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Isolation, Identification and Antimicrobial Activity of Unprecedented Lactic Acid Bacterial Isolates from Honeybees



A.A. El-Sohaimy, S.H.D. Masry, M.G. Shehata, S.N. Al-Kahtani, T.E. Abdelwahab, Y.A.T. Abdelmotaleb and M.E. Nour
 
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ABSTRACT

Background and Objective: Lactic acid bacteria are generally recognized as safe that could be beneficial for several uses in food industry to get their health benefits. The present study was focused on the isolation and identification of some new lactic acid bacteria that might be naturally occurred in the honeybees stomach and tried to explore their benefits. Materials and Methods: Twenty five isolates of lactic acid bacteria were isolated from the stomach of three different types of Egyptian bees (Apis mellifera lamarckii ), Carniolan bees (A.m. carnica) and hybrid Carniolan bees. Identification of isolates was carried out based on phenotypical tests and carbohydrate assimilation using API50 CHL and 16S rDNA sequencing. Results: In the present study, the results emphasized Lactobacillus plantarum to be the predominant species (62.5%), other strains were identified as Pediococcus pentosaceus (12.5%), Lactobacillus pentosus (12.5%) and Lactobacillus sakei (12.5%). Eight of 25 isolates showed a potential antibacterial activity especially against Salmonella senftenberg strain. The novel isolates (HBMSS1, HBMSS3, HBMSS4, HBMSS5, HBMSS6 and HBMSS8) showed a significant antimicrobial activity against C. botulinum, E. coli, S. Senftenberg and S. epidermidis as food borne pathogens and P. larvae and M. plutonius as honeybee pathogens. Conclusion: These promising findings might be beneficial for discovering novel preservatives in food industry and substitution of antibiotic drugs used in the treatment of honeybees’ infection.

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A.A. El-Sohaimy, S.H.D. Masry, M.G. Shehata, S.N. Al-Kahtani, T.E. Abdelwahab, Y.A.T. Abdelmotaleb and M.E. Nour, 2020. Isolation, Identification and Antimicrobial Activity of Unprecedented Lactic Acid Bacterial Isolates from Honeybees. Pakistan Journal of Biological Sciences, 23: 467-477.

DOI: 10.3923/pjbs.2020.467.477

URL: https://scialert.net/abstract/?doi=pjbs.2020.467.477
 
Copyright: © 2020. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

INTRODUCTION

Honeybees (Apis mellifera) perform a crucial function as pollinators of horticultural land and agricultural crops in USA, pollination of horticulture crops by honeybees was around 15 billion dollars per year in agricultural sector production1. However, pathogenic bacteria, fungi, viruses and protozoa could infect honeybee colonies. American foulbrood (AFB) and European foulbrood (EFB) are considered dangerous honeybee infections caused by the Gram-positive bacteria Paenibacillus larvae and Melissococcus plutonius2,3. Overall, the microbiota of the honeybee A. mellifera L. is convoluted and difficult to be resolved; nonetheless, it has been reported to be principally consists of yeasts and Gram-positive bacteria (such as Lactobacillus spp., Lactococcus spp., Bifidobacterium spp., Enterococcus spp., Bacillus spp. and Streptococcus spp.) and Gram negative bacteria4,5. Corby-Harris et al.6 described that these bacteria as being capable of residing in the bees' digestive tract and not dependent on seasonal or food factors. In addition, Bastos et al.7 has reported that the natural microorganisms of bees derive from pollen or direct contact with forager worker bees. In hibernating honeybees, Lyapunov et al.8 emphasized the presence of gut Enterobacteria. Interestingly, there are a handful studies focusing on the isolation of lactic acid bacteria (LAB) from the honeybee microbiota1,4,5,9-11. In few studies, Lactobacillus bacteria might be able to produce catalase and reduce nitrate, even though sometimes these bacteria might be not inline with these properties12. Thus, only the subsistence of culturable bee-gut (Lactobacillus, Enterococcus or Bifidobacterium) strains associated with the intestinal tract of the honeybee has been described. Although, it has always been the most powerful isolation of microorganisms from native sources to obtain useful and genetically stable strains. Olofsson and Vasquez10 were disclosed a remarkable growth in the number of LAB in honeybees from healthy honeybee colonies. Moreover, honeybees infected with P. larvae showed fewer Lactobacillus spp. content13. LAB are fundamental in food and dairy technology due to their capability to generate the organic acids, diacetyl compounds, hydrogen peroxide and bacteriocins which may act as natural preservatives that can play a decisive function in the extension of the shelf-life of foods. Indeed, LAB are becoming increasingly accepted as probiotics that stimulating the immune response, inhibiting bacterial infection and preventing diarrhoea14-17. There are different microorganisms seriously affecting honeybees, such as P. larvae and M. plutonius, as gram-positive bacilli causing AFB and EFB in bee larvae18,19. Moreover, random antibiotic use against these pathogens has not only resulted in chemo-resistant strains but also causing honey contamination20. So, LAB isolated from honeybees might perform a substantial role in conserving the adult bees and larvae against pathogens, with an in vitro inhibitory effect on the causative agents of AFB and EFB21,22. Hence, the present work was focused on screening, isolation and phenotypic and genotypic characterization of LAB from honeybees’ stomach and midgut honeybees. Furthermore, determine their in vitro antimicrobial potential against different Foulbrood and foodborne pathogens, which lead us to explore novel natural food preservatives and feasible biological control for honeybees’ infection.

MATERIAL AND METHODS

The present investigation was carried out in the Department ofFood Technology, Arid Lands Cultivation Research Institute, City of Scientific Research and Technological Applications, Alexandria, Egypt from June 2017, to December, 2018.

Sample collection: During the foraging season from April to June, 2017, 180 forager worker bees were collected from colonies of three races and hybrids in Egypt. Sixty forager worker bees from Egyptian honeybee (Apis mellifera lamarckii) colonies, 60 forager worker bees from Carniolan honeybee (A.m. carnica) colonies and 60 forager worker bees from hybrid Carniolan honeybee colonies were collected. The colonies of A.m. lamarckii were located at Upper Egypt (Assiut Governorate); those of A.m. carnica and hybrid Carniolan honeybees were located at New Borg El-Arab City, northwestern Egypt, at the apiary of City of Scientific Research and Technological Applications (SRTA-City). The samples of foraging worker bees were collected alive in wooden cages and immediately transported to the laboratory of Department of Food Technology (SRTA-City). Selected foraging worker bees were surface-sterilized with 70% ethanol for 60 sec, followed by 5% NaCl for 60 sec and then washed with sterile distilled water to isolate the honey stomach and gut bacteria. In a laminar flow hood, the worker bees were dissected and the honey stomach and mid-gut were separated from the alimentary canal23.

Table 1: Indicator strains and their growth conditions
YPD broth: Yeast peptone dextrose, TPGY broth: Tryptone-peptone-glucose-yeast extract, LB broth: Luria-Bertani medium, AFB: American foulbrood, EFB: European foulbrood

Isolation of honeybee stomach and mid-gut bacteria: In 100 μL of phosphate buffered saline (PBS), the honey stomach was homogenized with a plastic pestle. On Brain Heart Infusion (BHI) agar medium (Difco), the stomach and mid-gut homogenates were aerobically incubated at 35°C for 2 days. The size, color and morphology selected the colonies that grew on the plates. On solid agar, isolated colonies have been grown repeatedly.

Antimicrobial activity: The antimicrobial activity of the isolates against pathogenic bacteria was determined (Table 1) using the spot-on-lawn method24,25. LAB was grown in MRS for 16-18 h with 1% inoculum and cells were extracted from the medium by centrifugation (6,500 g for 10 min, 4°C) in order to obtain a supernatant free of cells. Indicator strain lawns have been prepared by adding 0.125 mL (2×107 cell mL1) of 10×-diluted overnight culture to 5 mL of corresponding soft agar (Table 1). The tubes contents are gently mixed and poured over pre-poured MRS agar plate’s surfaces. Ten microliters of each cell-free supernatant were spotted on the agar's surface and the plates were tested for an inhibition area after 24 h of incubation. Clear zones around spots show the isolated bacteria's antibacterial activity.

Identification of promising LAB isolates
Phenotypic characterization and biochemical analysis: Promising LAB isolates have been phenotypically identified using Bergey's Systematic Bacteriology Manual26,27. Microscopic examination has been used for phenotypic identification after gram staining. Furthermore, according to the manufacturer's instructions, fermentation patterns are determined using API 50 CHL and API 20 kits (Biomerieux SA, France).

Molecular identification
DNA extraction: Microbial DNA was extracted from LAB using the Fermentas DNA extraction kit (FDEK), according to the protocol described by the manufacturers. The DNA extracts are examined by 1% agarose gel electrophoresis and stained with ethidium bromide.

Polymerase chain reaction amplification: PCR reaction was carried out using universal 16S rDNA primers for invariant region that purchased from Sigma Scientific Company, Germany28. The PCR amplification was performed in a thermocycler (Santa Clara, California, USA) with the following program: Initial denaturation at 95°C for 5 min, 30 cycles of 95°C for 40 sec, 55°C for 40 sec and 72°C for 1 min, with a final extension at 72°C for 10 min. PCR product was identified by loading on 1% agarose gel containing 1 μg mL1 ethidium bromide. A 100-1500 bp DNA marker (TAKARA BIO INC., Shiga, Japan) was used as a DNA marker.

DNA sequencing: An automated DNA sequencer was used to perform DNA sequencing reactions. The National Center for Biotechnology Information (NCBI) website (http:/www.ncbi. nlm.nih.gov/BLAST29) used the latest release of the non-redundant DNA sequence database.

Nucleotide sequence accession numbers: The sequences from the 8 amplicon samples representing different combinations of extracted DNA and PCR amplification were uploaded to GenBank (NCBI) under the following accession numbers: MF662586, MF662587, MF662588, MF662589, MF662590, MF662591, MF662592, MF662593.

Phylogenetic analysis: BLAST-N searches (http:/blast.ncbi. nlm.nih.gov/) retrieved closely related 16S rRNA sequences from the GenBank/EMBL database. Several sequence alignments with ClustalW (http:/www.ebi.ac.uk/ Tools/msa/clustalo/) were performed and used to reconstruct phylogenetic relationships using neighbour-joining (NJ) algorithms with bootstrapping of 1000 times in MEGA7.

Statistical analysis: All assays were carried out in triplicate and the results are expressed as the mean with the standard deviation (mean±SD) using SPSS16 software. Values were compared by ANOVA with a general linear model followed by Duncan’s post hoc test and p<0.05 were considered significant.

RESULTS AND DISCUSSION

Isolation and phenotypic characterization of LAB: Twenty-five LAB isolates have been isolated from the honeybee stomach of 3 different types of Egyptian bees (A. mellifera lamarckii), Carniolan bees (A.m. carnica) and hybrid Carniolan bees (Table 2). All 25 isolates obtained from the honey stomach were found to be catalase negative and gram positive. Eight isolates exhibiting antimicrobial activity potential against food-borne and foulbrood pathogenic microorganisms were chosen for further characterization (Table 3). Morphologically, the cells of 7 isolates were rod shaped and arranged either in pairs or chains, colonies on MRS agar were orbicular and entirely low convex in shape and cream coloured. The remaining isolate was coccus shaped and arranged in pairs and tetrads. The phenotypic features of the isolates suggest their close resemblance to Lactobacilli spp. and Pediococcus spp.30,31. Previous studies on the association of LAB with the honeybee gut and honey stomach are limited. For instance, Mohr and Tebbe32 assured the existence of Lactobacillus in the bees gut over molecular analysis without isolation and the detected strains were uncultured. Mrazek et al.33 have examined a number of insect intestinal microbiota, only mentioning the possible presence of Bifidobacterium sp. without isolation in honeybees. Additionally, Lactobacillus sp., Enterococcus and some Bifidobacterium spp. have been isolated by Nora and Amel34 and the honeybee digestive tract microbiota has been investigated.

Antimicrobial activity against food born pathogens: The antimicrobial activity of the LAB isolates and their power to restrain the outgrowth of diverse pathogens lead to food deterioration have been examined by using nutrient agar media and a spot-on-lawn assay (Table 3). In a preliminary assay, indicator organisms were tested for antimicrobial activity by LAB under aerobic conditions: indicator organisms (Escherichia coli BA 12296, Candida albicans ATCCMYA-2876, Bacillus subtilis DB 100, S. senftenberg ATCC 8400, Staphylococcus aureus NCTC 10788, Clostridium botulinum ATCC 3584, Staphylococcus epidermidis and Streptococcus pyogenes) were inoculated individually and overlaid with a plain nutrient agar plate (control) or over plates with the supernatant of LAB grown in MRS broth. Ehb8 isolate was exhibited the highest antimicrobial activity against S. senftenberg ATCC 8400 (Fig. 1). Additionally, an anti microbial activity with considerable zone of inhibition against Salmonella senftenberg ATCC 8400 was noted with Ehb (2, 5, 8 and 9) isolates. However, negative results were found for other isolates, with no inhibitory zones against B. subtilis DB 100, C. albicans ATCCMYA-2876 or S. pyogenes (Table 3). Thus, current experimental data showed that some of the isolated strains of LAB inhibited the growth of various pathogenic bacteria and yeast especially those causing honeybee diseases. These findings might be due to the differences in the essence of the bioactive secondary metabolites produced by LAB (organic acids, hydrogen peroxide, acetaldehyde, diacetyl compounds and bacteriocins) that inhibit pathogenic and spoilage microorganisms in food25,35. Experimental data in the present work were proven by the literature in relation to the different susceptibility of Salmonella spp., Clostridium spp. and E. coli to various LAB16,36,37.

Antimicrobial activity against AFB and EFB: The antimicrobial potency of LAB isolates against P. larvae (causing AFB) and M. plutonius (causing EFB) have been investigated as shown in (Fig. 2). Five isolates out of 25 (Ehb2, Ehb8, Ehb9, Hhb11 and Chb19) showed inhibitory halo zones against P. larvae and M. plutonius (Fig. 1). This finding for Egyptian honeybees was as opposed to that for European honeybees that showed activity against a wide range of bacterial species. This may point out to variations in the gut's physiological conditions, such as differences in pH and antimicrobial substances in the bees diet.

Table 2: Isolation sources of lactic acid bacteria

Table 3: Antimicrobial activities of cell-free supernatants of 25 LAB isolates against various pathogens
Ehb: Egyptian honeybees, Hhb: Hybrid honeybees, Chb: Carniolan honeybees, HS: Honey stomach, HbM: Honey bee midgut, diameter of the zone of inhibition (mm) using the agar well diffusion assay method

Fig. 1(a-b): Inhibitory activity of the cell-free supernatant of isolates against (a) Salmonella senftenberg ATCC 8400 and (b) P. larvae

Fig. 2: Inhibitory activities of antagonistic bacteria isolated from the honey stomach against honeybee pathogens
  AFB: Paenibacillus larvae, EFB: Melissococcus plutonius

European honeybees A. mellifera, accommodate numerous species of gut bacteria9, including antagonistic bacteria to young larvae pathogens38. The existence of Lactobacilli in A. mellifera and A.m. scutellata have been reported6,32 and a novel bacterial flora of the A. mellifera honey stomach composed of LAB of the genera Lactobacillus and Bifidobacterium has recently been reported10. On the other hand, Evans and Armstrong38 failed to isolate of Lactobacillus species from A. mellifera, suggested that the bacterial gut population is not even constant in the same species. In the current investigation, the honey stomach of A. mellifera harboured Lactobacillus and Pediococcus species and we also detected several stomach LAB that had not previously been detected in the genus Apis. Honeybees visit several types of flowers that vary seasonally and geographically; in addition, honeybees of different species tend to visit similar species of flowers. Consequently, changes in feed source may be correlated with the lack of a characteristic honey stomach bacterial profile in Apis. Antibiotics such as oxytetracycline hydrochloride (OTC) and sulfathiazole are commonly used for controlling AFB and EFB worldwide, though P. larvae and M. plutonius have developed antibiotic resistance properties. Because the LAB strains isolated in the present work displayed defence activity against honeybee pathogens, a promising management strategy might be developed to prevent honeybee bacterial diseases to avoid antibiotic treatment.

Morphological and biochemical identification: Morphological classification of LAB isolates was carried out according to the schemes described by Systematic Bacteriology Manual of Bergey26,27. The existence of Lactobacillus was confirmed by tiny yellow colonies of similar sizes that appeared on MRS agar using the streak plate process, as previously reported30. Furthermore, biochemical analysis of LAB was conducted by the API50 strip kit and selected strains were identified as Gram-positive rods or coccus-shaped isolates most closely associated with Lactobacilli and Pediococcus spp., respectively (Table 4). The API web software confirmed that lactobacilli strains are typically able to utilize carbohydrates such as galactose, glucose, fructose, glycerol, D-xylose, mannose, mannitol, ribose, N-acetyl-glucosamine, amygdalin, α-methyl-D-glucoside, esculin, arbutin, cellobiose, salicin, maltose, sucrose, lactose, melibiose, melezitose, raffinose, trehalose, gentiobiose, D-arabitol, D-tagatose and gluconate. Moreover, the Pediococcus strain was able to ferment carbohydrates that included L-arabinose, glycerol, D-xylose, galactose, glucose, ribose, mannose, fructose, mannitol, gentiobiose, esculin, cellobiose, salicin, N-acetyl-glucosamine and D-tagatose (Table 4).

Table 4: Biochemical characterizations of lactic acid bacteria based on carbohydrate interpretation using the API 50 CHL kit
−: Bacterium does not use this carbohydrate, +: Bacterium uses this carbohydrate, Ehb2: Lactobacillus plantarum, Ehb3: Pediococcus acidilactici, Ehb5: Lactobacillus plantarum, Ehb8: Lactobacillus plantarum, Ehb9: Lactobacillus plantarum, Hhb11: Lactobacillus plantarum, Hhb15: Lactobacillus sakei, Chb19: Lactobacillus plantarum

Fig. 3: Agarose gel electrophoresis of PCR products amplified using 16S rDNA primers
  M: GeneRuler DNA ladder, Lane 1: HBMSS1, 2: HBMSS2, 3: HBMSS3, 4: HBMSS4, 5: HBMSS5, 6: HBMSS6, 7: HBMSS7, 8: HBMSS8

Table 5: Identification of isolates based on 16S rRNA sequences

The color change in the strip capsule from violet to yellow was indicated complete Pediococcus pentosaceus 4I1 fermentation39. Lactobacillus species isolated from fermented sausage using an API 50 kit have recently been described phenotypically and biochemically by Casaburi et al.40.

16S rRNA sequencing and phylogenetic analysis: Eight strains were chosen because of their antimicrobial potential and 16S rRNA sequence analysis was used to classify them (Fig. 3). Strains HBMSS1, HBMSS2, HBMSS3, HBMSS4, HBMSS5, HBMSS6, HBMSS7 and HBMSS8 exhibited 92, 90, 81, 96, 98, 94, 93 and 94% DNA sequence identity of known species Lactobacilli and Pediococcus registry entries (Fig. 4). In addition, the similarity of the BLAST 16S rRNA sequence and biochemical tests showed that these strains in GenBank were closely linked to LAB. The HBMSS1, HBMSS2, HBMSS3, HBMSS4, HBMSS5, HBMSS6, HBMSS7 and HBMSS8 nucleotide sequence data were deposited in GenBank (accession numbers MF662586, MF662587, MF662588, MF662589, MF662590, MF662591, MF662592 and MF662593, respectively) (http://www.ncbi.nlm.nih.gov). Using the 16S rRNA universal primers28, nearly full-length 16S rRNA gene regions were successfully amplified and sequenced and according to sequence similarity, these isolates were identified as LAB (Lactobacilli and Pediococcus) (Table 5). A phylogenetic tree representing different reference species of LAB with the isolates was generated using the NJ method (Fig. 4). Lactobacillus plantarum, Lactobacillus sakei and Pediococcus pentosaceus were clearly separated from different Lactobacilli and Pediococcus strains (Fig. 4-c). Lactobacillus plantarum isolates HBMSS1, -3, -4, -5, -6 and -8 clustered in the Lactobacilli linage, close to L. plantarum reference strains. The P. pentosaceus isolate grouped with the Pediococcus sp. lineage, with a high degree of genetic resemblance to Pediococcus acidilactici. In addition, Lactobacillus sakei grouped within the Lactobacillus spp. cluster.

Fig. 4(a-c): A phylogenetic relationship based on 16S rDNA sequences of honeybee stomach isolates, (a) Lactobacillus plantarum, (b) Lactobacillus sakei and (c) Pediococcus pentosaceu

CONCLUSION

The current study aimed to isolate and identify lactic acid bacteria from the stomach of Egyptian honeybees (Carniolan and hybrid honeybees). Furthermore, define the in vitro antibacterial activity of novel isolates against several pathogenic bacteria. The novel isolates (HBMSS1, HBMSS3, HBMSS4, HBMSS5, HBMSS6 and HBMSS8) showed a significant antimicrobial activity against C. botulinum, E. coli, S. senftenberg and S. epidermidis as foodborne pathogens and P. larvae and M. plutonius as honeybee pathogens. Accordingly, due to their good antimicrobial activity, these promising new strains might be used as natural preservatives in food industry to prevent or delay the deterioration of food products. Moreover, these promising LAB isolates might be used against honeybee pathogens to prevent honeybee bacterial diseases as natural and save antibiotic alternatives.

SIGNIFICANCE STATEMENT

This study discover the novel lactic acid bacteria isolates from honeybees stomach with good antimicrobial activity that can be beneficial for food industry and food safety as a natural preservatives instead of synthetic ones. In the same time, these isolates could be used for treating the honeybees’ infection with P. larvae and M. plutonius as honeybee pathogens. This study will help the researcher to uncover the critical areas of food safety and Honeybees infection that many researchers were not able to explore.

ACKNOWLEDGMENTS

Authors are thankful to the Department of Food Technology, Arid Lands Cultivation Research Institute at City for Scientific Research and Technological Application, Alexandria, Egypt, for carrying out all experiments and providing the chemicals and facilities for this research.

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