to distinguish similarly the narrow niche of bacterial species and the diversity of strains present in these matrices [120]. In some works, the complete genome [132, 133] or metagenome [114, 115] of the narrow range of species of microorganisms is accessed, enabling the search for specific functions of these bacteria for beehives by gene annotation, PCR screening [114], and Post-Light TM ion semiconductor sequencing [127]. Fluorescent in situ hybridization microscopy has also been used to characterize distribution and abundance of specific phyla across the life cycle and among gut organs [68]. Changes in the diversity of microbial populations found by these authors would be able to explain the transformations that occur in honey and pollen, as well as strategies of these insects to combat pathogens and invaders [11, 114, 116, 121] and beebread preservation [11, 120].

Several microorganisms present in the honey and in the gut of honeybees have antagonistic effects on honeybees and human pathogens, especially of *Bacillus* genus [123, 134], lactic acid bacteria as *Lactobacillus* [71, 121, 124, 130–132, 135], *Enterococcus* [130], *Bifidobacteria* [116, 132, 136–138], and *Acetobacteraceae* [117, 121, 133]. These same microorganisms can be accessed for other purposes, such as its potential as fermenters [116, 130, 133] or probiotics [116]. In this case, direct detection strategies of these microorganisms are not the analysis priority since their isolation is of interest to researchers for the antagonism studies. "This isolation is mainly done using traditional selective media, especially Man-Rogosa and Sharpe agar to *Lactobacillus*; *Streptococcus* selective medium and MTPY or Wilkins-Chalgren medium for *Bifidobacterium*" [71, 130, 136, 138, 139], with or without prior enrichment [133], and the identification of the isolates is mainly performed by sequencing 16S rRNA amplicons. However, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry profiling was used for acetobacterium identification from bumble bee crop [133] and clustering of lactic acid bacteria of a bumble bee gut microbiota [139]. Several studies have shown the effectiveness of these microorganisms to inhibit human pathogens such as *Staphylococcus aureus*, *Escherichia coli* O157: H7, *Salmonella*, and *Listeria* [130, 140] or pathogens of honeybees as *Melissococcus plutonius* [124, 138], the causative agent of European foulbrood and *Paenibacillus*  *larvae* [123, 124, 130, 134], and the causative agent of American foulbrood, among others. This effectiveness is generally associated with the production of acid, bacteriocins [130], and other antimicrobial molecules [140].

#### **7.2. Monitoring of the microbiological honey quality**

Traditional methods are often still used for monitoring the microbiological quality of honey used for human consumption, even as the rates established by the laws use these methods. Potato dextrose agar and yeast extract glucose chloramphenicol agar are media normally used for aerobic count and the total fungi (yeasts and molds), while Violet Red Bile and MacConkey medium agars are normally used for counting coliforms, which can also be done by the most probable number technique [119, 141, 142]. These media have recently been used to monitor the efficiency of a new filter-based method based in reducing the microbial burden and to improve the microbiological quality of honey [143]. Potato glucose agar in Brazil was also used for monitoring the honey contamination by yeast and fungi [144]. Standard plate count agar is used for monitoring of mesophilic bacteria, such as that was done in honey samples of Portugal [141, 142] and Argentina [119, 145].

## **7.3. Detection of honeybee pathogens in honey**

The honey is an important route of contamination of honeybees, spreading many microorganisms, particularly pathogens that infect the honeybees. Several molecular techniques have been developed for the detection of pathogens like *Paenibacillus larvae*, *Melissococcus plutonius*, *Nosema ceranae* and *Nosema apis* [129, 146, 147], *Ascophera apis* and *Ascophera ceranae*, and *A*. *flavus* [129, 148]. Among them can highlight the simple PCR [149–151], NESTED-PCR [152], RT-PCR [153, 154], immunology-based tests (ELISA), and probe-based hybridization analysis [155]. The main advantages of these techniques would be less needed for sample treatment which often can be applied directly to the honeybee products, fast technique, specificity, and sensitivity of detection.

The use of these techniques and the detection of this pathogen have allowed the control of mortality of honeybee populations around the world, restricting the dissemination of pathogens in bee products. For example, the diagnosis of American foulbrood and European foulbrood usually occurs through visual inspection of brood combs and detection of diseased larvae, subjective criteria that could be confused with other beehive conditions [155, 156]. The traditional methods of detection of these pathogens include the visualization by microscopy and detection in tissues [155]; culture on selective medium [151, 155, 156], including *P*. *larvae* agar [151]; bacteriophage sensitivity; immunotechniques; and microscopy of suspect bacterial strains have been considered adequate for routine identification purposes [151]; these methods are time-consuming and laborious but especially require that the infection is in progress so that the pathogen is detected and confirmed. The detection of pathogens before any clinical signs of disease to be visible in the colony would not only control these diseases but also the prevention of their consequences for the hive. That is, *M*. *plutonius* was detected in healthy colonies by RT-PCR in England and Wales, showing that the extent of the prevalence of this pathogen in hives goes beyond the clinical signs [157].

RT-PCR has been used to simultaneously detect multiple viruses such as in cases of honeybee parasitic mite syndrome where five out of seven viruses were detected in sample mite in Thailand [158]. Also, different multiplex RT-PCR were developed for the simultaneous detection of i) black queen cell virus (BQCV), deformed wing virus (DWV), Kashmir bee virus (KBV) and Sacbrood virus (SV) [159], ii) acute bee paralysis virus (ABPV), BQCV and SV [160], iii) ABPV and SV [161] iv) ABPV, chronic bee paralysis virus (CBPV), BQCV, DWV, KBV, and SV [162]. The effectiveness of this method in the detection of these pathogens was demonstrated in the simultaneous detection of these viruses in colonies [159, 160] and queens [162], where up to 93% of the queens have multiple infections [162].

Even more efficiently nine viruses (ABPV, BQCV, CBPV, DWV, KBV, SV, Israel acute paralysis virus (IAPV), *Varroa destructor* virus 1 (VDV-1), and slow paralysis virus (SPV)) were detected simultaneously in a single test developed by Glover and coworkers. These authors used a microarray technique with oligonucleotides based on DNA sequences of each of these viruses, but the time and cost of the technique are still unfeasible with its use for routine diagnosis [163].
