In Vitro Antagonistic Effect of Gut Bacteriota Isolated from Indigenous Honey Bees and Essential Oils against Paenibacillus Larvae.

The aim of study was to isolate and identify the gut bacteria of Apis mellifera and to evaluate antagonistic effect of the bacteriota against Paenibacillus larvae, which causes American foulbrood (AFB) in honeybees. The dilution plating method was used for the quantification of selected microbial groups from digestive tract of bees, with an emphasis on the bacteriota of the bees' intestines. Bacteria were identified using mass spectrometry (MALDI-TOF-MS Biotyper). Overall, five classes, 27 genera and 66 species of bacteria were identified. Genera Lactobacillus (10 species) and Bacillus (8 species) were the most abundant. Gram-negative bacteria were represented with 16 genera, whereas Gram-positive with 10 genera. Delftia acidovorans and Escherichia coli were the most abundant in the digestive tract of honey bee. Resistance to a selection of antimicrobials was assessed for the bacterial isolates from bee gut and confirmed against all antimicrobials included in the study, with the exception of cefepime. Lactobacillus spp., especially L. kunkeei, L. crispatus and L. acidophilus. showed the strongest antimicrobial activity against P. larvae, the causal pathogen of AFB. Antimicrobial activity of essential oils against isolated bacteria and two isolates of P. larvae were assessed. Application of a broad selection of plant essential oils indicated that Thymus vulgaris had the highest antimicrobial activity against P. larvae.

1. Introduction

The digestive tract of the worker bee is inhabited with a variety of microorganisms diverse in their morphology, physiology and metabolism. The microbiota of digestive tract consists of yeasts (1%), Gram-positive bacteria (29%) and Gram-negative and gram-variable bacteria (70%) [1]. The first research on microbiota of digestive tract of bees had been published in the beginning of the 20th century and Lactobacillus rigidus apis, Lactobacillus constellatus and Bacillus influenzoides apis were found the main representatives of digestive tract microbiota. Subsequent reports on microflora studies of bees and microorganisms in their diet were published in the 1960s [2,3]. It has been agreed that the only probiotic bacteria species present belonged to Bifidobacteria [4].

American foulbrood (AFB) is a disease caused by aerobic to microaerophilic, Gram-positive, spore-forming rod, Paenibacillus larvae. The disease causes huge economic losses to beekeepers around the world [5]. P. larvae affects honey bee larvae in period when it takes food, rendering the bee larvae more susceptible between 12 to 48 h of life. Bacterial spores germinate in the gut of larvae, bacteria multiply and kill the larvae at pre-pupal or pupal stage. Infected larvae are settled at the bottom of the cells with sunken sealed brood appearance. The disease is highly contagious as more than 2.5 billion oval spores could be produced in 10 days. AFB does not affect the adult bees, but they facilitate the spread of infection within a colony [6].

The use of antimicrobials in beekeeping is permitted in the United States and is also used in South America and some East Asian countries. In the European Union, the application of antimicrobials in beekeeping is banned in some countries [7].

In recent years, there has been a growing interest in application of natural substances, including for pathogen and pest control: chemical compounds of plant secondary metabolism, extracts or vegetable oils supporting green consumer behavior and healthy lifestyles trends. The diversity of plants stimulates the search and research of new plant-based chemical compounds. Some of identified compounds share antimicrobial activity against pathogenic microorganisms and have even appeared in controlled clinical trials [8]. In particular, the essential oils and mixtures of mono- and sesquiterpenes are known for their strong antimicrobial activity. The possible applications include, inter alia, food production, medicines or cosmetics industries [9].

Therefore, the aims of this study were: (i) to isolate and identify bacteria from the digestive tract of adult honeybee workers (Apis mellifera), (ii) evaluate the antagonistic effects of selected bacteria from the bee gut against the bacteria P. larvae and (iii) detect antimicrobial activity of essential oils against P. larvae.

2. Results

2.1. Bacteriota of Adult Worker Bees (Apis mellifera)

Groups of bacteria isolated from the digestive tract of summer and winter adult worker bees are shown in Table 1. The highest counts of aerobic microorganisms were found in the intestine of winter bees (5.39 ± 0.14 log cfu/g) and the lowest in the rectum of summer bees (4.48 ± 0.13 log cfu/g). The total counts of anaerobic microorganisms ranged from 8.12 ± 0.06 in the intestine of summer bees to 9.25 ± 0.15 log cfu/g in the rectum of winter bees. Anaerobic Gram-positive microorganisms counts ranged from 6.13 ± 0.09 for summer bees in the intestine to 7.10 ± 0.12 log cfu/g for winter bees in the rectum. The lowest counts of Bacillus spp. were found in the intestine of winter bees (2.48 ± 0.09 log cfu/g) and the highest were found in the winter bees in the rectum (3.53 ± 0.07 log cfu/g). The lowest counts of Lactobacillus spp. were found in the intestine of winter bees (7.14 ± 0.06) whereas the highest were found in the rectum of winter bees (8.27 ± 0.11). The coliform bacteria counts were the highest in the rectum of the winter bees (3.57 ± 0.13) whereas the lowest counts were in the intestines of the winter bees (2.52 ± 0.11). There were statistically significant differences among all groups of microorganisms (p ≤ 0.05, p ≤ 0.01).

Table 1

Isolated bacteriota of adult worker honeybee guts in in log cfu/g (mean ± SD).

	Bee Gut from Intestine		Bee Gut from Rectum
	Winter Bees Samples	Summer Bees Samples	Winter Bees Samples	Summer Bees Samples
TCAM *	5.39 ± 0.14 a	5.03 ± 0.16 ab	5.00 ± 0.22 abc	4.48 ± 0.13 abc
TCANM	8.38 ± 0.11 a	8.12 ± 0.06 b	9.25 ± 0.15 ab	9.05 ± 0.09 ab
AG+	6.49 ± 0.13 a	6.13 ± 0.09 ab	7.10 ± 0.12 abc	6.77 ± 0.11 abc
BS	2.48 ± 0.09 a	3.43 ± 0.16 ab	3.53 ± 0.07 ac	3.22 ± 0.10 abc
LS	7.14 ± 0.06 a	7.66 ± 0.14 ab	8.27 ± 0.11 ab	8.12 ± 0.06 ab
PS	2.55 ± 0.06 a	2.29 ± 0.13 ab	3.12 ± 0.07 abc	2.85 ± 0.15 abc
ES	3.21 ± 0.08 a	3.42 ± 0.12 ab	2.24 ± 0.10 abc	2.53 ± 0.15 abc
SS	3.22 ± 0.09 a	3.45 ± 0.08 ab	2.56 ± 0.19 abc	2.25 ± 0.07 abc
CB	2.52 ± 0.11 a	3.25 ± 0.13 ab	3.57 ± 0.13 abc	3.37 ± 0.14 ac

* TCAM—total counts of aerobic microorganisms, TCANM—total counts of anaerobic microorganisms, AG+—anaerobic Gram-positive bacteria, BS—Bacillus spp., LS—Lactobacillus spp., PS—Pseudomonas spp., ES—Enterococcus spp., SS—Staphylococcus spp., CB—coliform bacteria. a,b,c same letters in the raw show statistically significant differences among the groups.

2.2. Isolated Bacteria from Bees Gut

A total of five classes of bacteria were obtained from the gut of the honey bee: Actinobacteria, Alphaproteobacteria, Betaproteobacteria, Firmicutes and Gammaproteobacteria. A total of 27 genera were isolated from the honey bee bacteriota: Aeromonas, Arthrobacter, Bacillus, Citrobacter, Delftia, Enterobacter, Enterococcus, Escherichia, Fructobacillus, Hafnia, Klebsiella, Kocuria, Lactobacillus, Lactococcus, Microbacterium, Moraxella, Morganella, Paenibacillus, Pantotea, Proteus, Pseudomonas, Rahnella, Ralstonia, Raoultella, Serratia, Sphingomonas and Staphylococcus. A total of 66 species were isolated from bees, of which the genus Lactobacillus represented by 10 species and the genus Bacillus by eight species were the most numerous (Table 2).

Table 2

Isolated species of adult worker honeybee bacteriota from gastrointestinal tract.

Class	Genus	Species
Gammaproteobacteria	Aeromonas	Aeromonas salmonicida
Actinobacteria	Arthrobacter	Arthrobacter tumbae
Firmicutes	Bacillus	Bacillus cereus
Firmicutes	Bacillus	Bacillus circulans
Firmicutes	Bacillus	Bacillus licheniformis
Firmicutes	Bacillus	Bacillus megaterium
Firmicutes	Bacillus	Bacillus oleronius
Firmicutes	Bacillus	Bacillus spp.
Firmicutes	Bacillus	Bacillus subtilis
Firmicutes	Bacillus	Bacillus thuringiensis
Gammaproteobacteria	Citrobacter	Citrobacter spp.
Gammaproteobacteria	Citrobacter	Citrobacter braakii
Gammaproteobacteria	Citrobacter	Citrobacter koseri
Betaproteobacteria	Delftia	Delftia acidovorans
Gammaproteobacteria	Enterobacter	Enterobacter aerogenes
Gammaproteobacteria	Enterobacter	Enterobacter clocae
Gammaproteobacteria	Enterobacter	Enterobacter kobei
Firmicutes	Enterococcus	Enterococcus cloacae
Firmicutes	Enterococcus	Enterococcus faecalis
Gammaproteobacteria	Escherichia	Escherichia coli
Firmicutes	Fructobacillus	Fructobacillus fructosus
Gammaproteobacteria	Hafnia	Hafnia alvei
Gammaproteobacteria	Klebsiella	Klebsiella aerogenes
Gammaproteobacteria	Klebsiella	Klebsiella oxytoca
Gammaproteobacteria	Klebsiella	Klebsiella pneumoniae
Gammaproteobacteria	Klebsiella	Klebsiella variicola
Actinobacteria	Kocuria	Kocuria kristinae
Firmicutes	Lactobacillus	Lactobacillus acidophilus
Firmicutes	Lactobacillus	Lactobacillus agilis
Firmicutes	Lactobacillus	Lactobacillus apis
Firmicutes	Lactobacillus	Lactobacillus brevis
Firmicutes	Lactobacillus	Lactobacillus crispatus
Firmicutes	Lactobacillus	Lactobacillus jensenii
Firmicutes	Lactobacillus	Lactobacillus kunkeei
Firmicutes	Lactobacillus	Lactobacillus mellis
Firmicutes	Lactobacillus	Lactobacillus plantarum
Firmicutes	Lactobacillus	Lactobacillus spp.
Firmicutes	Lactococcus	Lactococcus garvieae
Firmicutes	Lactococcus	Lactococcus lactis
Actinobacteria	Microbacterium	Microbacterium pumilum
Actinobacteria	Microbacterium	Microbacterium testaceum
Gammaproteobacteria	Moraxella	Moraxella spp.
Gammaproteobacteria	Morganella	Morganella morgani
Firmicutes	Paenibacillus	Paenibacillus larvae
Gammaproteobacteria	Pantotea	Pantotea agglomerans
Gammaproteobacteria	Pantotea	Pantotea ananatis
Gammaproteobacteria	Pantotea	Pantotea vagans
Gammaproteobacteria	Proteus	Proteus mirabilis
Gammaproteobacteria	Pseudomonas	Pseudomonas marginalis
Gammaproteobacteria	Pseudomonas	Pseudomonas oryzihabitans
Gammaproteobacteria	Pseudomonas	Pseudomonas putida
Gammaproteobacteria	Rahnella	Rahnella aquatilis
Gammaproteobacteria	Rahnella	Rahnella terrigena
Betaproteobacteria	Ralstonia	Ralstonia picketii
Gammaproteobacteria	Raoultella	Raoultella ornithinolytica
Gammaproteobacteria	Raoultella	Raoultella planticola
Gammaproteobacteria	Serratia	Serratia fonticola
Gammaproteobacteria	Serratia	Serratia liquefaciens
Gammaproteobacteria	Serratia	Serratia marcescens
Alphaproteobacteria	Sphingomonas	Sphingomonas parapaucimobilis
Alphaproteobacteria	Sphingomonas	Sphingomonas melonis
Firmicutes	Staphylococcus	Staphylococcus capitis
Firmicutes	Staphylococcus	Staphylococcus epidermidis
Firmicutes	Staphylococcus	Staphylococcus hemolyticus
Firmicutes	Staphylococcus	Staphylococcus hominis
Firmicutes	Staphylococcus	Staphylococcus warneri

In total, there were 10 genera of the Gram-positive and 16 genera of the Gram-negative bacteria isolated in the study. MALDI-TOF-MS Biotyper identification score for Lactococcus garvieae ranged from 2.015 to 2.026, Kocuria kristinae from 2.035 to 2.563, Staphylococcus capitis from 2.035 to 2.503, Staphylococcus epidermidis from 2.050 to 2.445, Staphylococcus hemolyticus from 2.041 to 2.341, Staphylococcus hominis from 2.150 to 2.345, Staphylococcus warneri from 2.053 to 2.545, Hafnia alvei from 2.296 to 2.563, Morganella morganii from 2.198 to 2.578, Pantoea ananatis from 2.196 to 2.363, Pantoea agglomerans ranged 2.371 to 2.466, Raoultella ornithinolytica from 2.051 to 2.550, Raoultella planticola from 2.198 to 2.428 and Serratia fonticola from 2.190 to 2.251, indicating reliable identification of bacterial species. Similarly, high scores were achieved for the other identified species. From the taxonomic point of view, 42.8% of isolates belonged to the class Gammaproteobacteria, whereas 43.9% to Firmicutes, 4.8% to Betaproteobacteria, 4.3% to Actinobacteria and 4.2% to the class Alphaproteobacteria (Figure 1, Figure 2 and Figure 3). Isolates of Gram-negative bacteria belonged to the families Aeromonadaceae, Comamonadaceae, Enterobacteriaceae, Pseudomonadaceae, Ralstoniaceae and Sphingomonadaceae of Proteobacteria phylum. Gram-positive bacteria belonged to the families of Bacillaceae, Enterococcaceae, Lactobacillaceae, Lactococcaceae, Microbacteriaceae, Micrococcaceae, Paenibacillaceae, Staphylococcaceae of phyla Actinobacteria and Firmicutes.

Figure 1

Krona RSF display of total bacteriota isolated from bee digestive tracts. Presented are the frequencies of detected species, genera and classes, from the outer ring inwards.

Figure 2

Krona RSF display of Gram-negative bacteriota isolated from bee digestive tracts. Presented are the frequencies of detected species, genera and classes, from the outer ring inwards.

Figure 3

Krona RSF display of Gram-positive bacteriota isolated from bee digestive tracts. Presented are the frequencies of detected species, genera and classes, from the outer ring inwards.

A total of 66 species of bacteria from the digestive tract of bees were isolated, of which 33 were Gram-positive and 33 Gram-negative. Escherichia coli was isolated most frequently from all samples tested, but P. larvae was isolated from only one sample (Table 3).

Table 3

Frequency of isolated bacteriota (%) detected in the samples of bee digestive tract.

Species	No. of Isolates/No. of Samples	No. of Positive Samples (%)
Aeromonas salmonicida	15/12	6.00
Arthrobacter tumbae	21/15	7.50
Bacillus cereus	51/25	12.50
Bacillus circulans	35/10	5.00
Bacillus licheniformis	64/25	12.50
Bacillus megaterium	128/96	48.00
Bacillus oleronius	25/20	10.00
Bacillus spp.	125/52	26.00
Bacillus subtilis	56/35	17.50
Bacillus thuringiensis	68/42	21.00
Citrobacter spp.	188/112	56.00
Citrobacter braakii	37/15	7.50
Citrobacter koseri	60/30	15.00
Delftia acidovorans	150/200	100.00
Enterobacter aerogenes	136/110	55.00
Enterobacter clocae	126/99	49.50
Enterobacter kobei	59/32	16.00
Enterococcus cloacae	56/15	7.50
Enterococcus faecalis	150/100	50.00
Escherichia coli	350/200	100.00
Fructobacillus fructosus	29/11	5.50
Hafnia alvei	218/169	84.50
Klebsiella aerogenes	59/28	14.00
Klebsiella oxytoca	98/58	29.00
Klebsiella pneumoniae	36/12	6.00
Klebsiella variicola	45/15	7.50
Kocuria kristinae	186/125	62.50
Lactobacillus acidophilus	64/30	15.00
Lactobacillus agilis	55/20	10.00
Lactobacillus apis	123/69	34.50
Lactobacillus brevis	150//100	50.00
Lactobacillus crispatus	164//88	44.00
Lactobacillus jensenii	15/10	5.00
Lactobacillus kunkeei	135/120	60.00
Lactobacillus mellis	64/35	17.50
Lactobacillus plantarum	95/80	40.00
Lactobacillus spp.	167/150	75.00
Lactococcus garvieae	121/90	45.00
Lactococcus lactis	68/39	19.50
Microbacterium pumilum	15/5	2.50
Microbacterium testaceum	25/10	5.00
Moraxella spp.	55/15	7.50
Morganella morgani	115/100	50.00
Paenibacillus larvae	1/1	0.50
Pantotea agglomerans	52/40	20.00
Pantotea ananatis	65/30	15.00
Pantotea vagans	87/58	29.00
Proteus mirabilis	120/95	47.50
Pseudomonas marginalis	12/3	1.50
Pseudomonas oryzihabitans	65/50	25.00
Pseudomonas putida	35/15	7.50
Rahnella aquatilis	65/40	20.00
Rahnella terrigena	35/22	11.00
Ralstonia picketii	126/110	55.00
Raoultella ornithinolytica	69/52	26.00
Raoultella planticola	35/15	7.50
Serratia fonticola	95/95	47.50
Serratia liquefaciens	87/58	29.00
Serratia marcescens	64/30	15.00
Sphingomonas parapaucimobilis	125/100	50.00
Sphingomonas melonis	120/60	30.00
Staphylococcus capitis	136/120	60.00
Staphylococcus epidermidis	168/62	31.00
Staphylococcus haemolyticus	58/35	17.50
Staphylococcus hominis	112/90	45.00
Staphylococcus warneri	64/52	26.00

2.3. Antibiotic Resistance of A. mellifera Gut Bacteriota

A total of 5789 isolates were isolated from the digestive tract of 200 bees. Gram-positive and Gram-negative bacteria showed antimicrobial resistance to various classes of antimicrobials (Table 4).

Table 4

Antimicrobial resistance of bacteria isolated from bee digestive tracts.

Antimicrobial	CEF	CIP	–	–
Resistance/Sensitivity	R/S	R/S	–	–
Aeromonas salmonicida	0/15	0/15	–	–
Arthrobacter tumbae	ND	ND	–	–
Bacillus cereus	ND	ND	–	–
Bacillus circulans	ND	ND	–	–
Bacillus licheniformis	ND	ND	–	–
Bacillus megaterium	ND	ND	–	–
Bacillus oleronius	ND	ND	–	–
Bacillus spp.	ND	ND	–	–
Bacillus subtilis	ND	ND	–	–
Bacillus thuringiensis	ND	ND	–	–
	TIC	IMI	CIP	CHL
	R/S	R/S	R/S	R/S
Citrobacter spp.	8/188	25/188	0/188	45/188
Citrobacter braakii	6/37	15/37	5/37	10/37
Citrobacter koseri	16/60	10/60	5/60	14/60
Delftia acidovorans	ND	ND	ND	ND
	TIC	IMI	CIP	CHL
	R/S	R/S	R/S	R/S
Enterobacter aerogenes	61/136	25/136	10/136	22/136
Enterobacter clocae	28/126	5/136	1/136	6/136
Enterobacter kobei	9/59	5/59	0/59	0/59
	IMI	TEI	TIG	–
	R/S	R/S	R/S	–
Enterococcus cloacae	5/56	6/56	11/56	–
Enterococcus faecalis	58/150	10/150	25/150	–
	TIC	IMI	CIP	CHL
	R/S	R/S	R/S	R/S
Escherichia coli	53/350	26/350	12/350	10/350
Fructobacillus fructosus	ND	ND	ND	ND
Hafnia alvei	15/218	12/218	5/218	5/218
Klebsiella aerogenes	42/59	25/59	15/59	5/59
Klebsiella oxytoca	63/98	35/98	15/98	10/98
Klebsiella pneumoniae	14/36	10/36	5/36	1/36
Klebsiella variicola	5/45	10/45	4/45	5/45
Kocuria kristinae	ND	ND	ND	ND
	AMP	IMI	MER	CHL
	R/S	R/S	R/S	R/S
Lactobacillus acidophilus	4/64	0/64	0/64	0/64
Lactobacillus agilis	2/55	3/55	2/55	0/55
Lactobacillus apis	16/123	10/123	8/123	5/123
Lactobacillus brevis	15/150	20/150	10/150	15/150
Lactobacillus crispatus	25/164	38/164	5/164	6/164
Lactobacillus jensenii	0/15	0/15	0/15	0/15
Lactobacillus kunkeei	52/135	25/135	15/135	10/135
Lactobacillus mellis	2/64	0/64	1/64	0/64
Lactobacillus plantarum	50/95	20/95	10/95	10/95
Lactobacillus spp.	0/167	0/167	0/167	0/167
Lactococcus garvieae	ND	ND	ND	ND
Lactococcus lactis	ND	ND	ND	ND
Microbacterium pumilum	ND	ND	ND	ND
Microbacterium testaceum	ND	ND	ND	ND
Moraxella spp.	ND	ND	ND	ND
	TIC	IMI	CIP	CHL
	R/S	R/S	R/S	R/S
Morganella morgani	65/115	35/115	25/115	15/115
Paenibacillus larvae	ND	ND	ND	ND
Pantotea agglomerans	15/52	15/52	10/52	10/52
Pantotea ananatis	10/65	15/65	15/65	10/65
Pantotea vagans	37/87	30/87	15/87	10/87
Proteus mirabilis	25/120	15/120	16/120	10/120
	TIC	IMI	CIP	TOB
	R/S	R/S	R/S	R/S
Pseudomonas marginalis	5/12	4/12	2/12	0/12
Pseudomonas oryzihabitans	30/65	20/65	10/65	10/65
Pseudomonas putida	5/35	5/35	5/35	5/35
	TIC	IMI	CIP	CHL
	R/S	R/S	R/S	R/S
Rahnella aquatilis	24/65	20/65	12/65	8/65
Rahnella terrigena	5/35	0/35	0/35	0/35
Ralstonia picketii	ND	ND	ND	ND
Raoultella ornithinolytica	29/69	20/69	10/69	10/69
Raoultella planticola	15/35	20/35	10/35	5/35
Serratia fonticola	45/95	30/95	15/95	5/95
Serratia liquefaciens	25/87	32/87	16/87	10/87
Serratia marcescens	16/64	12/64	5/64	2/64
Sphingomonas parapaucimobilis	ND	ND	ND	ND
Sphingomonas melonis	ND	ND	ND	ND
	TIG	LIN	CIP	CHL
	R/S	R/S	R/S	R/S
Staphylococcus capitis	15/136	25/136	20/136	10/136
Staphylococcus epidermidis	60/168	30/168	15/168	5/168
Staphylococcus haemolyticus	28/58	15/58	10/58	5/58
Staphylococcus hominis	41/112	23/112	16/112	7/112
Staphylococcus warneri	5/64	15/64	10/64	5/64

CEF—cefepime; CIP—ciprofloxacin; TIC—ticarcillin; IMI—imipenem; CHL—chloramphenicol; TEI—teicoplanin; TIG—tigecycline; LIN—linezolid; TOB—tobramycin; AMP—ampicillin; MER–meropenem. ND—not defined. R– resistant; S—sensitive.

2.4. Antimicrobial Activity of Isolated Bee Digestive Tract Bacteriome against P. larvae

The interactions between intestinal bacteria and pathogens of A. mellifera, in particular the action of intestinal bacteria against P. larvae, are an area of great research interest. Research on microbial composition of digestive tract of A. mellifera are perspective from the bee’s health point of view. The research on antagonisms of P. larvae may promote the development of bee-friendly compounds, to protect the bees from infection with pathogens.

All microorganisms tested showed antimicrobial activity against P. larvae. The strongest antimicrobial activity was shown by Lactobacillus, whereas the weakest was typical for Enterobacteriaceae (Table 5). Among the species analyzed, L. kunkei, L. crispatus, L. acidophilus were the most active against P. larvae. Klebsiella variicola, Ralstonia picketii, Pantotea agglomerans, Pa. vagans and Serratia liquefaciens were less active against P. larvae isolated from bee intestines. The strongest antimicrobial activity of L. kunkei, L. acidophilus and L. crispatus and the weakest antimicrobial activity of Pa. ananatis and Rahnella aquatilis were found against P. larvae CCM 4483.

Table 5

Antimicrobial activity of individual isolates against P. larvae in mm (mean ± SD of three replicates).

Species	P. larvae	P. larvae CCM 4483
Aeromonas salmonicida	10.67 ± 0.58	10.33 ± 0.58
Arthrobacter tumbae	9.67 ± 1.15	8.67 ± 0.58
Bacillus cereus	14.33 ± 0.58	13.67 ± 0.58
Bacillus circulans	14.67 ± 1.15	14.33 ± 0.58
Bacillus licheniformis	15.67 ± 0.58	16.33 ± 1.15
Bacillus megaterium	11.67 ± 0.58	11.33 ± 0.58
Bacillus oleronius	10.33 ± 1.15	10.67 ± 0.58
Bacillus spp.	9.33 ± 0.58	8.67 ± 0.58
Bacillus subtilis	12.33 ± 0.58	11.67 ± 0.58
Bacillus thuringiensis	12.33 ± 1.15	11.67 ± 1.15
Citrobacter spp.	8.67 ± 0.58	6.67 ± 1.53
Citrobacter braakii	8.33 ± 1.53	7.33 ± 1.15
Citrobacter koseri	6.33 ± 1.53	7.67 ± 0.58
Delftia acidovorans	11.67 ± 1.15	11.33 ± 0.58
Enterobacter aerogenes	8.67 ± 0.58	6.67 ± 1.53
Enterobacter clocae	8.33 ± 1.53	7.33 ± 1.15
Enterobacter kobei	6.33 ± 1.53	7.67 ± 0.58
Enterococcus cloacae	14.67 ± 0.58	14.33 ± 0.58
Enterococcus faecalis	16.33 ± 1.53	16.33 ± 0.58
Escherichia coli	15.67 ± 0.58	15.33 ± 0.58
Fructobacillus fructosus	18.67 ± 0.58	18.33 ± 0.58
Hafnia alvei	8.33 ± 1.53	7.33 ± 1.15
Klebsiella aerogenes	6.33 ± 1.53	7.67 ± 0.58
Klebsiella oxytoca	7.67 ± 0.58	8.33 ± 0.58
Klebsiella pneumoniae	7.33 ± 0.58	6.67 ± 0.58
Klebsiella variicola	5.33 ± 0.58	4.67 ± 0.58
Kocuria kristinae	11.33 ± 0.58	10.67 ± 0.58
Lactobacillus acidophilus	23.33 ± 0.58	22.67 ± 0.58
Lactobacillus agilis	18.67 ± 0.58	18.33 ± 0.58
Lactobacillus apis	20.33 ± 0.58	20.67 ± 0.58
Lactobacillus brevis	19.33 ± 0.58	19.00 ± 1.00
Lactobacillus crispatus	20.33 ± 1.15	19.67 ± 1.15
Lactobacillus jensenii	20.33 ± 0.58	20.33 ± 1.15
Lactobacillus kunkeei	25.67 ± 1.15	24.33 ± 0.58
Lactobacillus mellis	18.67 ± 1.15	17.67 ± 0.58
Lactobacillus plantarum	22.33 ± 0.58	21.67 ± 0.58
Lactobacillus spp.	17.00 ± 1.00	17.33 ± 0.58
Lactococcus garvieae	16.67 ± 0.58	16.33 ± 0.58
Lactococcus lactis	17.67 ± 0.58	17.33 ± 0.58
Microbacterium pumilum	13.67 ± 0.58	13.33 ± 0.58
Microbacterium testaceum	12.67 ± 0.58	12.33 ± 0.58
Moraxella spp.	8.67 ± 0.58	6.67 ± 1.53
Morganella morgani	8.33 ± 1.53	7.33 ± 1.15
Pantotea agglomerans	6.33 ± 1.53	7.67 ± 0.58
Pantotea ananatis	8.67 ± 0.58	6.67 ± 1.53
Proteus mirabilis	8.33 ± 1.53	7.33 ± 1.15
Pantotea vagans	6.33 ± 1.53	7.67 ± 0.58
Pseudomonas marginalis	11.33 ± 0.58	10.67 ± 0.58
Pseudomonas oryzihabitans	11.33 ± 1.15	11.00 ± 1.00
Pseudomonas putida	10.67 ± 0.58	10.33 ± 0.58
Rahnella aquatilis	8.67 ± 0.58	6.67 ± 1.53
Rahnella terrigena	8.33 ± 1.53	7.33 ± 1.15
Ralstonia picketii	6.33 ± 1.53	7.67 ± 0.58
Raoultella ornithinolytica	8.67 ± 0.58	6.67 ± 1.53
Raoultella planticola	8.33 ± 1.53	7.33 ± 1.15
Serratia fonticola	8.67 ± 0.58	6.67 ± 1.53
Serratia liquefaciens	8.33 ± 1.53	7.33 ± 1.15
Serratia marcescens	6.33 ± 1.53	7.67 ± 0.58
Sphingomonas parapaucimobilis	11.67 ± 1.15	11.33 ± 0.58
Sphingomonas melonis	10.67 ± 0.58	10.33 ± 0.58
Staphylococcus capitis	13.67 ± 0.58	13.33 ± 0.58
Staphylococcus epidermidis	14.67 ± 0.58	14.33 ± 0.58
Staphylococcus haemolyticus	13.67 ± 0.58	13.33 ± 0,58
Staphylococcus hominis	12.67 ± 0.58	12.33 ± 0.58
Staphylococcus warneri	11.67 ± 0.58	11.33 ± 0.58

2.5. Antimicrobial Activity of Essential Oils against P. larvae

The next aim of the work was to determine the antimicrobial activity of essential oils against two strains of P. larvae. The highest antimicrobial activity (Table 6) was recorded for Thymus vulgaris (19.67 ± 1.53 mm and 15.67 ± 1.53), Origanum vulgare (18.67 ± 1.15 and 19.00 ± 1.00 mm, respectively) and Pinus montana (17.67 ± 0.58 and 17.33 ± 0.58 mm, respectively). The lowest antimicrobial activity was recorded for Citrus sinensis (2.00 ± 1.00 mm).

Table 6

Antimicrobial activity of essential oils against P. larvae in mm.

Essential Oil	P. larvae	P. larvae CCM 4483
Lavandula angustifolia Mill.	14.33 ± 1.15	15.33 ± 0.58
Cinnamomum zeylanicum L.	10.00 ± 1.00	12.33 ± 2.52
Pinus montana Mill.	17.67 ± 0.58	17.33 ± 0.58
Mentha piperita L.	7.33 ± 0.58	7.00 ± 2.00
Foeniculum vulgare Mill.	14.66 ± 0.58	14.00 ± 0.57
Pinus sylvestris L.	17.00 ± 1.00	17.67 ± 0.57
Satureja hortensis L.	12.33 ± 0.58	17.67 ± 1.53
Origanum vulgare L.	18.67 ± 1.15	19.00 ± 1.00
Pimpinella anisum L.	12.33 ± 0.58	11.67 ± 0.58
Rosmarinus officinalis L.	14.67 ± 0.58	10.00 ± 1.00
Salvia officinalis L.	14.33 ± 0.58	13.00 ± 1.00
Abies alba Mill.	17.33 ± 0.58	18.00 ± 1.00
Citrus aurantium var. dulce L.	4.33 ± 0.58	3.00 ± 1.00
Citrus sinensis L. Osbeck.	2.00 ± 1.00	5.33 ± 0.58
Cymbopogon nardus L.	8.67 ± 0.58	8.00 ± 1.00
Mentha spicata var. crispa L.	9.67 ± 1.53	9.33 ± 0.57
Thymus vulgaris L.	19.67 ± 1.53	15.67 ± 1.53
Carvum carvi L.	7.67 ± 0.58	5.00 ± 0.58
Thymus serpyllum L.	4.33 ± 0.58	7.33 ± 0.58
Amyris balsamifera	9.33 ± 0.58	9.67 ± 0.58
Ocimum basilicum	13.67 ± 1.15	14.00 ± 1.00
Canarium luzonicum Miq.	11.33 ± 1.15	12.33 ± 0.58
Eucalyptus globulus	16.33 ± 1.15	17.33 ± 0.58
Gaultheria procumbens	8.33 ± 0.58	7.33 ± 0.58
Pelargonium graveolens	6.67 ± 0.58	7.33 ± 0.58
Cinnamomum caphora var. linalolifera	16.00 ± 1.73	15.67 ± 1.15
Boswellia carterii	7.67 ± 1.15	7.00 ± 1.00
Melaleuca leucadendron	9.67 ± 0.58	9.33 ± 0.58
Litsea cubeba Pers.	10.33 ± 0.58	10.66 ± 0.58
Melaleuca ericifolia Smith.	9.67 ± 0.58	10.00 ± 1.00

3. Discussion

The highest counts of the aerobic microorganisms, Bacillus spp., Lactobacillus spp. and coliform bacteria were found in the intestine of winter bees and the lowest in the rectum of summer bees. Similar results of bacterial counts have been reported previously [10,11,12,13]. The microbiome of bees represents not only the microorganisms present in the adult worker bees, but also reflects the hive microbiota. The origin of hive microorganisms are nectar, pollen, dust and other airborne and soilborne environmental contaminants [12,13,14]. The excrement of honey bees and animals could be a source of microbiota during nectar harvesting. A wide variation in bacteria associated with bees have been ascribed to the external environment [15]. The bacteriota of the digestive tract of the Japanese eastern bee (Apis cerana japonica) revealed that Bacillus species could be potential antagonists for biologic control of P. larvae [16].

Non-culture studies of bee microbiome were conducted on the digestive tract or only on the middle and posterior parts of the intestines [17,18,19,20,21,22,23,24,25] and revealed that the pollination-based environmental microbiota and the four nectar-bearing ones are an important source of the beneficiary and potentially beneficiary microorganisms for bees [26,27,28]. Lactobacillus spp. were frequently found in the bee intestines and were considered the most important genus of lactic acid bacteria (LAB) in promoting animal and human health [11,29,30,31]. Lactobacillus spp. play significant role in feed digestibility in animals and they are important for functioning of gastrointestinal tract and accompanied immunological responses [32,33,34,35,36,37]. In our study, we did not identify species from the Bifidobacterium genus.

Antimicrobial resistance of the bacterial isolates varied in our study, depending on the genus and strain properties. Kačániová et al. [38] found resistance to tigecycline (12.5%) and amikacin (18.2%), gentamicin (9.5%) and chloramphenicol (7.2%) in their bacteriome of honey bees. Administration of antimicrobials triggers changes in the microbiome of humans and livestock, therefore, assessment of the effect of the antimicrobials on bee intestinal microorganisms is important for their health prognosis [23,24,39,40] and a possible explanation of unexpected bee colony deaths [41]. The studies on microbiome diversity and its antimicrobial resistance can provide an overview on nutritional and health problems of honey bees [42].

American foulbrood (AFB) is the most destructive bacterial disease of honey bee larvae [43]. AFB is a contagious infection that begins in an individual bee larva and can cause the collapse of the entire colony because only a few spores of P. larvae are necessary to initiate the disease [44].

The use of antimicrobials, especially oxytetracycline, could protect the bees hives against infection, however, P. larvae resistance to oxytetracycline has been identified in the USA, Argentina and Canada [5,45]. Use of antimicrobials in beekeeping poses a serious risk to human health as their residues may persist in honey and other bee products [46]. Adverse effects of application of antimicrobials on the honey of honey bees [47] and on the beneficial intestinal bacteria [48] have been described.

The biologic control of AFB pathogen is considered an environmentally conscious and bee-friendly perspective. Evans and Armstrong [49,50] found that certain intestinal bacteria of A. mellifera showed antagonistic activity against P. larvae. Eastern Japanese bee (Apis cerana japonica), native to Japan, exhibited resistance against parasitic and microbial pathogens, including mite and AFB pathogen [51]. The antagonistic effect of bacteria may also depend on bacterial communities present or strains properties, including production of antimicrobial substances, e.g., bacteriocins and lysozyme and changes in pH as a result of organic acids production [52]. Bacteria with antagonistic properties enhance control or inhibition of pathogens. Bacillus spp. were found to exhibit bactericidal and fungicidal effects in the host gut as a result of production of various antimicrobial compounds [53,54]. Apis mellifera jemenitica was shown as biologically better adapted to harsh environment with higher productivity [55,56].

Several natural compounds were studied for antagonistic activity against P. larvae in vitro [57,58,59], however, the identified cytotoxic effects on bees had limited their practical application. Alternatives, such as prevention and control methods of the AFB pathogen are an area of great interest. Since the ancient times, the herbal medicine and herbal extracts were applied for treatment of human and animal diseases [60]. Biologically active compounds of honey, propolis, essential oils, agents from spore of bacteria of honey and fungal extract of pollen were tested against AFB pathogen [61,62,63,64,65]. Of these, essential oils showed the strongest antibacterial activity against microorganisms responsible for bee diseases without toxicity on bees in vitro. The main complication in those studies is to obtain the results applicable to beekeeping related to the antimicrobial activity of essential oils and their effect on bees [66,67]. In our study, Thymus vulgaris was the most effective essential oil against both species of P. larvae, whereas the most effective essential oils against P. larvae CCM4483 were those from Pinus silvestris and Abies alba.

Tests of Melaleuca viridiflora and Cymbopogon nardus essential oils against P. larvae have shown an inhibition at 320 mg/L in vitro [68]. Almost all essential oils of Achyrocline satureioides, Chenopodium ambrosioide, Eucalyptus cinerea, Gnaphalium gaudichaudianum, Lippia turbinata, Marrubium vulgare, Minthostachys verticillata, Origanum vulgare, Tagetes minuta and Thymus vulgaris were effective against P. larvae strains. Eucalyptus cinerea and M. verticillata essential oils exhibited 100% efficiency in inhibiting the growth of all P. larvae strains [69]. Essential oils of Schinus molle var. areira L., Acantholippia seriphioides A. Gray, Mintosthachys mollis, Tagetes minuta L. and Lippia turbinata Griseb grown in wild in Argentina shared minimum and maximum MIC and MBC values of 200–250 mg/L and 200–300 mg/L for Andean thyme and 800–1000 mg/L and 850–1100 mg/L. Andean thyme has been shown to be the most effective in vitro against P. larvae and could be a perspective natural alternative to the traditional antimicrobial treatment of AFB pathogen [61].

4. Materials and Methods

4.1. Samples of Bees

A total of 200 samples of Apis mellifera carnica workers were examined. Samples of bees were taken from hives from the eastern Slovakia in the Košice area (48.7164° N, 21.2611° E). Bees were sampled in winter and summer, with samples from the digestive tract (intestines and rectum). examined separately. Workers of honey bees were anesthetized on ice and washed in 86% ethanol before dissection. The head or thorax of a honeybee was fixed and the entire intestine was removed by pulling the stinger using sterile dissecting forceps. The intestines and rectum were separated and collected into sterile, separate microcentrifuge tubes.

The basic dilution (10−2) was obtained by homogenizing 0.1 g of the digestive tract contents of five bees and 9.9 mL of peptone saline (0.89%). Selection for groups of microorganisms followed as shown in Table 7. All agars were purchased from Oxoid (Basingstoke, United Kingdom).

Table 7

Incubation conditions of bacteriota of the intestine of honey bees.

Microorganisms Group	Dilution	Agar	Inoculation Method	Cultivation Condition
				Relation of O2	Temperature	Time
TCAM	10−5–10−7	PCA	surface	aerobic	30 °C	48 h
TCANM	10−5–10−7	PCA	surface	anaerobic	25 °C	48 h
AG+	10−3–10−6	AA	surface	anaerobic	37 °C	48 h
Bacillus spp.	10−3–10−5	PCA	surface	aerobic	30 °C	48 h
Lactobacillus spp.	10−2–10−6	MRS	surface	aerobic	37 °C	48 h
Pseudomonas spp.	10−3–10−5	Pseudomonas agar	surface	aerobic	30 °C	48 h
Enterococcus spp.	10−3–10−5	Enterococcus selective agar	surface	aerobic	37 °C	48 h
Staphylococcus spp.	10−2–10−4	Blood agar	surface	aerobic	37 °C	48 h
CB	10−4–10−6	McC	surface	aerobic	37 °C	48 h

TCAM—total counts of aerobic microorganisms; TCNANM—total counts of anaerobic microorganisms; AG+—anaerobic Gram-positive bacteria; CB—coliform bacteria; PCA—plate count agar; AA—anaerobic agar; MRS—Main Rogosa agar; McC—MacConkey agar.

4.2. Identification of Bacteria

Identification of bacteriota was performed using MALDI-TOF-MS Biotyper (Bruker Daltonics, Bremen, Germany). All the preparatory stages for the samples were carried out according to the MALDI-TOF-MS Biotyper manufacturer’s recommendations. Bacterial colonies were transferred into 300 μL of distilled water and 900 µL of ethanol in Eppendorf tubes, which were centrifuged for 2 min at 14,000 rpm. The supernatant was removed, and centrifugation was repeated for the pellet, which was subsequently allowed to dry. Ten microliters of 70% formic acid and 10 μL of acetonitrile were added to the dried pellet. Tubes were centrifuged for 2 min at 14,000 rpm and 1 μL of the supernatant was applied for identification with the MALDI-TOF. Matrix, α-cyano-4-hydroxycinnamic acid in a volume of 1 μL, was added to that 1 µL of supernatant and allowed to dry. The analysis was performed with a Microflex LT (Bruker Daltonics, Bremen, Germany) instrument and Flex Control 3.4 software and Biotyper Realtime Classification 3.1 with BC specific software. Confidence scores of ≥2.0 and ≥1.7 were the criteria for successful identification at the levels of species and genus, respectively [70].

4.3. Antimicrobial Resistance Testing

Antimicrobial susceptibility tests were carried out using the disc diffusion method, whereas the antimicrobial resistance of Lactobacillus spp. was assessed using MIC E-tests. Antimicrobial resistance against cefepime (CEF, 30 μg), ciprofloxacin (CIP, 10 μg), ticarcillin (TIC, 10 μg), imipenem (IMI, 10 μg), chloramphenicol (CHL, 10 μg), teicoplanin (TEI, 30 μg), tigecycline (TIG,15 μg), linezolid (LIN, 10 μg), tobramycin (TOB, 10 μg), ampicillin (AMP, 10 μg) or meropenem (MER, 10 μg) (Oxoid, Basingstoke, UK) was examined. Bacteria strains were cultured on Muller Hinton agar for 24 h at 37 °C, suspended in sterile distilled water at approximately 105 cells/mL (A620 = 0.388, equivalent to a McFarland standard) and used for testing. The diameters of inhibition zones were measured after incubation. Three replicates were tested for each isolate strain.

For Lactobacillus spp. strains, the MICs (μg/mL) of AMP, MER, IMI and CHL were evaluated using the commercial E-test® (Oxoid, Basingstoke, UK). The concentrations of antimicrobials ranged from 0.016 to 256 μg/mL. Bacterial cultures in exponential growth phase were adjusted to a suitable turbidity (106 to 107 CFU/mL) and used for inoculation of iso-sensitized agar (90% w/v, Oxoid, UK) supplemented with main Rogosa agar (MRS) or TPY agar (10% w/v) (Oxoid, Basingstoke, UK). E-test strips were placed on the surface of the inoculated agar and incubated at 37 °C for 24 h microaerophilically. The MIC test result was interpreted as the point at which the ellipse intersected the E-test strip as described in the E-test technical guide.

4.4. Antimicrobial Activity of Bacterial Suspensions against P. larvae

Bacterial strains after 24 h of incubation on MRS and tryptone soya agar (TSA) medium were centrifuged at 5500× g for 10 min at 4 °C and 0.1 mL of the supernatant was used for detection of activity against P. larvae. A suspension (0.1 mL, 105 CFU/mL) was plated on Mueller–Hinton agar. Filter paper discs (6 mm diameter) were impregnated with 15 μL of supernatant from each bacteria and placed on the P. larvae-inoculated agar. The agars were incubated initially at 4 °C for 2 h and then at 37 °C for 16 h. All tests were performed in triplicate. Filter discs impregnated with 10 μL of distilled water were used as a negative control and antibiotics (amikacin, 10 μg and gentamicin, 10 μg) were used as a positive control [71]. Two P. larvae isolates were tested in this study: one isolate was from bee hive and second isolate was purchased (P. larvae CCM 4483) from the Czech collection of microorganisms (Brno, Czech Republic).

4.5. Antimicrobial Activity of Essential Oils against P. larvae

For testing their antimicrobial activity, 30 essential oils purchased from Hanus s.r.o., Slovakia were used in the present study: Lavandula angustifolia Mill., Cinnamomum zeylanicum L., Pinus montana Mill., Mentha piperita L., Foeniculum vulgare Mill., Pinus sylvestris L., Satureja hortensis L., Origanum vulgare L., Pimpinella anisum L., Rosmarinus officinalis L., Salvia officinalis L., Abies alba Mill., Citrus aurantium var. dulce L., Citrus sinensis L. Osbeck., Cymbopogon nardus L., Mentha spicata var. crispa L., Thymus vulgaris L., Carvum carvi L., Thymus serpyllum L., Amyris balsamifera, Ocimum basilicum, Canarium luzonicum Miq., Eucalyptus globulus, Gaultheria procumbens, Pelargonium graveolens, Cinnamomum caphora var. Linalolifera, Boswellia carterii, Melaleuca leucadendron, Litsea cubeba Pers. and Melaleuca ericifolia Smith. The inoculation and testing technique was as described in Section 4.3.

4.6. Statistical Analyses

All measurements were made in triplicate. Statistical processing of data of the bacterial counts was performed using Microsoft Excel® software. Bacterial counts and measurements of inhibition zones were expressed as the means and standard deviation (SD). Student’s t-test was used for calculation of significance of variability in distribution of bacteria among seasons as well as among different parts of bee gut for individual groups of analysed microorganisms. Significance of the results was considered at the following thresholds: p ≤ 0.05, p ≤ 0.01, p ≤ 0.001.

5. Conclusions

Understanding of bacteriome inhabiting the intestine of bees has a potential to help beekeepers and promote bee health. Apis mellifera is the most important pollinator insect in means of global food security. Our studies on characterization and functional role of the bee’s intestinal microbiota reveal the unique properties of A. mellifera bacteriota. EU prohibited antibiotics in beekeeping practice and P. larvae after antibiotics treatments can develop resistance. Natural antimicrobials as probiotic bacteria and essential oils can play the biggest role in control of bee pathogens.

The antimicrobials may cause an alteration in bee gut microbiota so the studies of beneficiary intestinal bacteria, which may increase colony resistance to various bee’s pathogens, is a promising alternative to bee’s antimicrobial treatment. Essential oils showed the inhibitory effect on P. larvae isolated from bees, so the application of essential oils may be expanded in beekeeping. Therefore, the present results on the antimicrobial activity of bee-beneficial bacteria and essential oils from plants can help increase the beekeepers’ awareness of these possibilities and possibly reduce bee colony mortality on a global scale.