Journal of Apicultural Research

ISSN: 0021-8839 (Print) 2078-6913 (Online) Journal homepage: https://www.tandfonline.com/loi/tjar20

In vitro antagonistic potential of gut bacteria

isolated from indigenous honey bee race of Saudi
Arabia against Paenibacillus larvae

Ahmad Al-Ghamdi, Amal Abdullah Al-Abbadi, Khalid Ali Khan, Hamed Ali
Ghramh, Ashraf M. Ahmed & Mohammad Javed Ansari

To cite this article: Ahmad Al-Ghamdi, Amal Abdullah Al-Abbadi, Khalid Ali Khan, Hamed Ali
Ghramh, Ashraf M. Ahmed & Mohammad Javed Ansari (2020): In�vitro antagonistic potential of
gut bacteria isolated from indigenous honey bee race of Saudi Arabia against Paenibacillus�larvae,
Journal of Apicultural Research, DOI: 10.1080/00218839.2019.1706912

To link to this article: https://doi.org/10.1080/00218839.2019.1706912

Published online: 14 Jan 2020.

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Journal of Apicultural Research, 2020
https://doi.org/10.1080/00218839.2019.1706912

ORIGINAL RESEARCH ARTICLE

In vitro antagonistic potential of gut bacteria isolated from indigenous
honey bee race of Saudi Arabia against Paenibacillus larvae
Ahmad Al-Ghamdia§, Amal Abdullah Al-Abbadib, Khalid Ali Khanc,d,e§, Hamed Ali Ghramhc,d,e§,
Ashraf M. Ahmedf and Mohammad Javed Ansaria,g
a
Bee Research Chair, Department of Plant Protection, College of Food and Agriculture Sciences, King Saud University, Riyadh, Saudi Arabia;
b
Department of Plant Production and Protection, Faculty of Agricultural Technology, Al-Balqa Applied University, Salt, Jordan; cUnit of Bee
Research and Honey Production, Faculty of Science, King Khalid University, Abha, Saudi Arabia; dResearch Center for Advanced Materials
Science (RCAMS), King Khalid University, Abha, Saudi Arabia; eBiology Department, Faculty of Science, King Khalid University, Abha, Saudi
Arabia; fDepartment of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia; gDepartment of Botany, Hindu College
Moradabad, Moradabad, Uttar Pradesh, India

(Received 19 May 2018; accepted 26 September 2019)

Paenibacillus larvae is one of the major bacterial pathogens of honey bee broods and the causative agent of American foul-
brood (AFB) disease. The factors responsible for the pathogenesis of AFB disease are still not fully understood, and the
increasing resistance of P. larvae to commonly used antibiotics requires a search for new agents to control this disease. An
in vitro screen was carried out to determine the antagonistic activity of gut bacteria isolated from indigenous honey bees,
Apis mellifera jemenitica of Saudi Arabia against P. larvae. The gut bacterial isolates were evaluated individually against P. larvae
ATCC9545 strain by the disc diffusion method. Seven of the 100 evaluated gut bacterial isolates, Fructobacillus fructosus
(KY027123); Proteus mirabilis (KY027132); Bacillus licheniformis (KY027142); Lactobacillus kunkeei (KY027158); Bacillus subtilis
(KY027169); Enterobacter kobei (KY027178); and Morganella morganii (KY027186) showed strong inhibitory effects. To our
knowledge, this the first demonstration of antagonistic activity of F. fructosus, P. mirabilis, E. kobei, and M. morganii isolated
from honey bee gut against P. larvae. The tested gut isolates exhibited significant antimicrobial activities against P. larvae, and
they could play an important role in the treatment or prevention of AFB disease.
Keywords: antagonistic activity; gut bacteria; Paenibacillus larvae; Apis mellifera jemenitica

Introduction the peritrophic matrix (Garcia-Gonzalez & Genersch,

Honey bees (Apis mellifera L.) are the most valuable
insects, both economically and ecologically, to mankind,
as they are important pollinators throughout the world.
Sustainable agriculture and vigorous non-agricultural eco-
systems are directly linked to their fitness. In most coun-
tries, the health of this important insect has become a
serious concern, as it has been threatened by numerous
pathogens including fungi, bacteria, viruses, and protozoa
(Genersch, 2010). American foulbrood (AFB), caused by
P. larvae, a gram-positive spore producing bacterium, is
the most devastating bacterial disease in honey bee larvae
(Genersch et al., 2006). AFB is contagious, and infection,
which starts in an individual bee larva, can cause an entire
colony to collapse (Ansari et al., 2017).
Infection is initiated by spores of this bacterium, and
the bee larva is most vulnerable to AFB during the first
36 hours after eclosion. During this period, just a small
number of spores is sufficient to start disease
(Genersch, Ashiralieva, & Fries, 2005). The spores start
to germinate in the larval midgut after ingestion and
grow rapidly for several days without breaking down
the epithelium of the midgut (Yue, Nordhoff, Wieler, &
Genersch, 2008). Then, the invading bacteria destroy
Corresponding author. Email: mjavedansari@gmail.com
§
Al-Ghamdi and Khalid Ali Khan contributed equally in this Research Paper.
2013) and the epithelial membrane in the later stages of
infection, and the bacteria enter the hemocoel (Djukic
et al., 2014). Rupturing of the epithelial membrane
results in larval death (Yue et al., 2008).
AFB disease spreads rapidly throughout the hive
through the exchange of hive materials among bee colo-
nies, handling numerous hives in a limited area, trading
of packaged bees, and bee products, including honey
(Genersch, 2010). The spores are viable for long peri-
ods and can survive in unfavorable environmental condi-
tions (Hansen & Brødsgaard, 1999), hence the control
of AFB is very difficult. In many countries, burning
infected bee colonies and hive materials is the only
practical measure to control this disease. Application of
antibiotics, especially oxytetracycline (OTC), is another
management practice for infected beehives. However,
OTC-resistance P. larvae has been reported to emerge
in the USA, Argentina, and Canada (Evans, 2003; Miyagi
et al., 2000). The use of antibiotics in apiculture is a ser-
ious concern for human health because residues can
persist in honey and other bee products (Mutinelli,
2003). Antibiotics have also adverse effects on honey
bee brood (Thompson et al., 2005) and beneficial gut

ß 2020 International Bee Research Association

2 A. Al-Ghamdi et al.

Table 1. Bacterial isolates from gut of Apis mellifera jemenitica, sampling areas, culture media, and zone of inhibition.
Accession Identification based Culture Sampling ZOI mean diameter
Isolates No. on 16S rRNA broth area (mm) ± SD
Highly AMJ 210 KY027142 Bacillus licheniformis TSB Al Baha 7.67 ± 0.5b
effective AMJ 226 KY027158 Lactobacillus kunkeei MRS Al Baha 7.33 ± 0.5b
AMJ 237 KY027169 Bacillus subtilis TSB Al Baha 7.33 ± 1.1b
AMJ 124 KY027123 Fructobacillus fructosus MRS Riyadh 6.67 ± 1.1b
AMJ 246 KY027178 Enterobacter kobei TSB Al Baha 6.33 ± 2.1b
AMJ 254 KY027186 Morganella morganii TSB Al Baha 5.33 ± 0.5bc
AMJ 133 KY027132 Proteus mirabilis BHI Riyadh 5.00 ± 1.7bcd
OTC — — — — 14.33 ± 2.1a
Moderately AMJ 215 KY027147 Lactobacillus kunkeei MRS Al Baha 3.00 ± 1.7cde
effective AMJ 204 KY027136 Lactobacillus kunkeei MRS Al Baha 2.33 ± 0.5f
AMJ 223 KY027155 Lactobacillus kunkeei MRS Al Baha 2.33 ± 0.5def
AMJ 127 KY027126 Fructobacillus fructosus MRS Riyadh 2.00 ± 1.7ef
Minimally AMJ 229 KY027161 Morganella morganii BHI Al Baha 1.67 ± 0.5ef
effective AMJ 261 KY027193 Morganella morganii BSM Al Baha 1.67 ± 2.1ef
AMJ 110 KY027110 Bacillus thuringiensis MRS Riyadh 1.33 ± 1.1ef
AMJ 118 KY027117 Enterobacter cloacae BHI Riyadh 1.33 ± 1.1ef
AMJ 221 KY027153 Microbacterium pumilum BHI Al Baha 1.33 ± 0.5ef
AMJ 239 KY027171 Proteus mirabilis BHI Al Baha 1.33 ± 1.5ef
AMJ 245 KY027177 Bacillus circulans TSB Al Baha 1.33 ± 0.5ef
Amj-1 KX268228 Bacillus subtilis TSB Al Baha 1.33 ± 1.1ef
AMJ 102 KY027102 Bacillus circulans TSB Riyadh 1.00 ± 1.0ef
AMJ 116 KY027115 Fructobacillus fructosus MRS Riyadh 1.00 ± 1.0ef
AMJ 121 KY027120 Bacillus thuringiensis MRS Riyadh 1.00 ± 1.1ef
AMJ 125 KY027124 Bacillus anthracis MRS Riyadh 1.00 ± 1.1ef
AMJ 126 KY027125 Enterobacter cloacae BSM Riyadh 1.00 ± 0.0ef
AMJ 132 KY027131 Klebsiella pneumoniae BSM Riyadh 1.00 ± 1.0ef
AMJ 234 KY027166 Lactobacillus kunkeei MRS Al Baha 1.00 ± 1.7ef
AMJ 253 KY027185 Lactococcus lactis MRS Al Baha 1.00 ± 1.7ef
AMJ 260 KY027192 Enterobacteriaceae bacterium TSB Al Baha 1.00 ± 1.0ef
AMJ 262 KY027194 Enterococcus faecalis MRS Al Baha 1.00 ± 1.7ef
Amj-2 KT901802 Bacillus cereus BHI Riyadh 1.00 ± 1.7ef
Ineffective AMJ 107 KY027107 Bacillus thuringiensis MRS Riyadh 0.67 ± 1.1f
AMJ 114 KY027100 Lactobacillus kunkeei MRS Riyadh 0.67 ± 1.1f
AMJ 131 KY027130 Proteus mirabilis BHI Riyadh 0.67 ± 0.5f
AMJ 225 KY027157 Fructobacillus fructosus MRS Al Baha 0.67 ± 1.1f
AMJ 228 KY027160 Lactobacillus kunkeei MRS Al Baha 0.67 ± 1.1f
AMJ 232 KY027164 Enterobacter kobei BSM Al Baha 0.67 ± 1.1f
AMJ 243 KY027175 Bacillus circulans BHI Al Baha 0.67 ± 1.1f
AMJ 250 KY027182 Bacillus circulans BHI Al Baha 0.67 ± 1.1f
AMJ 252 KY027184 Enterobacter xiangfangensis BSM Al Baha 0.67 ± 1.1f
AMJ 257 KY027189 Enterobacter kobei BSM Al Baha 0.67 ± 0.5f
AMJ 258 KY027190 Morganella morganii BHI Al Baha 0.67 ± 1.1f
Amj-3 KT901804 Planomicrobium okeanokoites BHI Riyadh 0.67 ± 0.5f
AMJ 106 KY027106 Fructobacillus fructosus MRS Riyadh 0.33 ± 0.5f
AMJ 109 KY027109 Klebsiella pneumoniae BHI Riyadh 0.33 ± 0.5f
AMJ 112 KY027112 Exiguobacterium acetylicum BHI Riyadh 0.33 ± 0.5f
AMJ 122 KY027121 Staphylococcus haemolyticus BHI Riyadh 0.33 ± 0.5f
AMJ 227 KY027159 Proteus mirabilis BSM Al Baha 0.33 ± 0.5f
AMJ 242 KY027174 Enterobacter xiangfangensis TSB Al Baha 0.33 ± 0.5f
AMJ 244 KY027176 Enterobacteriaceae bacterium TSB Al Baha 0.33 ± 0.5f
AMJ 248 KY027180 Morganella morganii BSM Al Baha 0.33 ± 0.5f
AMJ 101 KY027101 Enterobacter aerogenes MRS Riyadh 0.00 ± 0.0f
AMJ 103 KY027103 Enterobacteriaceae bacterium MRS Riyadh 0.00 ± 0.0f
AMJ 104 KY027104 Enterobacter hormaechei TSB Riyadh 0.00 ± 0.0f
AMJ 105 KY027105 Klebsiella variicola MRS Riyadh 0.00 ± 0.0f
AMJ 108 KY027108 Staphylococcus haemolyticus MRS Riyadh 0.00 ± 0.0f
AMJ 111 KY027111 Enterobacter aerogenes MRS Riyadh 0.00 ± 0.0f
AMJ 113 KY027113 Enterobacter cloacae BSM Riyadh 0.00 ± 0.0f
AMJ 115 KY027114 Klebsiella pneumoniae MRS Riyadh 0.00 ± 0.0f
AMJ 117 KY027116 Klebsiella pneumoniae TSB Riyadh 0.00 ± 0.0f
AMJ 119 KY027118 Proteus mirabilis BHI Riyadh 0.00 ± 0.0f
AMJ 120 KY027119 Enterobacter hormaechei BSM Riyadh 0.00 ± 0.0f
(Continued)
 Antagonistic potential of gut bacteria against P. larvae 3

Table 1. (Continued).
Accession Identification based Culture Sampling ZOI mean diameter
Isolates No. on 16S rRNA broth area (mm) ± SD
AMJ 123 KY027122 Proteus mirabilis BHI Riyadh 0.00 ± 0.0f
AMJ 128 KY027127 Enterobacter cloacae BSM Riyadh 0.00 ± 0.0f
AMJ 129 KY027128 Klebsiella pneumoniae MRS Riyadh 0.00 ± 0.0f
AMJ 130 KY027129 Pantoea vagans TSB Riyadh 0.00 ± 0.0f
AMJ 201 KY027133 Morganella morganii BSM Al Baha 0.00 ± 0.0f
AMJ 202 KY027134 Acetobacteraceae bacterium MRS Al Baha 0.00 ± 0.0f
AMJ 203 KY027135 Fructobacillus fructosus MRS Al Baha 0.00 ± 0.0def
AMJ 205 KY027137 Proteus vulgaris BSM Al Baha 0.00 ± 0.0f
AMJ 206 KY027138 Bacillus cereus MRS Al Baha 0.00 ± 0.0f
AMJ 207 KY027139 Proteus mirabilis TSB Al Baha 0.00 ± 0.0f
AMJ 208 KY027140 Microbacterium pumilum BHI Al Baha 0.00 ± 0.0f
AMJ 209 KY027141 Proteus mirabilis BSM Al Baha 0.00 ± 0.0f
AMJ 211 KY027143 Proteus mirabilis TSB Al Baha 0.00 ± 0.0f
AMJ 212 KY027144 Fructobacillus fructosus MRS Al Baha 0.00 ± 0.0f
AMJ 213 KY027145 Proteus mirabilis TSB Al Baha 0.00 ± 0.0f
AMJ 214 KY027146 Morganella morganii BHI Al Baha 0.00 ± 0.0f
AMJ 216 KY027148 Pantoea agglomerans TSB Al Baha 0.00 ± 0.0f
AMJ 217 KY027149 Proteus mirabilis TSB Al Baha 0.00 ± 0.0f
AMJ 218 KY027150 Aeromonas salmonicida TSB Al Baha 0.00 ± 0.0f
AMJ 219 KY027151 Fructobacillus fructosus MRS Al Baha 0.00 ± 0.0f
AMJ 220 KY027152 Enterobacter xiangfangensis TSB Al Baha 0.00 ± 0.0f
AMJ 222 KY027154 Proteus mirabilis BSM Al Baha 0.00 ± 0.0f
AMJ 224 KY027156 Enterobacteriaceae bacterium BHI Al Baha 0.00 ± 0.0f
AMJ 230 KY027162 Proteus mirabilis BHI Al Baha 0.00 ± 0.0f
AMJ 231 KY027163 Citrobacter sp. BHI Al Baha 0.00 ± 0.0f
AMJ 233 KY027165 Proteus mirabilis TSB Al Baha 0.00 ± 0.0f
AMJ 235 KY027167 Enterobacteriaceae bacterium BHI Al Baha 0.00 ± 0.0f
AMJ 236 KY027168 Microbacterium testaceum BHI Al Baha 0.00 ± 0.0f
AMJ 238 KY027170 Morganella morganii TSB Al Baha 0.00 ± 0.0f
AMJ 240 KY027172 Providencia vermicola MRS Al Baha 0.00 ± 0.0f
AMJ 241 KY027173 Lactococcus lactis MRS Al Baha 0.00 ± 0.0f
AMJ 247 KY027179 Lactobacillus kunkeei MRS Al Baha 0.00 ± 0.0f
AMJ 249 KY027181 Proteus mirabilis BHI Al Baha 0.00 ± 0.0f
AMJ 251 KY027183 Lactobacillus kunkeei MRS Al Baha 0.00 ± 0.0f
AMJ 255 KY027187 Proteus mirabilis BHI Al Baha 0.00 ± 0.0f
AMJ 256 KY027188 Proteus mirabilis BHI Al Baha 0.00 ± 0.0f
AMJ 259 KY027191 Proteus mirabilis BSM Al Baha 0.00 ± 0.0f
AMJ 263 KY027195 Morganella morganii TSB Al Baha 0.00 ± 0.0f
Amj-6 KT901803 Arthrobacter tumbae BHI Riyadh 0.00 ± 0.0f
Note: ZOI, zone of inhibition; OTC, oxytetracycline used as control; BHI, brain heart infusion broth; MRS, Lactobacilli deMan, Rogosa and Sharpe
broth; TSB, tryptic soy broth; BSM, bifidus selective medium broth. Results were expressed as mean values ± SD of three replicates. Means with
same superscript letters are not significantly different (p < 0.05).

bacteria (Vasquez et al., 2012). Some natural com-
pounds and essential oils are tested to treat P. larvae
in vitro (Ansari et al., 2016; Erler & Moritz, 2016;
Kuzy
sinova, Mudro
nova, Topor
cak, Molnar, & Javorsky,
2016) but most of them are not recommended because
of their cytotoxic effects to honey bees. Thus, there is
great interest in exploring alternative, proficient control
measures against AFB. Evans and Armstrong (2006)
showed that some of the gut bacteria isolated from A.
mellifera exhibit antagonistic activity against P. larvae, and
may offer a novel method for combating AFB.
Many factors contribute to the antagonistic activity
of bacteria against pathogens, including the production
of antibiotics, bacteriocins, lysozymes, proteases, and
hydrogen peroxide as well as variations in pH values
caused by the production of organic acids by either a
single colony or a combination of strains (De Vuyst &
Leroy, 2007). Benign bacteria that exist in the gut of
honey bees and secrete antimicrobial substances are
important for confining or inhibiting various pathogens.
Bacteria belonging to the genus Bacillus play a significant
role in the host gut as studies have shown that many
species belonging to the genus Bacillus have bactericidal
and fungicidal effects due to the secretion of antibiotics,
bacteriocins or antifungal compounds (Alippi, 2000;
Martinari, Varcamonti, Naclerio, & De Felice, 2002).
A. m. jemenitica, the smallest race of A. mellifera, is an
indigenous honey bee of Saudi Arabia (Alqarni, Hannan,
Owayss, & Engel, 2011). It is biologically and behavior-
ally well adapted to the extremely harsh environmental
conditions of the country and performs better in
worker brood rearing, pollen storage, and foraging than
the imported race, A. m. carnica (Alqarni et al., 2011).
This local bee is also more tolerant of the pest mite,
Varroa destructor (Al-Ghamdi, 2002). We may be able to
exploit the qualities of this local honey bee to control
4 A. Al-Ghamdi et al.
Figure 1. Inhibition zones surrounding discs impregnated with gut bacteria isolated from Apis mellifera jemenitica on a lawn of
Paenibacillus larvae grown on BHI agar plates. The identities of the isolates, according to 16S rRNA sequencing, were (A) Lactobacillus
kunkeei, (B) Fructobacillus fructosus, (C) Morganella morganii, (D) Bacillus subtilis, (E) Enterobacter kobei, (F) Bacillus licheniformis, and (G)
Proteus mirabilis. Oxytetracycline (OTC) (center) was used as a control.
bee pathogens in apiculture. Some of the previous stud-
ies have explored the interactions between the gut bac-
teria of A. mellifera and pathogens, however, the roles
of gut bacteria isolated from A. m. jemenitica still remain
elusive. Because of all these reasons, in order to better
understand the role of these microbial communities as
biological control agents, the antagonistic activities of
bacterial strains isolated from the gut of A. m. jemenitica
against P. larvae were evaluated in this study.
Materials and methods
The antagonistic effects of one hundred bacterial strains
previously isolated by our working group from the gut of
local honey bees (A. m. jemenitica) were evaluated (Khan
et al., 2017). Cellular suspensions of each bacterial isolate
were used to screen for activity against a P. larvae
ATCC9545 by disc diffusion assay as described by Forsgren,
Olofsson, VAsquez, and Fries (2010), with some modifica-
tions. Each gut bacterium was picked with a sterile loop
from selective agar plates and inoculated in 10 mL of differ-
ent broths (Table 1). To grow these bacteria, tubes were
incubated at 34  C for 24 hours. The bacterial suspensions
were diluted in sterilized distilled water and their micro-
scopic count was made in a cell counting chamber (Thoma)
under the light microscope (40). Suspensions were
adjusted to approximately 1  106 colony forming units per
mL and used in inhibitory assays.
Similarly, an individual colony of P. larvae was picked
up from brain heart infusion BHI (Oxoid Ltd.,
Hampshire, UK) agar plate, inoculated in a tube contain-
ing 10 mL of BHI broth, and incubated at 34  C over-
night to serve as preculture. To induce sporulation of P.
larvae, bacterial preculture was heat shocked at 80  C
for 10 minutes to kill non spore forming bacteria
(Shimanuki & Knox, 2000) and then 100 mL of this sus-
pension was cultured on BHI agar plates at 34  C for 10
to 14 days. Spore suspensions of P. larvae were made by
harvesting bacterial colonies in sterilized distilled water
and adjusted to 1  106 spores/mL.
Disc diffusion assay
The antagonistic activity of gut bacterial isolates was
determined by disc diffusion assays (Brown & Kothari,
1975). OTC paper discs (BioanalyseV R ) were used as a
control and placed in the center of BHI agar plates
while blank filter paper discs (Arcomex-Arab Company
for Medical Diagnostics) were placed around the out-
side in a circle. BHI agar plates were fortified with thia-
mine hydrochloride (BDH Chemicals, Ltd., Poole, UK)
to encourage in vitro spore germination (Evans, 2003)
and inoculated with P. larvae spores prior to placement
of paper discs. Each blank filter paper disc was impreg-
nated with 10 mL of the gut bacterial suspension and
Petri plates were incubated at 34  C for 24 hours. The
radii of the inhibition zones were observed and meas-
ured in mm from the center of the paper discs to the
first line of P. larvae growth. On the basis of a mean
zone of inhibition (ZOI) of gut bacterial isolates against
P. larvae, we categorized the isolates as highly effective,
moderately effective, minimally effective and ineffective.
All samples were tested in triplicate (Figure 1; Table 1)
Phylogenetic analysis
A phylogenetic tree of the seven best antagonistic bac-
terial isolates obtained in our study and retrieved
sequences (https://www.ncbi.nlm.nih.gov) of antagonistic
honey bee gut bacteria from different countries was
constructed based on 16S rRNA sequences by the
Neighbor-Joining method using MEGA version 6.0.6.
The 16S rRNA sequence of the seven bacteria has been
taken from our previous study (Khan et al., 2017).
Numbers at the nodes of branches are bootstrap val-
ues, expressed as a percentage of 1000 replicates.
GenBank accession numbers are shown in parentheses.

Figure 2. Average diameter of the zones of inhibition produced
by gut bacteria isolated from Apis mellifera jemenitica against
Paenibacillus larvae. Error bars show standard deviations.
Statistical analysis
The antagonistic activity of bacterial isolates was ana-
lyzed using one-way ANOVA, followed by post-hoc
Tukey’s HSD tests with Statistix 8.1. The means and
standard error of the diameter of ZOI were calculated
from three independent replicates.
Results
One hundred isolated gut bacteria were screened for
antagonistic activity against P. larvae, the causative agent
of AFB. Out of the 100 bacterial isolates, seven isolates
exhibited antibacterial activities, with ZOI ranging from
5 to 7.67 mm (Table 1; Figure 2). The ZOI produced by
seven bacteria were more than 3.00 mm, four isolates
ranged from 2.00 to 2.33 mm (moderately effective),
whereas those produced by 19 isolates ranged from
1.00 to 1.67 mm (minimally effective). The other tested
isolates (70 isolates) were found to be ineffective with
ZOI ranged from 0.00 to 0.67 mm. The ZOI produced
by the OTC hydrochloride was 14.33 mm, which was
almost double to the ZOI produced by B. licheniformis
(isolate: AMJ210). The largest ZOI was obtained using
B. licheniformis that shows it has high antibacterial activ-
ity against P. larvae. Our findings demonstrated that
these bacterial isolates might not only have the poten-
tial to control P. larvae infections but may also represent
possible alternatives to the use of OTC to control AFB.
Phylogenetic analysis
A phylogenetic tree was constructed to evaluate the
relationships among some of the isolated gut bacteria in
different studies (sequences previously deposited at
NCBI database) with antagonistic effects against P. larvae
with seven antagonistic bacteria isolated from A. m.
jemenitica gut in our previous study (Khan et al., 2017).
The Neighbor Joining tree showed that AMJ226 isolate
Antagonistic potential of gut bacteria against P. larvae 5
was clustered in Clade-I of Firmicutes phylum, AMJ237,
and AMJ210 isolates were clustered in Clade-II of the
phylum Firmicutes, whereas AMJ124 isolates are
grouped in a separate branch in Firmicutes phylum and
not included in either Clade-I or Clade-II. The remaining
three isolates (AMJ246, AMJ133, and AMJ254) together
were grouped Clade-I of phylum Gammaproteobacteria.
In Clade-I of phylum Firmicutes, AMJ226 isolate was
closely related to L. kunkeei (EF187239), isolated from
Sweden, whereas AMJ237 and AMJ210 isolates were
grouped with B. subtilis strains isolated from Japan and
Argentina, whereas AMJ 124, do not show any hom-
ology with any isolate and make a separate branch in
phylum Firmicutes in phylogenetic tree. In Clade-I of
phylum Gammaproteobacteria, AMJ246 isolate was
closely related to E. coli strain (AB480753) reported
from Japan, whereas AMJ133 and AMJ254 were on sep-
arate branches and placed close to each other
(Figure 3)
Discussion
Bacteria of the genus Bacillus are well known to pro-
duce some antimicrobial compounds. B. subtilis produces
the greatest number of antibiotics (as many as 66), and
4%–5% of the bacterial genome is involved in antibiotic
synthesis (Stein, 2005). B. licheniformis exhibits anti-
microbial activity because of the production of proticin,
a phosphorous containing triene (Katz & Demain,
1977). Members of the genus Bacillus are found in honey
bees and in honey (Alippi, Reynaldi, L opez, De Giusti, &
Aguilar, 2004; Evans & Armstrong, 2006). A B. subtilis
strain isolated from the gut of honey bees (A. cerana
japonica) from Japan showed antibacterial activity against
P. larvae (Yoshiyama & Kimura, 2009), and Melissococcus
plutonius, the causal agent of European foulbrood (Wu
et al., 2014). Sabate, Carrillo, and Audisio (2009) dem-
onstrated that a B. subtilis isolate from the gut of A. mel-
lifera in Argentina showed antagonistic effects against P.
larvae by producing surfactin. B. subtilis isolated from the
gut of a hybrid Carniolan honey bee, A. m. carnica, in
Egypt showed antagonistic activity against Ascosphaera
apis, the causal organism of chalkbrood disease (Omar
et al., 2014). Alippi and Reynaldi (2006) found that a B.
licheniformis isolate from an apiarian source in Argentina
showed the strongest antagonistic effects against P. lar-
vae among tested isolates. Minnaard and Alippi (2016)
found that B. cereus inhibited the growth of P. larvae and
bacteriocin-like compounds secreted by these two bac-
terial strains were responsible for their inhibi-
tory activity
B. subtilis and B. licheniformis showed inhibitory effects
against P. larvae and in the phylogenetic tree, these bac-
teria showed closely relatedness with B. subtilis strainsis
olated from Japan and Argentina. The antimicrobial
activity assay results for the Bacillus isolates are in
accordance with previous studies (Alippi & Reynaldi,
2006; Sabate et al., 2009; Wu et al., 2014; Yoshiyama &
6 A. Al-Ghamdi et al.

Figure 3. Phylogenetic tree based on the 16S rRNA gene sequences of gut bacteria with strong antibacterial activity against
Paenibacillus larvae isolated from local honey bees in Saudi Arabia and the sequences of antagonistic bacterial strains found in Apis melli-
fera retrieved from the NCBI database. The tree was constructed by the neighbor-joining method using MEGA (V. 6.0.6). Bootstrap
values, expressed as percentages of 1000 replicates, are shown at each branch. GenBank accession numbers are shown in parentheses.

Kimura, 2009). This clearly indicates that Bacillus isolates
AMJ237 and AMJ210, may produce some antimicrobial
substances that inhibit the growth of pathogens.
In disc diffusion methods, tested samples diffuse out
from disc to agar medium by creating a circular concen-
tration gradient that decreases logarithmically with
increasing distance from the disc. As the tested sample
diffuses out from the disc, the bacteria multiply creating
a lawn of visible growth on the agar except in area
(zones) around the disc where diffused molecules pos-
sessed properties to inhibit bacterial growth (Kiska,
1998). The movement of diffusing molecules mostly
depends on the molecular weight and concentration
gradient. In our study, the oxytetracycline was applied
as positive control which was a pure single compound
with an average mass of 460.434 Da. On the other
hand, filter paper discs were impregnated with 10 mL of
the gut bacterial suspension. The gut bacteria that may
have released some antibacterial compounds would be
imagined in small quantity compared to positive control
that why their inhibition zones were smaller than antibi-
otics. Further, the molecular mass of putative antibac-
terial compounds released by gut bacteria were also
unknown. However, instead of low quantity and
unknown molecular weight of antibacterial compounds
released by gut bacterial their antibacterial activity was
comparable with positive control. If we will be able to
isolate and purify these compounds from the gut bac-
teria there is the possibility to manage AFB in the
future. Therefore, a further study is needed for the
characterization of antimicrobial compounds released by
these seven gut bacteria.
Lactic acid bacteria (LAB) are beneficial bacteria used
as probiotics for both humans and animals because they
modulate the bacterial composition of the gut and
defend their host against different pathogens
(Ouwehand, Salminen, & Isolauri, 2002; Audisio,
Torresa, Sabate, Ibargurena, & Apella, 2011; Killer,
Dubna, Sedla
cek, &
Svec, 2014). The L. kunkeei strain
AMJ 226 (KY027158) isolated in our previous study
(Khan et al., 2017) was shown to have in vitro antagonis-
tic activity against P. larvae. Forsgren et al. (2010), eval-
uated the antagonistic activity of LAB, isolated from the
honey stomach of A. mellifera in Sweden and identified
L. kunkeei strain and other LAB with strong inhibitory
effects against P. larvae. In addition, an L. kunkeei isolate
from A. mellifera in Finland showed inhibitory effects
against Melissococcus plutonius (Endo & Salminen, 2013).
The phylogenetic tree showed that the isolate AMJ226
(KY027158) was related to L. kunkeei (EF187239) strain
from Sweden, which indicates that these antagonistic
bacteria are of the same lineage. A special group of LAB
that favor fructose over glucose as a media supplement
is known as Fructophilic LAB (Endo, Futagawa-Endo, &
Dicks, 2009). In this study F. fructosus, AMJ124
(KY027123) showed antagonistic activity against P. lar-
vae. Forsgren et al., 2010 evaluated the in vitro
Antagonistic potential of gut bacteria against P. larvae 7
antagonistic activity against P. larvae of LAB isolated
from the honey crop of A. mellifera in Sweden. The pos-
sible reasons for the antagonistic activity of F. fructosus
may be related to the secretion of antimicrobial sub-
stances such as organic acids, hydrogen peroxide, and/
or antimicrobial peptides (De Vuyst &
Vandamme, 1994).
Bacteria belonging to the family Enterobacteriaceae
are associated with the dietary requirement of honey
bees and are in high numbers when the bees are col-
lecting nectar (Ludvigsen et al., 2015) or during the fall
season (Corby-Harris, Maes, & Anderson, 2014;
Lyapunov, Kuzyaev, Khismatullin, & Bezgodova, 2008).
Different genera of the Enterobacteriaceae have been
previously isolated from the gut of honey bees
(Anderson et al., 2013; Jeyaprakash, Hoy, & Allsopp,
2003; Yoshiyama & Kimura, 2009). Escherichia coli
Acj105 (AB480753), which is a member of the
Enterobacteriaceae isolated from the gut of the
Japanese honey bees showed antimicrobial activity
against P. larvae. In our study, E. kobei isolate AMJ246
(KY027178), showed antagonistic activity against P lar-
vae. In the phylogenetic tree, E. kobei (KY027178) was
clustered with E. coli (AB480753), and both showed
antagonistic effects. We can assume that these bacteria
have common genes expression, which is responsible
for antimicrobial activity against P. larvae. M. morganii
belongs to the Enterobacteriaceae, and is a typical resi-
dent of the physical environment, human gut, and ali-
mentary tracts of some other mammals and reptiles (Di
Ianni et al., 2015). Members of the genera Morganella
and Proteus were also identified in the midgut of A. melli-
fera (Anderson et al., 2013; Lyapunov et al., 2008). M.
morganii showed strong inhibitory effects against
Helicobacter pylori, a human pathogen that colonizes the
stomach (Krausse, Piening, & Ullmann, 2005). M. morga-
nii (KY027186) and P. mirabilis (KY027132), both
belongstoEnterobacteriaceae showed antimicrobial
activities against P. larvae. In the phylogenetic tree, these
two isolates were clustered together near Providencia
alcalifaciens (AB480755). P. alcalifaciens was isolated
from the gut of the Japanese honey bees by Yoshiyama
and Kimura (2009) and was reported to have strong
antimicrobial activity against P. larvae. Our data (Table
1) clearly demonstrated that some bacterial isolates
inhibited the growth of P. larvae, which is in accordance
to the results of earlier studies on bacterial isolate and
their antagonistic effect on P. larvae (Evans &
Armstrong, 2006; Forsgren et al., 2010)
Conclusions
Some bacterial isolates from the gut of A. m. jemenitica
have the ability to inhibit P. larvae. The bacteria inhabit-
ing the honey bee gut have adapted to the specific
environmental conditions of the gut, where no other
bacteria can grow normally. The antagonistic bacterial
isolates from the indigenous honey bee race of Saudi
8 A. Al-Ghamdi et al.
Arabia, for instance, F. fructosus (KY027123); P. mirabilis
(KY027132); B. licheniformis (KY027142); L. kunkeei
(KY027158); B. subtilis (KY027169); E. kobei
(KY027178); and M. morganii (KY027186) may secretes
some antibiotics or the compounds with similar effects
or bacteriocins, which may be responsible for antagonis-
tic effects against P. larvae. The results of this study
should serve as a basis for additional investigation on
the isolation and characterization of antagonistic sub-
stances from the gut microbiota of local honey bees of
Saudi Arabia for possible use in disease control not only
against honey bee pathogens but also against some
multi-drug human pathogens.
Authors contributions
AAG, MJA and KAK conceived this research and
designed experiments; KAK, AAG, AAA, HAG and MJA
wrote the paper and participated in the revisions of it.
All authors read and approved the final manuscript.
Disclosure statement
The authors confirm that there is no conflict of interests and
are also liable for the content and writing of this article.
Funding
The work was funded by the National Plan for Science
Technology and Innovation (MAARIFAH), King Abdul-Aziz
City for Science and Technology, Kingdom of Saudi Arabia,
Grant Number 3-17-07-001-0007.
ORCID
Ashraf M. Ahmed http://orcid.org/0000-0001-5459-5255
Mohammad Javed Ansari http://orcid.org/0000-0002-
8718-3078
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