1887

Abstract

The present study was done to explore the diversity of lactic acid bacteria (LAB) associated with the gastrointestinal tract (GIT) of honeybee species endemic to northeastern Pakistan. Healthy worker bees belonging to and were collected from hives and the surroundings of a major apiary in the region. The 16S rRNA amplicon sequencing revealed a microbial community in that was distinct from the others in having an abundance of and . However, this was not reflected in the culturable bacteria obtained from these species. The isolates were characterized for safety parameters, and 20 LAB strains deemed safe were evaluated for resistance to human GIT stresses like acid and bile, adhesion and adhesiveness, and anti-pathogenicity. The five most robust strains, NPL780a, NPL782a, NPL783a, and NPL784, and NPL783b, were identified through normalized Pearson (n) principal components analysis (PCA). These strains were checked for inhibition of human pathogens, antibiotic resistance, osmotic tolerance, metabolic and enzymatic functions, and carbohydrate utilization, along with antioxidative and cholesterol-removing potential. The findings suggest at least three strains (NPL 783a, 784 and 782a) as candidates for further and investigations of their potential health benefits and application as novel probiotic adjuncts.

Funding
This study was supported by the:
  • Ministry of Planning, Development & Special Initiatives, Govt. of Pakistan (Award PSDP-Development of a National Probiotics Lab at NIBGE')
    • Principle Award Recipient: ZaidiArsalan
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.001032
2021-02-15
2024-12-08
Loading full text...

Full text loading...

/deliver/fulltext/micro/167/3/micro001032.html?itemId=/content/journal/micro/10.1099/mic.0.001032&mimeType=html&fmt=ahah

References

  1. Granato D, Branco GF, Nazzaro F, Cruz AG, Faria JAF. Functional foods and nondairy probiotic food development: trends, concepts, and products. Compr Rev Food Sci Food Saf 2010; 9:292–302 [View Article][PubMed]
    [Google Scholar]
  2. Tripathi MK, Giri SK. Probiotic functional foods: survival of probiotics during processing and storage. J Funct Foods 2014; 9:225–241 [View Article]
    [Google Scholar]
  3. Sybesma W, Kort R, Lee Y-K. Locally sourced probiotics, the next opportunity for developing countries?. Trends Biotechnol 2015; 33:197–200 [View Article][PubMed]
    [Google Scholar]
  4. Linares DM, Ross P, Stanton C. Beneficial microbes: the pharmacy in the gut. Bioengineered 2016; 7:11–20 [View Article][PubMed]
    [Google Scholar]
  5. Dillon RJ, Dillon VM. The gut bacteria of insects: nonpathogenic interactions. Annu Rev Entomol 2004; 49:71–92 [View Article][PubMed]
    [Google Scholar]
  6. Hughes DP, Pierce NE, Boomsma JJ. Social insect symbionts: evolution in homeostatic fortresses. Trends Ecol Evol 2008; 23:672–677 [View Article][PubMed]
    [Google Scholar]
  7. Endo A, Maeno S, Tanizawa Y, Kneifel W, Arita M et al. Fructophilic lactic acid bacteria, a unique group of fructose-fermenting microbes. Appl Environ Microbiol 2018; 84: 01 10 2018 [View Article][PubMed]
    [Google Scholar]
  8. C HC, T R K, Honey Chandran C, Keerthi T R. Probiotic potency of Lactobacillus plantarum KX519413 and KX519414 isolated from honey bee gut. FEMS Microbiol Lett 2018; 365: 01 02 2018 [View Article][PubMed]
    [Google Scholar]
  9. Vergalito F, Testa B, Cozzolino A, Letizia F, Succi M et al. Potential application of Apilactobacillus kunkeei for human use: evaluation of probiotic and functional properties. Foods 2020; 9:1535 [View Article][PubMed]
    [Google Scholar]
  10. Akhtar T, Aziz MA, Naeem M, Ahmed MS, Bodlah I. Diversity and relative abundance of pollinator fauna of canola (Brassica napus L. var Chakwal Sarsoon) with managed Apis mellifera L. in Pothwar region, Gujar Khan, Pakistan. Pak J Zool 2018; 50: [View Article]
    [Google Scholar]
  11. Waghchoure-Camphor E, Martin S. Beekeeping in Pakistan: a bright future in a troubled land. Am Bee J 2008; 148:726–728
    [Google Scholar]
  12. Pachla A, Wicha M, Ptaszyńska AA, Borsuk G, Trokenheim Łucja Łaniewska et al. The molecular and phenotypic characterization of fructophilic lactic acid bacteria isolated from the guts of Apis mellifera L. derived from a Polish apiary. J Appl Genet 2018; 59:503–514 [View Article][PubMed]
    [Google Scholar]
  13. Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Lozupone CA et al. Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc Natl Acad Sci U S A 2011; 108 Suppl 1:4516–4522 [View Article][PubMed]
    [Google Scholar]
  14. Zhao LL, Yin HC, Lu TF, Niu YJ, Zhang YY et al. Application of high-throughput sequencing for microbial diversity detection in feces of specific-pathogen-free ducks. Poult Sci 2018; 97:2278–2286 [View Article][PubMed]
    [Google Scholar]
  15. Sim K, Cox MJ, Wopereis H, Martin R, Knol J et al. Improved detection of bifidobacteria with optimised 16S rRNA-gene based pyrosequencing. PLoS One 2012; 7:e32543 [View Article][PubMed]
    [Google Scholar]
  16. Li M, Wang Y, Cui H, Li Y, Sun Y et al. Characterization of lactic acid bacteria isolated from the gastrointestinal tract of a wild boar as potential probiotics. Front Vet Sci 2020; 7:10 [View Article][PubMed]
    [Google Scholar]
  17. Botta C, Langerholc T, Cencič A, Cocolin L. In vitro selection and characterization of new probiotic candidates from table olive microbiota. PLoS One 2014; 9:e94457 [View Article][PubMed]
    [Google Scholar]
  18. Garcia-Gonzalez N, Prete R, Battista N, Corsetti A. Adhesion properties of food-associated Lactobacillus plantarum strains on human intestinal epithelial cells and modulation of IL-8 release. Front Microbiol 2018; 9:2392 [View Article][PubMed]
    [Google Scholar]
  19. Leccese Terraf MC, Mendoza LM, Juárez Tomás MS, Silva C, Nader-Macías MEF. Phenotypic surface properties (aggregation, adhesion and biofilm formation) and presence of related genes in beneficial vaginal lactobacilli. J Appl Microbiol 2014; 117:1761–1772 [View Article][PubMed]
    [Google Scholar]
  20. Halder D, Mandal M, Chatterjee SS, Pal NK, Mandal S. Indigenous probiotic Lactobacillus isolates presenting antibiotic like activity against human pathogenic bacteria. Biomedicines 2017; 5:31 [View Article][PubMed]
    [Google Scholar]
  21. Ren D, Li C, Qin Y, Yin R, Du S et al. In vitro evaluation of the probiotic and functional potential of Lactobacillus strains isolated from fermented food and human intestine. Anaerobe 2014; 30:1–10 [View Article][PubMed]
    [Google Scholar]
  22. EFSA-panel Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance. EFSA J 2012; 10:2740
    [Google Scholar]
  23. Turpin W, Humblot C, Noordine M-L, Thomas M, Guyot J-P. Lactobacillaceae and cell adhesion: genomic and functional screening. PLoS One 2012; 7:e38034 [View Article][PubMed]
    [Google Scholar]
  24. Cebrián R, Baños A, Valdivia E, Pérez-Pulido R, Martínez-Bueno M et al. Characterization of functional, safety, and probiotic properties of Enterococcus faecalis UGRA10, a new AS-48-producer strain. Food Microbiol 2012; 30:59–67 [View Article][PubMed]
    [Google Scholar]
  25. Jayamanne V, Adams M, survival Dof. identity and stress resistance of probiotic Bifidobacteria in bio‐yoghurts. Lett Appl Microbiol 2006; 42:189–194
    [Google Scholar]
  26. Raymann K, Shaffer Z, Moran NA. Antibiotic exposure perturbs the gut microbiota and elevates mortality in honeybees. PLoS Biol 2017; 15:e2001861 [View Article][PubMed]
    [Google Scholar]
  27. Tajabadi N, Ebrahimpour A, Baradaran A, Rahim RA, Mahyudin NA et al. Optimization of γ-aminobutyric acid production by Lactobacillus plantarum Taj-Apis362 from honeybees. Molecules 2015; 20:6654–6669 [View Article][PubMed]
    [Google Scholar]
  28. Potts SG, Imperatriz-Fonseca V, Ngo HT, Aizen MA, Biesmeijer JC et al. Safeguarding pollinators and their values to human well-being. Nature 2016; 540:220–229 [View Article][PubMed]
    [Google Scholar]
  29. Rokop ZP, Horton MA, Newton ILG. Interactions between cooccurring lactic acid bacteria in honey bee hives. Appl Env Microbiol 2015; 81:7261–7270 [View Article][PubMed]
    [Google Scholar]
  30. Anguita-Maeso M, Olivares-García C, Haro C, Imperial J, Navas-Cortés JA et al. Culture-dependent and culture-independent characterization of the olive xylem microbiota: effect of SAP extraction methods. Front Plant Sci 2019; 10:10 [View Article][PubMed]
    [Google Scholar]
  31. Alenezi H. Microbiological analysis of root canal infections using high throughput sequencing on the Illumina MiSeq platform. PhD, University of Leeds; 2015
  32. Loman NJ, Misra RV, Dallman TJ, Constantinidou C, Gharbia SE et al. Performance comparison of benchtop high-throughput sequencing platforms. Nat Biotechnol 2012; 30:434–439 [View Article][PubMed]
    [Google Scholar]
  33. Romero S, Nastasa A, Chapman A, Kwong WK, Foster LJ. The honey bee gut microbiota: strategies for study and characterization. Insect Mol Biol 2019; 28:455–472 [View Article][PubMed]
    [Google Scholar]
  34. Fettweis JM, Serrano MG, Brooks JP, Edwards DJ, Girerd PH et al. The vaginal microbiome and preterm birth. Nat Med 2019; 25:1012–1021 [View Article][PubMed]
    [Google Scholar]
  35. Hilton SK, Castro-Nallar E, Pérez-Losada M, Toma I, McCaffrey TA et al. Metataxonomic and metagenomic approaches vs. culture-based techniques for clinical pathology. Front Microbiol 2016; 7:12 [View Article]
    [Google Scholar]
  36. Jones JC, Fruciano C, Hildebrand F, Al Toufalilia H, Balfour NJ et al. Gut microbiota composition is associated with environmental landscape in honey bees. Ecol Evol 2018; 8:441–451 [View Article][PubMed]
    [Google Scholar]
  37. Ali M, Sajjad A, Saeed S. Yearlong association of Apis dorsata and Apis florea with flowering plants: planted forest vs. agricultural landscape. Sociobiology 2017; 64:18 [View Article]
    [Google Scholar]
  38. Somanathan H, Warrant EJ, Borges RM, Wallén R, Kelber A. Resolution and sensitivity of the eyes of the Asian honeybees Apis florea, Apis cerana and Apis dorsata . J Exp Biol 2009; 212:2448–2453 [View Article][PubMed]
    [Google Scholar]
  39. Hroncova Z, Havlik J, Killer J, Doskocil I, Tyl J et al. Variation in honey bee gut microbial diversity affected by ontogenetic stage, age and geographic location. PLoS One 2015; 10:e0118707 [View Article][PubMed]
    [Google Scholar]
  40. Anderson KE, Sheehan TH, Mott BM, Maes P, Snyder L et al. Microbial ecology of the hive and pollination landscape: bacterial associates from floral nectar, the alimentary tract and stored food of honey bees (Apis mellifera). PLoS One 2013; 8:e83125 [View Article][PubMed]
    [Google Scholar]
  41. Harada T, Dang VC, Nguyen DP, Nguyen TAD, Sakamoto M et al. Enterococcus saigonensis sp. nov., isolated from retail chicken meat and liver. Int J Syst Evol Microbiol 2016; 66:3779–3785 [View Article][PubMed]
    [Google Scholar]
  42. Anjum SI, Aldakheel F, Shah AH, Khan S, Ullah A et al. Honey bee gut an unexpected niche of human pathogen. J King Saud Univ Sci 2021; 33:101247 [View Article]
    [Google Scholar]
  43. Kešnerová L, Emery O, Troilo M, Liberti J, Erkosar B et al. Gut microbiota structure differs between honeybees in winter and summer. Isme J 2020; 14:801–814 [View Article][PubMed]
    [Google Scholar]
  44. Lagier J-C, Million M, Hugon P, Armougom F, Raoult D. Human gut microbiota: repertoire and variations. Front Cell Inf Microbiol 2012; 2:136 [View Article][PubMed]
    [Google Scholar]
  45. Ellegaard KM, Tamarit D, Javelind E, Olofsson TC, Andersson SGE et al. Extensive intra-phylotype diversity in lactobacilli and bifidobacteria from the honeybee gut. BMC Genomics 2015; 16:284 [View Article][PubMed]
    [Google Scholar]
  46. Salvetti E, Harris HMB, Felis GE, Toole PW. Comparative genomics of the genus Lactobacillus reveals robust phylogroups that provide the basis for reclassification. Appl Env Microbiol 2018; 84:e00993–00918
    [Google Scholar]
  47. Johnson JS, Spakowicz DJ, Hong B-Y, Petersen LM, Demkowicz P et al. Evaluation of 16S rRNA gene sequencing for species and strain-level microbiome analysis. Nat Commun 2019; 10:5029 [View Article][PubMed]
    [Google Scholar]
  48. Vuong HQ. Function and transmission of novel wild bee symbionts from the Lactobacillus micheneri clade PhD, UC Riverside; 2019
  49. Wang S, Yang B, Ross RP, Stanton C, Zhao J et al. Comparative genomics analysis of Lactobacillus ruminis from different niches. Genes 2020; 11:70 [View Article][PubMed]
    [Google Scholar]
  50. Bottacini F, Milani C, Turroni F, Sánchez B, Foroni E et al. Bifidobacterium asteroides PRL2011 genome analysis reveals clues for colonization of the insect gut. PLoS One 2012; 7:e44229 [View Article][PubMed]
    [Google Scholar]
  51. Ribière C, Hegarty C, Stephenson H, Whelan P, O'Toole PW. Gut and whole-body microbiota of the honey bee separate thriving and non-thriving hives. Microb Ecol 2019; 78:195–205 [View Article][PubMed]
    [Google Scholar]
  52. Anderson KE, Ricigliano VA. Honey bee gut dysbiosis: a novel context of disease ecology. Curr Opin Insect Sci 2017; 22:125–132 [View Article][PubMed]
    [Google Scholar]
  53. Filannino P, Di Cagno R, Addante R, Pontonio E, Gobbetti M. Metabolism of Fructophilic lactic acid bacteria isolated from the Apis mellifera L. bee gut: Phenolic acids as external electron acceptors. Appl Environ Microbiol 2016; 82:6899–6911 [View Article][PubMed]
    [Google Scholar]
  54. Lamei S, Hu YOO, Olofsson TC, Andersson AF, Forsgren E et al. Improvement of identification methods for honeybee specific lactic acid bacteria; future approaches. PLoS One 2017; 12:e0174614 [View Article][PubMed]
    [Google Scholar]
  55. Meihua W. Isolation and characterization of European foulbrood antagonistic bacteria from the gastrointestine of the Japanese honeybee Apis cerana japonica PhD, University of Tsukuba; 2013
  56. Gupta M, Bajaj BK. Functional characterization of potential probiotic lactic acid bacteria isolated from Kalarei and development of probiotic fermented oat flour. Probiotics Antimicrob Proteins 2018; 10:654–661 [View Article][PubMed]
    [Google Scholar]
  57. Iorizzo M, Lombardi SJ, Ganassi S, Testa B, Ianiro M et al. Antagonistic activity against Ascosphaera apis and functional properties of Lactobacillus kunkeei strains. Antibiotics 2020; 9:E262262 18 05 2020 [View Article][PubMed]
    [Google Scholar]
  58. de Albuquerque TMR, Garcia EF, de Oliveira Araújo A, Magnani M, Saarela M et al. In vitro characterization of Lactobacillus strains isolated from fruit processing by-products as potential probiotics. Probiotics Antimicrob Proteins 2018; 10:704–716 [View Article][PubMed]
    [Google Scholar]
  59. Ł G, Collado MC, Salminen S. Evaluation of aggregation abilities between commensal fish bacteria and pathogens. Aquaculture 2012; 356-357:412–414
    [Google Scholar]
  60. Ohland CL, Macnaughton WK. Probiotic bacteria and intestinal epithelial barrier function. Am J Physiol Gastrointest Liver Physiol 2010; 298:G807–G819 [View Article][PubMed]
    [Google Scholar]
  61. Karched M, Bhardwaj RG, Asikainen SE. Coaggregation and biofilm growth of Granulicatella spp. with Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans . BMC Microbiol 2015; 15:114 [View Article][PubMed]
    [Google Scholar]
  62. Kumar KV, Pal A, Bai P, Kour A, E S et al. Co-aggregation of bacterial flora isolated from the human skin surface. Microb Pathog 2019; 135:103630 [View Article][PubMed]
    [Google Scholar]
  63. Cheng Z, Meng X, Wang H, Chen M, Li M. Isolation and characterization of broad spectrum coaggregating bacteria from different water systems for potential use in bioaugmentation. PLoS One 2014; 9:e94220 [View Article][PubMed]
    [Google Scholar]
  64. Kaškonienė V, Stankevičius M, Bimbiraitė-Survilienė K, Naujokaitytė G, Šernienė L et al. Current state of purification, isolation and analysis of bacteriocins produced by lactic acid bacteria. Appl Microbiol Biotechnol 2017; 101:1323–1335 [View Article][PubMed]
    [Google Scholar]
  65. Tirloni E, Cattaneo P, Ripamonti B, Agazzi A, Bersani C et al. In vitro evaluation of Lactobacillus animalis SB310, Lactobacillus paracasei subsp. paracasei SB137 and their mixtures as potential bioprotective agents for raw meat. Food Control 2014; 41:63–68 [View Article]
    [Google Scholar]
  66. Chen Y-T, Hsieh P-S, Ho H-H, Hsieh S-H, Kuo Y-W et al. Antibacterial activity of viable and heat-killed probiotic strains against oral pathogens. Lett Appl Microbiol 2020; 70:310–317 [View Article][PubMed]
    [Google Scholar]
  67. Barache N, Ladjouzi R, Belguesmia Y, Bendali F, Drider D. Abundance of Lactobacillus plantarum strains with beneficial attributes in blackberries (Rubus sp.), fresh figs (Ficus carica), and prickly pears (Opuntia ficus-indica) grown and harvested in Algeria. Probiotics Antimicrob Proteins 2020; 12:1514–1523 [View Article][PubMed]
    [Google Scholar]
  68. Zheng H, Nishida A, Kwong WK, Koch H, Engel P et al. Metabolism of toxic sugars by strains of the bee gut symbiont Gilliamella apicola.. mBio 2016; 7:e01326–01316 [View Article][PubMed]
    [Google Scholar]
  69. Fiocco D, Longo A, Arena MP, Russo P, Spano G et al. How probiotics face food stress: they get by with a little help. Crit Rev Food Sci Nutr 2020; 60:1552–1580 [View Article][PubMed]
    [Google Scholar]
  70. Yebra MJ, Zúñiga M, Beaufils S, Pérez-Martínez G, Deutscher J et al. Identification of a gene cluster enabling Lactobacillus casei BL23 to utilize myo-inositol. Appl Environ Microbiol 2007; 73:3850–3858 [View Article][PubMed]
    [Google Scholar]
  71. Theilmann MC, Goh YJ, Nielsen KF, Klaenhammer TR, Barrangou R et al. Lactobacillus acidophilus metabolizes dietary plant glucosides and externalizes their bioactive phytochemicals. mBio 2017; 8:e01421–01417 [View Article][PubMed]
    [Google Scholar]
  72. Plaza-Diaz J, Ruiz-Ojeda FJ, Gil-Campos M, Gil A. Mechanisms of action of probiotics. Adv Nutr 2019; 10:S49–S66 [View Article][PubMed]
    [Google Scholar]
  73. Shokryazdan P, Jahromi MF, Liang JB, Sieo CC, Kalavathy R et al. In vitro assessment of bioactivities of Lactobacillus strains as potential probiotics for humans and chickens. J Food Sci 2017; 82:2734–2745 [View Article][PubMed]
    [Google Scholar]
  74. Maeno S, Tanizawa Y, Kanesaki Y, Kubota E, Kumar H et al. Genomic characterization of a fructophilic bee symbiont Lactobacillus kunkeei reveals its niche-specific adaptation. Syst Appl Microbiol 2016; 39:516–526 [View Article][PubMed]
    [Google Scholar]
  75. Gustaw K, Michalak M, Polak-Berecka M, Waśko A. Isolation and characterization of a new fructophilic Lactobacillus plantarum FPL strain from honeydew. Ann Microbiol 2018; 68:459–470 [View Article][PubMed]
    [Google Scholar]
  76. Sakandar HA, Kubow S, Sadiq FA. Isolation and in-vitro probiotic characterization of fructophilic lactic acid bacteria from Chinese fruits and flowers. LWT 2019; 104:70–75 [View Article]
    [Google Scholar]
  77. Di Cagno R, Filannino P, Vincentini O, Cantatore V, Cavoski I et al. Fermented Portulaca oleracea L. juice: A novel functional beverage with potential ameliorating effects on the intestinal inflammation and epithelial injury. Nutrients 2019; 11:248 23 Jan 2019 [View Article][PubMed]
    [Google Scholar]
  78. Domingos-Lopes MFP, Stanton C, Ross PR, Dapkevicius MLE, Silva CCG. Genetic diversity, safety and technological characterization of lactic acid bacteria isolated from artisanal Pico cheese. Food Microbiol 2017; 63:178–190 [View Article][PubMed]
    [Google Scholar]
  79. Salvetti E, O'Toole PW. When regulation challenges innovation: The case of the genus Lactobacillus . Trends in Food Sci Technol 2017; 66:187–194 [View Article]
    [Google Scholar]
  80. Casarotti SN, Carneiro BM, Todorov SD, Nero LA, Rahal P et al. In vitro assessment of safety and probiotic potential characteristics of Lactobacillus strains isolated from water buffalo mozzarella cheese. Ann Microbiol 2017; 67:289–301 [View Article]
    [Google Scholar]
  81. Fernández MF, Boris S, Barbés C. Safety evaluation of Lactobacillus delbrueckii subsp. lactis UO 004, a probiotic bacterium. Res Microbiol 2005; 156:154–160 [View Article][PubMed]
    [Google Scholar]
  82. Zúñiga M, Monedero V, Yebra MJ. Utilization of host-derived glycans by intestinal Lactobacillus and Bifidobacterium species. Front Microbiol 2018; 9:9 [View Article][PubMed]
    [Google Scholar]
  83. García-Solache M, Rice LB. The Enterococcus: A model of adaptability to its environment. Clin Microbiol Rev 2019; 32: 20 03 2019 [View Article][PubMed]
    [Google Scholar]
  84. Berríos P, Fuentes JA, Salas D, Carreño A, Aldea P et al. Inhibitory effect of biofilm-forming Lactobacillus kunkeei strains against virulent Pseudomonas aeruginosa in vitro and in honeycomb moth (Galleria mellonella) infection model. Benef Microbes 2018; 9:257–268 [View Article][PubMed]
    [Google Scholar]
  85. Salas-Jara MJ, Ilabaca A, Vega M, García A. Biofilm forming Lactobacillus: new challenges for the development of probiotics. Microorganisms 2016; 4:E3535 20 09 2016 [View Article][PubMed]
    [Google Scholar]
/content/journal/micro/10.1099/mic.0.001032
Loading
/content/journal/micro/10.1099/mic.0.001032
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error