In Vitro Antagonistic Effect of Gut Bacteriota Isolated From Indigenous Honey Bees and Essential Oils Against Paenibacillus Larvae

Abstract

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.

Full text

Int. J. Mol. Sci. 2020, 21, 6736; doi:10.3390/ijms21186736 www.mdpi.com/journal/ijms
Article
In Vitro Antagonistic Effect of Gut Bacteriota Isolated
From Indigenous Honey Bees and Essential Oils
Against Paenibacillus Larvae
Miroslava Kačániová 1,2,*, Margarita Terentjeva 3, Jana Žiarovská 4 and
Przemysław Łukasz Kowalczewski 5,*
1 Department of Fruit Science, Viticulture and Enology, Faculty of Horticulture and Landscape Engineering,
Slovak University of Agriculture, Tr. A. Hlinku 2, 94976 Nitra, Slovakia
2 Department of Bioenergetics, Food Analysis and Microbiology, Institute of Food Technology and
Nutrition, University of Rzeszow, Cwiklinskiej 1, 35-601 Rzeszow, Poland
3 Institute of Food and Environmental Hygiene, Faculty of Veterinary Medicine, Latvia University of Life
Sciences and Technologies, K. Helmaņaiela 8, LV-3004 Jelgava, Latvia; margarita.terentjeva@llu.lv
4 Department of Plant Genetics and Breeding, Faculty of Agrobiology and Food Resources,
Slovak University of Agriculture, Tr. A. Hlinku 2, 94976 Nitra, Slovakia; jana.ziarovska@uniag.sk
5 Department of Food Technology of Plant Origin, Poznań University of Life Sciences,
31 Wojska Polskiego St., 60-624 Poznań, Poland
* Correspondence: miroslava.kacaniova@gmail.com (M.K.);
przemyslaw.kowalczewski@up.poznan.pl (P.Ł.K.); Tel.: +421-641-4715 (M.K.); +48-61-848-7297 (P.Ł.K.)
Received: 27 August 2020; Accepted: 12 September 2020; Published: 14 September 2020
Abstract: 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.
Keywords: Lactobacillus spp.; rectum; intestine; antimicrobial activity; antimicrobial resistance;
essential oils
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

Int. J. Mol. Sci. 2020, 21, 6736 2 of 19
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
TCAN
M 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

Int. J. Mol. Sci. 2020, 21, 6736 3 of 19
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).
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 (Figures 1–3). 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.
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

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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
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).

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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

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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
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.

Int. J. Mol. Sci. 2020, 21, 6736 7 of 19
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.

Int. J. Mol. Sci. 2020, 21, 6736 8 of 19
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

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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

Int. J. Mol. Sci. 2020, 21, 6736 10 of 19
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

Int. J. Mol. Sci. 2020, 21, 6736 11 of 19
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

Int. J. Mol. Sci. 2020, 21, 6736 12 of 19
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 [10–13]. 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 [12–14]. 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 [17–25] 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 [26–28]. 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 [11,29–31]. Lactobacillus spp. play significant role in feed
digestibility in animals and they are important for functioning of gastrointestinal tract and
accompanied immunological responses [32–37]. 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

Int. J. Mol. Sci. 2020, 21, 6736 13 of 19
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–
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
[61–65]. 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

Int. J. Mol. Sci. 2020, 21, 6736 14 of 19
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

Int. J. Mol. Sci. 2020, 21, 6736 15 of 19
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.
Author Contributions: Conceptualization, M.K.; data curation, M.K., M.T., J.Ž. and P.Ł.K.; investigation, M.K.,
M.T. and J.Ž.; methodology, M.K.; supervision, M.K.; writing—original draft, M.K., M.T., J.Ž. and P.Ł.K. All
authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by the grant APVV SK-BY-RD-19–0014 “The formulation of novel
compositions and properties study of the polysaccharides based edible films and coatings with antimicrobial
and antioxidant plant additives.”
Acknowledgments: The study was supported by the project Ņo. 26220220180: Building Research Center
“Agribiotech”. Marcin Nowicki (University of Tennessee, Knoxville, USA) is gratefully acknowledged for
copyediting and critical reading of this manuscript.
Conflicts of Interest: The authors declare no conflicts of interest.

Int. J. Mol. Sci. 2020, 21, 6736 16 of 19
References
1. Jeyaprakash, A.; Hoy, M.A.; Allsopp, M.H. Bacterial diversity in worker adults of Apis mellifera capensis and
Apis mellifera scutellata (Insecta: Hymenoptera) assessed using 16S rRNA sequences. J. Invertebr. Pathol. 2003,
84, 96–103, doi:10.1016/j.jip.2003.08.007.
2. Tysset, C.; Durand, C. Contribution to the study of the intestinal microbism of healthy worker bees (Apis
mellifica); reckoning and study of the constitutive groups. Bull Apic 1968, 2, 117–118.
3. Tysset, G.; Rousseau, M.; Durand, G. La présence des streptocoques du groupe D de Lancefield chez les
abeilles butineuses saines (Alpis mellifica L.) L’interprétation de leur présence en bactériologie alimentaire.
Bull. Acad. Vet. Fr. 1969, XLII, 173–186, doi:10.4267/2042/66877.
4. Scardovi, V. Genus Bifidobacterium. In Bergey’s Manual of Systematic Bacteriology; Williams and Wilkins:
Baltimore, MD, USA, 1986.
5. Miyagi, T.; Peng, C.Y.S.; Chuang, R.Y.; Mussen, E.C.; Spivak, M.S.; Doi, R.H. Verification of
Oxytetracycline-Resistant American Foulbrood Pathogen Paenibacillus larvae in the United States. J.
Invertebr. Pathol. 2000, 75, 95–96, doi:10.1006/jipa.1999.4888.
6. Shimanuki, H.; Knox, D.A. Susceptibility of Bacillus larvae to Terramycin. Am. bee J. 1994, 134, 125–126.
7. Michaud, V. Antibiotic residues in honey-the FEEDM view. Apiacta 2005, 40, 52–54.
8. Iwu, M.W.; Duncan, A.R.; Okunji, C.O.; others New antimicrobials of plant origin. In Perspectives on New
Crops and New Uses; ASHS Press: Alexandria, VA, USA, 1999; pp. 457–462.
9. Dorman, H.J.D.; Deans, S.G. Antimicrobial agents from plants: antibacterial activity of plant volatile oils. J.
Appl. Microbiol. 2000, 88, 308–316, doi:10.1046/j.1365-2672.2000.00969.x.
10. Rada, V.; Máchová, M.; Huk, J.; Marounek, M.; Dušková, D. Microflora in the honeybee digestive tract:
counts, characteristics and sensitivity to veterinary drugs. Apidologie 1997, 28, 357–365,
doi:10.1051/apido:19970603.
11. Killer, J.; Kopečný, J.; Mrázek, J.; Rada, V.; Dubná, S.; Marounek, M. Bifidobacteria in the digestive tract of
bumblebees. Anaerobe 2010, 16, 165–170, doi:10.1016/j.anaerobe.2009.07.007.
12. Kačániová, M.; Chlebo, R.; Kopernický, M.; Trakovická, A. Microflora of the honeybee gastrointestinal tract.
Folia Microbiol. (Praha) 2004, 49, 169–171, doi:10.1007/BF02931394.
13. Grubbs, K.J.; Scott, J.J.; Budsberg, K.J.; Read, H.; Balser, T.C.; Currie, C.R. Unique Honey Bee (Apis
mellifera) Hive Component-Based Communities as Detected by a Hybrid of Phospholipid Fatty-Acid and
Fatty-Acid Methyl Ester Analyses. PLoS ONE 2015, 10, e0121697, doi:10.1371/journal.pone.0121697.
14. Popa, M.; Vica, M.; Axinte, R.; Glevitzky, M.; Varvara, S. Study concerning the honey qualities in
Transylvania region. Ann. Univ. Apulensis Ser. Oeconomica 2009, 11, 1034–1040.
15. Nora, C.; Mahamed, A.L. Contribution to identification of the microflora of the digestive tract and pollen
of Algerian honeybees: Apis mellifera intermissa and Apis mellifera sahariensis. Int. J. Curr. Microbiol. Appl. Sci.
2014, 3, 601–607.
16. Yoshiyama, M.; Kimura, K. Bacteria in the gut of Japanese honeybee, Apis cerana japonica, and their
antagonistic effect against Paenibacillus larvae, the causal agent of American foulbrood. J. Invertebr. Pathol.
2009, 102, 91–96, doi:10.1016/j.jip.2009.07.005.
17. Mohr, K.I.; Tebbe, C.C. Diversity and phylotype consistency of bacteria in the guts of three bee species
(Apoidea) at an oilseed rape field. Environ. Microbiol. 2006, 8, 258–272, doi:10.1111/j.1462-2920.2005.00893.x.
18. Babendreier, D.; Joller, D.; Romeis, J.; Bigler, F.; Widmer, F. Bacterial community structures in honeybee
intestines and their response to two insecticidal proteins. FEMS Microbiol. Ecol. 2007, 59, 600–610,
doi:10.1111/j.1574-6941.2006.00249.x.
19. Cox-Foster, D.L.; Conlan, S.; Holmes, E.C.; Palacios, G.; Evans, J.D.; Moran, N.A.; Quan, P.-L.; Briese, T.;
Hornig, M.; Geiser, D.M.; et al. A Metagenomic Survey of Microbes in Honey Bee Colony Collapse
Disorder. Science (80-) 2007, 318, 283–287, doi:10.1126/science.1146498.
20. Martinson, V.G.; Danforth, B.N.; Minckley, R.L.; Rueppell, O.; Tingek, S.; Moran, N.A. A simple and
distinctive microbiota associated with honey bees and bumble bees. Mol. Ecol. 2011, 20, 619–628,
doi:10.1111/j.1365-294X.2010.04959.x.
21. Disayathanoowat, T.; Young, J.P.W.; Helgason, T.; Chantawannakul, P. T-RFLP analysis of bacterial
communities in the midguts of Apis mellifera and Apis cerana honey bees in Thailand. FEMS Microbiol. Ecol.
2012, 79, 273–281, doi:10.1111/j.1574-6941.2011.01216.x.

Int. J. Mol. Sci. 2020, 21, 6736 17 of 19
22. Ahn, J.-H.; Hong, I.-P.; Bok, J.-I.; Kim, B.-Y.; Song, J.; Weon, H.-Y. Pyrosequencing analysis of the bacterial
communities in the guts of honey bees Apis cerana and Apis mellifera in Korea. J. Microbiol. 2012, 50, 735–
745, doi:10.1007/s12275-012-2188-0.
23. Engel, P.; Martinson, V.G.; Moran, N.A. Functional diversity within the simple gut microbiota of the honey
bee. Proc. Natl. Acad. Sci. USA 2012, 109, 11002–11007, doi:10.1073/pnas.1202970109.
24. Schwarz, R.S.; Moran, N.A.; Evans, J.D. Early gut colonizers shape parasite susceptibility and microbiota
composition in honey bee workers. Proc. Natl. Acad. Sci. USA 2016, 113, 9345–9350,
doi:10.1073/pnas.1606631113.
25. Sabree, Z.L.; Hansen, A.K.; Moran, N.A. Independent Studies Using Deep Sequencing Resolve the Same
Set of Core Bacterial Species Dominating Gut Communities of Honey Bees. PLoS ONE 2012, 7, e41250,
doi:10.1371/journal.pone.0041250.
26. Singh, S.; Saini, K.; Jain, K.L. Quantitative comparison of lipids in some pollens and their phagostimulatory
effects in honey bees. J. Apic. Res. 1999, 38, 87–92, doi:10.1080/00218839.1999.11100999.
27. Singh, R.; Levitt, A.L.; Rajotte, E.G.; Holmes, E.C.; Ostiguy, N.; VanEngelsdorp, D.; Lipkin, W.I.;
DePamphilis, C.W.; Toth, A.L.; Cox-Foster, D.L. RNA Viruses in Hymenopteran Pollinators: Evidence of
Inter-Taxa Virus Transmission via Pollen and Potential Impact on Non-Apis Hymenopteran Species. PLoS
ONE 2010, 5, e14357, doi:10.1371/journal.pone.0014357.
28. McFrederick, Q.S.; Wcislo, W.T.; Taylor, D.R.; Ishak, H.D.; Dowd, S.E.; Mueller, U.G. Environment or kin:
whence do bees obtain acidophilic bacteria? Mol. Ecol. 2012, 21, 1754–1768, doi:10.1111/j.1365-
294X.2012.05496.x.
29. Olofsson, T.C.; Vásquez, A. Detection and Identification of a Novel Lactic Acid Bacterial Flora Within the
Honey Stomach of the Honeybee Apis mellifera. Curr. Microbiol. 2008, 57, 356–363, doi:10.1007/s00284-008-
9202-0.
30. Reuter, G. The Lactobacillus and Bifidobacterium microflora of the human intestine: composition and
succession. Curr. Issues Intest. Microbiol. 2001, 2, 43–53.
31. Ouwehand, A.C.; Salminen, S.; Isolauri, E. Probiotics: an overview of beneficial effects. Antonie Van
Leeuwenhoek 2002, 82, 279–89.
32. Sazawal, S.; Hiremath, G.; Dhingra, U.; Malik, P.; Deb, S.; Black, R.E. Efficacy of probiotics in prevention of
acute diarrhoea: a meta-analysis of masked, randomised, placebo-controlled trials. Lancet Infect. Dis. 2006,
6, 374–382, doi:10.1016/S1473-3099(06)70495-9.
33. Rafter, J.; Bennett, M.; Caderni, G.; Clune, Y.; Hughes, R.; Karlsson, P.C.; Klinder, A.; O’Riordan, M.;
O’Sullivan, G.C.; Pool-Zobel, B.; et al. Dietary synbiotics reduce cancer risk factors in polypectomized and
colon cancer patients. Am. J. Clin. Nutr. 2007, 85, 488–496, doi:10.1093/ajcn/85.2.488.
34. Younts-Dahl, S.M.; Galyean, M.L.; Loneragan, G.H.; Elam, N.A.; Brashears, M.M. Dietary Supplementation
with Lactobacillus- and Propionibacterium-Based Direct-Fed Microbials and Prevalence of Escherichia coli
O157 in Beef Feedlot Cattle and on Hides at Harvest. J. Food Prot. 2004, 67, 889–893, doi:10.4315/0362-028X-
67.5.889.
35. Younts-Dahl, S.M.; Osborn, G.D.; Galyean, M.L.; Rivera, J.D.; Loneragan, G.H.; Brashears, M.M. Reduction
of Escherichia coli O157 in Finishing Beef Cattle by Various Doses of Lactobacillus acidophilus in Direct-Fed
Microbials. J. Food Prot. 2005, 68, 6–10, doi:10.4315/0362-028X-68.1.6.
36. Nocek, J.E.; Kautz, W.P. Direct-Fed Microbial Supplementation on Ruminal Digestion, Health, and
Performance of Pre- and Postpartum Dairy Cattle. J. Dairy Sci. 2006, 89, 260–266, doi:10.3168/jds.S0022-
0302(06)72090-2.
37. Chaucheyras-Durand, F.; Madic, J.; Doudin, F.; Martin, C. Biotic and Abiotic Factors Influencing In Vitro
Growth of Escherichia coli O157:H7 in Ruminant Digestive Contents. Appl. Environ. Microbiol. 2006, 72, 4136–
4142, doi:10.1128/AEM.02600-05.
38. Kačániová, M.; Gasper, J.; Brindza, J.; Schubertová, Z.; Ivanišová, E. Bacteria Of Apis Mellifera
Gastrointestinal Tract: Counts, Identification And Their Antibiotic Resistance. In Agrobiodiversity for
Improving Nutrition, Health and Life Quality; Agrobionet: Nitra, Slovakia, 2017; pp. 210–215.
39. Koch, H.; Schmid-Hempel, P. Socially transmitted gut microbiota protect bumble bees against an intestinal
parasite. Proc. Natl. Acad. Sci. USA 2011, 108, 19288–19292, doi:10.1073/pnas.1110474108.
40. Li, J.; Powell, J.E.; Guo, J.; Evans, J.D.; Wu, J.; Williams, P.; Lin, Q.; Moran, N.A.; Zhang, Z. Two gut
community enterotypes recur in diverse bumblebee species. Curr. Biol. 2015, 25, R652–R653,
doi:10.1016/j.cub.2015.06.031.

Int. J. Mol. Sci. 2020, 21, 6736 18 of 19
41. Jones, B.M.; Wcislo, W.T.; Robinson, G.E. Developmental Transcriptome for a Facultatively Eusocial Bee,
Megalopta genalis. G3 Genes Genomes Genet. 2015, 5, 2127–2135, doi:10.1534/g3.115.021261.
42. Potts, S.G.; Biesmeijer, J.C.; Kremen, C.; Neumann, P.; Schweiger, O.; Kunin, W.E. Global pollinator
declines: trends, impacts and drivers. Trends Ecol. Evol. 2010, 25, 345–353, doi:10.1016/j.tree.2010.01.007.
43. Genersch, E.; Forsgren, E.; Pentikäinen, J.; Ashiralieva, A.; Rauch, S.; Kilwinski, J.; Fries, I. Reclassification
of Paenibacillus larvae subsp. pulvifaciens and Paenibacillus larvae subsp. larvae as Paenibacillus larvae without
subspecies differentiation. Int. J. Syst. Evol. Microbiol. 2006, 56, 501–511, doi:10.1099/ijs.0.63928-0.
44. Genersch, E.; Ashiralieva, A.; Fries, I. Strain- and Genotype-Specific Differences in Virulence of Paenibacillus
larvae subsp. larvae, a Bacterial Pathogen Causing American Foulbrood Disease in Honeybees. Appl.
Environ. Microbiol. 2005, 71, 7551–7555, doi:10.1128/AEM.71.11.7551-7555.2005.
45. Evans, J.D. Diverse origins of tetracycline resistance in the honey bee bacterial pathogen Paenibacillus larvae.
J. Invertebr. Pathol. 2003, 83, 46–50, doi:10.1016/S0022-2011(03)00039-9.
46. Mutinelli, F.; Rademacher, E. The use of drugs to control varroosis in honey bee colonies and European
legislation: the current situation. Bee World 2003, 84, 55–59, doi:10.1080/0005772X.2003.11099577.
47. Thompson, H.M.; Waite, R.J.; Wilkins, S.; Brown, M.A.; Bigwood, T.; Shaw, M.; Ridgway, C.; Sharman, M.
Effects of European foulbrood treatment regime on oxytetracycline levels in honey extracted from treated
honeybee (Apis mellifera) colonies and toxicity to brood. Food Addit. Contam. 2005, 22, 573–578,
doi:10.1080/02652030500089986.
48. Vásquez, A.; Forsgren, E.; Fries, I.; Paxton, R.J.; Flaberg, E.; Szekely, L.; Olofsson, T.C. Symbionts as Major
Modulators of Insect Health: Lactic Acid Bacteria and Honeybees. PLoS ONE 2012, 7, e33188,
doi:10.1371/journal.pone.0033188.
49. D Evans, J.; Armstrong, T.-N. Inhibition of the American foulbrood bacterium, Paenibacillus larvae larvae, by
bacteria isolated from honey bees. J. Apic. Res. 2005, 44, 168–171, doi:10.1080/00218839.2005.11101173.
50. Evans, J.D.; Armstrong, T.-N. Antagonistic interactions between honey bee bacterial symbionts and
implications for disease. BMC Ecol. 2006, 6, 4, doi:10.1186/1472-6785-6-4.
51. Chen, Y.-W.; Wang, C.-H.; An, J.; Kai-Kuang, H. Susceptibility of the Asian honey bee, Apis cerana, to
American foulbrood, Paenibacillus larvae larvae. J. Apic. Res. 2000, 39, 169–175,
doi:10.1080/00218839.2000.11101038.
52. De Vuyst, L.; Leroy, F. Bacteriocins from Lactic Acid Bacteria: Production, Purification, and Food
Applications. J. Mol. Microbiol. Biotechnol. 2007, 13, 194–199, doi:10.1159/000104752.
53. Alippi, A.M. Is Terramycin®losing its effectiveness against AFB? Bee Biz 2000, 11, 27–29.
54. Martirani, L.; Varcamonti, M.; Naclerio, G.; De Felice, M. Purification and partial characterization of
bacillocin 490, a novel bacteriocin produced by a thermophilic strain of Bacillus licheniformis. Microb. Cell
Fact. 2002, 1, 1, doi:10.1186/1475-2859-1-1.
55. Alqarni, A.; Hannan, M.; Owayss, A.; Engel, M. The indigenous honey bees of Saudi Arabia (Hymenoptera,
Apidae, Apis mellifera jemenitica Ruttner): Their natural history and role in beekeeping. Zookeys 2011, 134,
83–98, doi:10.3897/zookeys.134.1677.
56. Alghamdi, A. The Comprehensive Study of the Mite, Varroa Destructor on Honeybees Apis mellifera; Indigenous
and Imported; Bee Research Unit, PhD Department, College of Food and Agriculture Sciences, King Saud
University: Riyadh, Saudi Arabia, 2002;
57. Ansari, M.J.; Al-Ghamdi, A.; Usmani, S.; Al-Waili, N.; Nuru, A.; Sharma, D.; Khan, K.A.; Kaur, M.; Omer,
M. In vitro evaluation of the effects of some plant essential oils on Paenibacillus larvae, the causative agent
of American foulbrood. Biotechnol. Biotechnol. Equip. 2016, 30, 49–55, doi:10.1080/13102818.2015.1086690.
58. Erler, S.; Moritz, R.F.A. Pharmacophagy and pharmacophory: mechanisms of self-medication and disease
prevention in the honeybee colony (Apis mellifera). Apidologie 2016, 47, 389–411, doi:10.1007/s13592-015-
0400-z.
59. Kuzyšinová, K.; Mudroňová, D.; Toporčák, J.; Molnár, L.; Javorský, P. The use of probiotics, essential oils
and fatty acids in the control of American foulbrood and other bee diseases. J. Apic. Res. 2016, 55, 386–395,
doi:10.1080/00218839.2016.1252067.
60. Kaufman, P.; Dayanandan, P.; Li, C.; McKenzie, M.; Hoyt, J.; Kirakosyan, A. The Uses of Plant Natural
Products by Humans and Risks Associated with Their Use. In Natural Products from Plants, 2nd ed.; CRC
Press: Boca Raton, FL, USA, 2006; pp. 441–473.
61. Fuselli, S.R. Antimicrobial activity of some Argentinean wild plant essential oils against Paenibacillus larvae
larvae, causal agent of American foulbrood (AFB). J. Apic. Res. 2006, 6, 2–7, doi:10.3896/IBRA.1.45.1.02.

Int. J. Mol. Sci. 2020, 21, 6736 19 of 19
62. Fuselli, S.R.; de la Rosa, S.B.G.; Eguaras, M.J.; Fritz, R. Susceptibility of the Honeybee Bacterial Pathogen
Paenibacillus larvae to Essential Oils Distilled from Exotic and Indigenous Argentinean Plants. J. Essent. Oil
Res. 2008, 20, 464–470, doi:10.1080/10412905.2008.9700060.
63. Gende, L.B.; Floris, I.; Fritz, R.; Eguaras, M.J. Antimicrobial activity of cinnamon (Cinnamomum zeylanicum)
essential oil and its main components against Paenibacillus larvae from Argentine. Bull. Insectol. 2008, 61, 1–
4.
64. Antúnez, K.; Harriet, J.; Gende, L.; Maggi, M.; Eguaras, M.; Zunino, P. Efficacy of natural propolis extract
in the control of American Foulbrood. Vet. Microbiol. 2008, 131, 324–331, doi:10.1016/j.vetmic.2008.04.011.
65. Alippi, A.M.; Reynaldi, F.J. Inhibition of the growth of Paenibacillus larvae, the causal agent of American
foulbrood of honeybees, by selected strains of aerobic spore-forming bacteria isolated from apiarian
sources. J. Invertebr. Pathol. 2006, 91, 141–146, doi:10.1016/j.jip.2005.12.002.
66. Colin, M.E.; de Lahitte, J.D.; Larribau, E.; Boué, T. Activité des huiles essentielles de Labiées sur Ascophaera
apis et traitement d’un rucher. Apidologie 1989, 20, 221–228.
67. Floris, I.; Carta, C. In vivo activity of Cinnamomum zeylanicum Nees essential oil against Bacillus larvae White.
Apicoltura 1990, 57–61.
68. Fuselli, S.R.; García de la Rosa, B.; Eguaras, M.J.; Fritz, R. In vitro antibacterial effect of exotic plants
essential oils on the honeybee pathogen Paenibacillus larvae, causal agent of American foulbrood. Spanish J.
Agric. Res. 2010, 8, 651–657, doi:10.5424/sjar/2010083-1261.
69. González, M.J.; Marioli, J.M. Antibacterial activity of water extracts and essential oils of various aromatic
plants against Paenibacillus larvae, the causative agent of American Foulbrood. J. Invertebr. Pathol. 2010, 104,
209–213, doi:10.1016/j.jip.2010.04.005.
70. Rovná, K.; Ivanišová, E.; Žiarovská, J.; Ferus, P.; Terentjeva, M.; Kowalczewski, P.Ł.; Kačániová, M.
Characterization of Rosa canina Fruits Collected in Urban Areas of Slovakia. Genome Size, iPBS Profiles and
Antioxidant and Antimicrobial Activities. Molecules 2020, 25, 1888, doi:10.3390/molecules25081888.
71. Kačániová, M.; Gasper, J.; Terentjeva, M.; Kunová, S.; Kluz, M.; Puchalski, C. Antibacterial Activity of Bees
Gut Lactobacilli against Paenibacillus Larvae In Vitro. Adv. Res. Life Sci. 2018, 2, 7–10, doi:10.1515/arls-2018-
0020.
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