First report of Kurthia gibsonii in the digestive system of Apis mellifera in an indigenous community in the south of Mexico

.

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 com petitive (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.
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.
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 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.

Site
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 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.

Figure 2 .
Figure 2. Comparatives from physical properties of some selected bees.

Figure 3 .
Figure3.16S 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.

Table 2 .
List of bacteria isolated by conventional and molecular methods.