EUNK - Revista Científica de Abejas y Apicultores
Editorial EUNK
www.eunk.org
ISSN en línea: 2953-3740
Volumen 2 (2023), Número 2
https://doi.org/10.52559/eunk.v2i2.42/
NOTA CIENTÍFICA
First report of Kurthia gibsonii in the digestive system of Apis
mellifera in an indigenous community in the south of Mexico
David Francisco López1, Yanet Romero Ramírez2, Miguel Ángel Rodríguez Barrera2, Arturo Ramírez Peralta3,
Renato León Rodríguez4, Diana Iveth Orbe Díaz2, Alberto Patricio Hernández5, Angela Victoria Forero Forero6,
Jeiry Toribio Jiménez2
1 Maestría en Recursos Naturales y Ecología, Facultad de Ecología Marina, Universidad Autónoma de Guerrero, Av. Gran Vía Tropical
20, Playas, 39390 Acapulco de Juárez, Gro., México.
2 Laboratorio de Microbiología Molecular y Biotecnología Ambiental, Facultad de Ciencias Químico-Biológicas, Universidad Autónoma de
Guerrero, Av. Lázaro Cárdenas s/n. Ciudad Universitaria. Apdo. Postal 39070, Chilpancingo, Guerrero, México.
3 Laboratorio de Investigación en Patometabolismo Microbiano, Facultad de Ciencias Químico-Biológicas, Universidad Autónoma de
Guerrero, Chilpancingo, Guerrero, México. Av. Lázaro Cárdenas s/n, Cd. Universitaria, Chilpancingo, Guerrero, México.
4 Laboratorio de Contención Biológica (BSL-3). Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México.
Ciudad Universitaria. C. P. 04510. Ciudad de México.
5 Laboratorio de Agrobiotecnología, Departamento de Biotecnología, Universidad Politécnica de Pachuca, carretera Pachuca-Cd.
Sahagún, km. 20, Rancho Luna, Ex Hacienda de Sta. Bárbara, C.P. 43830. Zempoala, Hidalgo, México.
6 Facultad de Ciencias, Universidad Nacional Autónoma de México. Av. Universidad 3000, Circuito Exterior S/N. Delegación Coyoacán,
C.P. 04510. Ciudad Universitaria, CDMx. México.
E-mail de contacto: [email protected]nam.mx
Recibido: 18 julio 2023; aceptado: 8 noviembre 2023; publicado: 31 diciembre 2023
Abstract
The insect digestive system is an outstanding niche for recognizing and understanding features and interactions with unique
microbiota. Apis mellifera play an important role in honey production and pollination, but their populations have decreased
over time, so their study and comprehension are vital. Specimens were collected to study the culturable microbiota of the
digestive system in an indigenous community in southern Mexico. Digestive samples of bees from four apiaries were randomly
analyzed and cultured, and Gram-positive bacilli were identified through conventional biochemical tests and with molecular
markers. Kurthia gibsonii was identified in 7.5% (6/80) of the cases, and the sequences obtained were reported in GenBank.
This is the first report of K. gibsonii in the digestive system of bees in apiaries in southern Mexico.
Keywords: Apis mellifera, digestive system, Kurthia gibsonii
Primer reporte de Kurthia gibsonii en el aparato digestivo de Apis mellifera en una
comunidad indígena del sur de México
Resumen
El sistema digestivo de los insectos es un nicho excepcional para reconocer y comprender características e interacciones
particulares con una microbiota única. Apis mellifera juega un papel importante en la producción y polinización de la miel,
pero sus poblaciones han disminuido con el tiempo por lo que su estudio y comprensión es vital. Se recolectaron especímenes
para estudiar la microbiota cultivable del sistema digestivo en una comunidad indígena del sur de México. Se analizaron
aleatoriamente muestras digestivas de abejas de cuatro apiarios, se cultivaron e identificaron bacilos Gram positivos mediante
pruebas bioquímicas convencionales y marcadores moleculares. Se identificó Kurthia gibsonii en el 7,5% (6/80) de los casos
y las secuencias obtenidas se reportaron en Genbank. Este es el primer reporte de K. gibsonii en el sistema digestivo de abejas
en apiarios en el sur de México.
David Francisco López et al.
Julio - diciembre 2023 • Volumen 2 • Número 2
4
Palabras clave: Apis mellifera, Kurthia gibsonii, sistema digestivo
Beekeeping is an essential socioeconomic and
ecological activity worldwide, highlighting the
studies in Mexico. Indigenous communities have
practiced ancestral and sustainable beekeeping for
thousands of years and using modern modifications,
like substituting native bees for European bees
(Gupta, 2014). The Amuzgos are one of the most
representative ethnic groups of Mexico; they are in
Oaxaca and the area in Costa Chica from the State
of Guerrero. They inhabit a region with enriched
vegetation and template climate. Their economy
depends on the trade of agricultural products and,
particularly in beekeeping, an element of their
ancestral and cultural use and custom. This last
practice has been adapted into modern elements only
to increase production and be competitive (INPI,
2020).
Currently, because of the notable consequences
of anthropogenic activities, the conservation and
study of the local species of honeybees have
increased in importance. Moreover, the
comprehension of the bacterial microbiota in honey
bees has been a significant factor in their health and
conservation. For this reason, cultivable bacteria
isolated from bee gut microbiota have been an
unexpected result in various studies. Bacterial
genera such as Pseudomonas, Streptococci,
Micrococci, Lactobacillus, Klebsiella, Proteus,
Yersinia, Bifidobacteria, Corynebacterium,
Bacillus, and Clostridium (García et al., 2006) have
been detected. The current literature links Kurthia
gibsonii as an emerging pathogen in secondary
infections, together with other well-known
pathogenic genera causing severe damage in farm
animals (Lozica et al., 2022) and other insects.
In this study, 95 beehives were analyzed in four
sites from the northern area of the Amuzga
indigenous community of Zacualpan in Southern
Mexico (place 1: 16°45'11.5" N, 98°17'25.7" W;
place 2: 16°45'02.2" N, 98°17'19.5" W; place 3:
16°45'26.1" N, 98°18'53.0" W, and place 4:
16°45'07.1" N, 98°16'52.9" W) (Figure 1). For each
place, three beehives were considered, according to
the NOM-001-ZOO-1994 norm. A morphological
comparison was made using the size and proportion
of the bees, according to the Ortiz et al. (2004)
Figure 1. Characteristics of the apiaries of Apis mellifera in Southern México. a) Place 1, b)
Place 2, c) Place 3, and d) Place 4.
First report of Kurthia gibsonii in the digestive system of Apis mellifera in an indigenous community in the south of
Mexico
5 Julio - diciembre 2023 • Volumen 2 • Número 2
criteria.
The extraction method of the intestinal tract of
the bee was made according to Carreck et al. (2013).
The tissue was macerated in 0.9% saline solution
into a mortar under sterility conditions. Only 600 µl
of the solution was transferred into plastic tubes with
glycerol solution at 10% v/v (1 ml) and stored at -80
°C to produce a backup of the sample.
Each sample was diluted 1:1000 into trypticase
soy agar by mass streaking and made in duplicate.
Petri dishes were incubated from 24 to 48 h at 28-
30°C temperature. Colony-forming units were
counted and measured 24 to 48 hours after
inoculations. All colonies were studied by
macroscopic and microscopic morphology using
Gram stain. Solely Gram-positive strains were
studied by biochemical tests based on carbohydrate
metabolism: galactose, lactose, raffinose, mannose,
cellobiose, saccharose, maltose, glucose, sorbitol,
and trehalose, according to Dhameliya et al., (2020).
Genomic DNA from the selected strains was
extracted according to Adame-Gómez et al., (2019).
Subsequently, PCR (Polymerase Chain Reactions)
with the universal primers 27F (5'-
AGAGTTTTGATCCTGG CTCAG-3') and 1492R
(5'-GGTTACCTTGTTACGACTT-3') were made
according to previous reference (Tajabadi et al.
2013). A total volume of 13 µl of the master MIX
was prepared with 6.25 µl, 4.75 µl of water-free
nucleases, 0.5 µl of primers, and 1 µl of the DNA
sample using the protocol described in Adame-
Gómez et al., (2019). The PCR products were
analyzed and visualized in an agarose gel
electrophoresis. PCR products were extracted using
GeneJET extraction gel (Thermo Scientific®) and
sequenced in the Institute of Biotechnology from the
National Autonomous University of Mexico.
The obtained sequences were analyzed as Fasta
formatted in MEGAX program version 10.1 (Wong
et al., 2021) to correct undistinguished sequences.
The resulting sequences were compared with other
genes obtained from BLASTn
(https://blast.ncbi.nlm.nih.gov/Blast.cgi) (Altschul
et al. 1990) and downloaded from GenBank
(www.ncbi.nlm.nih.gov/genbank).
Phylogenetic analysis was made using the
sequences obtained and similar sequences of
BLAST naming genera and species (Table 1), using
the Neirghbor-Joining method with ~ 658
nucleotides from each sequence. The Jukes and
Cantor (1969) matrix was applied, and all the
processes were computed using MEGA (Wong et
al., 2021).
Four apiaries were visited and analyzed in the
Zacualpan community, where the honey production
was evaluated: 150 liters in place one, 180 liters in
Table 2. List of bacteria isolated by conventional and molecular methods.
Site
Strain
UFC/ml
Carbohydrate
Fermentation
Identity
Value (%)
GenBank
Access number
1
S1I
1x103
-
99.58
OP824654
2
S2H
1x103
Glucose
99.88
OP824658
3
S3GA
1x103
-
99.76
OP824657
S3GB
>15x103
Glucose/Lactose
99.79
OP824659
4
S4D
3x103
Glucose
99.83
OP824656
S4F
1x103
-
99.35
OP824653
S4G
>15x103
-
99.68
OP824655
Figure 2. Comparatives from physical properties of some selected bees.
David Francisco López et al.
Julio - diciembre 2023 • Volumen 2 • Número 2
6
place two, 320 liters in place three, and only 6 liters
in place four. In the review and macroscopic
analysis of the bee population, four adult specimens
were randomly sampled and observed to compare
their morphometric structure and sizes. Images of
each specimen are shown in Figure 2. According to
previous references (Ortiz et al., 2004), the
specimen from Apiary 1 is the only one with regular
proportions.
Seven of 80 bacterial strains were selected and
isolated from the bee gut microbiome for their macro
and microscopic characteristics, as well as by
biochemical tests. These unknown strains appeared
white, shiny, convex, and round 1 to 2 mm growing
on LB and nutrient agar. Characterized as Gram-
positive bacillus of short morphology, motile, with
positive catalase and urease activity, growing at
room temperature, some of these strains were
capable of fermenting glucose and lactose (Table 1).
Of the strains isolated, S2H was identified as
Bacillus cereus with an identity value of 99.88%,
Besides, S1I, S3GA, S3GB, S4D, S4F, and S4G
strains correspond to K. gibsonii with a 99% identity
value.
According to the phylogenetic analysis (Figure
3), the reported strains show low differences
between the strains of K. gibsonii and K. zopfii,
unlike the species B. cereus and B. thuringiensis
where it is notable differences and substitution
changes. This brings a discussion because
concerning their microscopical aspects and
properties Kurthia and Bacillus genera, have similar
elements and properties.
Kurthia spp. is associated with the microbiome
of some farm animals as chickens and pigs. The
study of this relation has been recently reported as
significative due to its biotechnological use in the
processing and decoloring of triphenylmethane in
textile effluents (Sani & Banerjee, 1999), into its
infective role, producing zoonotic secondary
infections (Kövesdi et al., 2016), and as a symbiont
in the carotenoids C30 production for the intestine
of Sympetrum frequens (Koyanagi et al., 2021).
Kurthia gibsonii has been reported as an
emerging opportunistic pathogen in secondary
coinfection in farm birds together with E. coli in
pathologies that could cause chondronecrosis and
osteomyelitis in lower joints (Lozica et al., 2022).
Due to the above, K. gibsonii species isolated in this
study could become a bee emerging pathogen, or
bees could be asymptomatic carriers. However,
more studies are recommended to recognize its
biology in bees.
The importance of the bee microbiota and its
health has previously been reported. However, the
finding of Kurthia genus in the hives may be a risk
factor that compromises health or leads to stress, as
suggested by Lorizzo et al. (2020). Understanding
the bacterial microbiome of bees is an essential
element in their health and relationship with the farm
Figure 3. 16S rRNA Neighbor-joining phylogenetic tree
from isolated strains in the digestive system from Apis
mellifera. These sequences are obtained from the
GenBank database, E. coli DH5α strain was added as an
outgroup. The node value is presented as a percent after
involving a bootstrap of 1000 replicates. The scale under
the tree indicates the number of substitutions per
nucleotide substitution. (*) Represent the strains obtained
in these studies.
First report of Kurthia gibsonii in the digestive system of Apis mellifera in an indigenous community in the south of
Mexico
7 Julio - diciembre 2023 • Volumen 2 • Número 2
itself because these bees are mainly involved in the
pollination of many crops. Its population decline
could cause severe problems in food production.
This is the first report of K. gibsonii in the
digestive system of bees cultivated by the
indigenous community in Southern Mexico.
DISCLOSURE STATEMENT
No potential conflict of interest was reported by
the authors.
FUNDING
The authors would like to thank CONACYT for
the scholarship in support (number 56728).
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David Francisco López et al.
Julio - diciembre 2023 • Volumen 2 • Número 2
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Derechos de Autor (c) 2023 David Francisco López, Yanet Romero Ramírez, Miguel Ángel Rodríguez
Barrera, Arturo Ramírez Peralta, Renato León Rodríguez, Diana Iveth Orbe Díaz, Alberto Patricio
Hernández, Angela Victoria Forero Forero, Jeiry Toribio Jiménez
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