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Evaluation of the presence of Paenibacillus larvae in commercial bee pollen using PCR amplification of the gene for tRNACys

  • Vicente Daniel Moreno Andrade
  • José Luis Hernández Flores
  • Miguel Angel Ramos López
  • Andrés Cruz Hernández
  • Sergio Romero Gómez
  • Rosa Paulina Medina Calvillo
  • Ana Gabriel Estrada Martínez
  • Juan Caballero Pérez
  • Iván Arvizu Hernández
  • Erika Álvarez Hidalgo
  • Claudia Álvarado Osuna
  • George H. Jones
  • Juan Campos GuillénEmail author
Environmental Microbiology - Research Paper

Abstract

American foulbrood (AFB) caused by Paenibacillus larvae is the most destructive honeybee bacterial disease and its dissemination via commercial bee pollen is an important mechanism for the spread of this bacterium. Because Mexico imports bee pollen from several countries, we developed a tRNACys-PCR strategy and complemented that strategy with MALDI-TOF MS and amplicon-16S rRNA gene analysis to evaluate the presence of P. larvae in pollen samples. P. larvae was not detected when the tRNACys-PCR approach was applied to spore-forming bacterial colonies obtained from three different locations and this result was validated by bacterial identification via MALDI-TOF MS. The genera identified in the latter analysis were Bacillus (fourteen species) and Paenibacillus (six) species. However, amplicon-16S rRNA gene analysis for taxonomic composition revealed a low presence of Paenibacillaceae with 0.3 to 16.2% of relative abundance in the commercial pollen samples analyzed. Within this family, P. larvae accounted for 0.01% of the bacterial species present in one sample. Our results indicate that the tRNACys-PCR, combined with other molecular tools, will be a useful approach for identifying P. larvae in pollen samples and will assist in controlling the spread of the pathogen.

Keywords

Paenibacillus larvae tRNACys-PCR MALDI-TOF MS Amplicon-16S rRNA 

Notes

Funding information

This study was partially financed by the Universidad Autónoma de Querétaro (PFCE 2018 and FOPER 2017).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. 1.
    De Luca PA, Vallejo-Marín M (2013) What’s the “buzz” about? The ecology and evolutionary significance of buzz-pollination. Curr Opin Plant Biol 16(4):429–435CrossRefGoogle Scholar
  2. 2.
    Roldán Serrano A, Guerra-Sanz JM (2006) Quality fruit improvement in sweet pepper culture by bumblebee pollination. Sci Hortic 110(2):160–166CrossRefGoogle Scholar
  3. 3.
    Velthuis HHW, Doorn AV (2006) A century of advances in bumblebee domestication and the economic and environmental aspects of its commercialization for pollination. Apidologie 37(4):421–451CrossRefGoogle Scholar
  4. 4.
    Graystock P, Yates K, Evison S, Darvill B, Goulson D, Hughes W (2013) The Trojan hives: pollinator pathogens, imported and distributed in bumblebee colonies. J Appl Ecol 50(5):1207–1215Google Scholar
  5. 5.
    Hansen H, Brødsgaard CJ (1999) American foulbrood: a review of its biology, diagnosis and control. Bee World 80(1):5–23CrossRefGoogle Scholar
  6. 6.
    Genersch E, Forsgren E, Pentikäinen J et al (2006) Reclassification of Paenibacillus larvae subsp. pulvifaciens and Paenibacillus larvae subsp. larvae as Paenibacillus larvae without subspecies differentiation. Int J Syst Evol Microbiol 56(3):501–511CrossRefGoogle Scholar
  7. 7.
    Yue D, Nordhoff M, Wieler LH, Genersch E (2008) Fluorescence in situ hybridization (FISH) analysis of the interactions between honeybee larvae and Paenibacillus larvae, the causative agent of American foulbrood of honeybees (Apis mellifera). Environ Microbiol 10(6):1612–1620CrossRefGoogle Scholar
  8. 8.
    Hutchison EA, Miller DA, Angert ER (2014) Sporulation in bacteria: beyond the standard model. Microbiol Spectr 2(5)Google Scholar
  9. 9.
    Ebeling J, Knispel H, Hertlein G, Fünfhaus A, Genersch E (2016) Biology of Paenibacillus larvae, a deadly pathogen of honey bee larvae. Appl Microbiol Biotechnol 100(17):7387–7395CrossRefGoogle Scholar
  10. 10.
    Bisson LF, Walker G, Ramakrishnan V et al (2016) The two faces of Lactobacillus kunkeei: wine spoilage agent and bee probiotic. CatalystGoogle Scholar
  11. 11.
    Djordjevic S, Ho-Shon M, Hornitzky M (1994) DNA restriction endonuclease profiles and typing of geographically diverse isolates of Bacillus larvae. J Apic Res 33(2):95–103CrossRefGoogle Scholar
  12. 12.
    Peréz de la Rosa D, Pérez de la Rosa JJ, Cossio-Bayugar R et al (2015) Complete genome sequence of Paenibacillus larvae MEX14, isolated from honey bee larvae from the Xochimilco quarter in Mexico City. Genome Announc 3(4):e00968–e00915CrossRefGoogle Scholar
  13. 13.
    De Guzman ZM, Cervancia CR, Dimasuay KG et al (2011) Radiation inactivation of Paenibacillus larvae and sterilization of American foul brood (AFB) infected hives using co-60 gamma rays. Appl Radiat Isot 69(10):1374–1379CrossRefGoogle Scholar
  14. 14.
    Graystock P, Jones JC, Pamminger T, Parkinson JF, Norman V, Blane EJ, Rothstein L, Wäckers F, Goulson D, Hughes WOH (2016) Hygienic food to reduce pathogen risk to bumblebees. J Invertebr Pathol 136:68–73CrossRefGoogle Scholar
  15. 15.
    Alippi AM, Aguilar OM (1998) Unique DNA fingerprint patterns of Paenibacillus larvae subsp. larvae strains. J Apic Res 37(4):273–280CrossRefGoogle Scholar
  16. 16.
    Govan VA, Allsopp MH, Davison S (1999) A PCR detection method for rapid identification of Paenibacillus larvae. J Appl Environ Microbiol 65(5):2243–2245Google Scholar
  17. 17.
    Dobbelaere W, Graaf DC, Peeters JE (2001) Development of a fast and reliable diagnostic method for American foulbrood disease (Paenibacillus larvae subsp. larvae) using a 16S rRNA gene based PCR. Apidologie 32(4):363–370CrossRefGoogle Scholar
  18. 18.
    Genersch E, Otten C (2003) The use of repetitive element PCR fingerprinting (rep-PCR) for genetic subtyping of German field isolates of Paenibacillus larvae subsp. larvae. Apidologie 34(3):195–206CrossRefGoogle Scholar
  19. 19.
    Antúnez K, D'Alessandro B, Piccini C, Corbella E, Zunino P (2004) Paenibacillus larvae larvae spores in honey samples from Uruguay: a nationwide survey. J Invertebr Pathol 86(1):56–58CrossRefGoogle Scholar
  20. 20.
    Schäfer MO, Genersch E, Fünfhaus A, Poppinga L, Formella N, Bettin B, Karger A (2014) Rapid identification of differentially virulent genotypes of Paenibacillus larvae, the causative organism of American foulbrood of honey bees, by whole cell MALDI-TOF mass spectrometry. Vet Microbiol 170(3):291–297CrossRefGoogle Scholar
  21. 21.
    Morrissey BJ, Helgason T, Poppinga L, Fünfhaus A, Genersch E, Budge GE (2015) Biogeography of Paenibacillus larvae, the causative agent of American foulbrood, using a new multilocus sequence typing scheme. Environ Microbiol 17(4):1414–1424CrossRefGoogle Scholar
  22. 22.
    Erban T, Ledvinka O, Kamler M, Nesvorna M, Hortova B, Tyl J, Titera D, Markovic M, Hubert J (2017) Honeybee (Apis mellifera)-associated bacterial community affected by American foulbrood: detection of Paenibacillus larvae via microbiome analysis. Sci Rep 7:5084CrossRefGoogle Scholar
  23. 23.
    Campos-Guillén J, Arvizu-Gómez JL, Jones GH, Olmedo-Alvarez G (2010) Characterization of tRNACys processing in a conditional Bacillus subtilis CCase mutant reveals the participation of RNase R in its quality control. Microbiology 156(7):2102–2111CrossRefGoogle Scholar
  24. 24.
    Kanaya S, Yamada Y, Kudo Y, Ikemura T (1999) Studies of codon usage and tRNA genes of 18 unicellular organisms and quantification of Bacillus subtilis tRNAs: gene expression level and species-specific diversity of codon usage based on multivariate analysis. Gene 238(1):143–155CrossRefGoogle Scholar
  25. 25.
    Dingman DW, Stahly DP (1983) Medium promoting sporulation of Bacillus larvae and metabolism of medium components. Appl Environ Microbiol 46(4):860–869Google Scholar
  26. 26.
    Lowe TM, Eddy SR (1997) tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 25(5):955–964CrossRefGoogle Scholar
  27. 27.
    Wang Q, Garrity G, Tiedje J, Cole JR (2007) Naïve Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73(16):5261–5267CrossRefGoogle Scholar
  28. 28.
    Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98Google Scholar
  29. 29.
    Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23(21):2947–2948CrossRefGoogle Scholar
  30. 30.
    Gouy M, Guindon S, Gascuel O (2010) SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol Biol Evol 27(2):221–224CrossRefGoogle Scholar
  31. 31.
    Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O (2010) New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol 59(3):307–321CrossRefGoogle Scholar
  32. 32.
    Lefort V, Longueville JE, Gascuel O (2017) SMS: smart model selection in PhyML. Mol Biol Evol 34(9):2422–2424CrossRefGoogle Scholar
  33. 33.
    Guimarães-Cestaro L, Serrão JE, Message D, Martins MF, Teixeira EW (2016) Simultaneous detection of Nosema spp., Ascosphaera apis and Paenibacillus larvae in honey bee products. J Hymenopt Res 49:43–50CrossRefGoogle Scholar
  34. 34.
    Koch H, Schmid-Hempel P (2012) Gut microbiota instead of host genotype drive the specificity in the interaction of a natural host-parasite system. Ecol Lett 15(10):1095–1103CrossRefGoogle Scholar
  35. 35.
    Engel P, Moran NA (2013) The gut microbiota of insects – diversity in structure and function. FEMS Microbiol Rev 37(5):699–735CrossRefGoogle Scholar
  36. 36.
    Cariveau DP, Elijah PJ, Koch H, Winfree R, Moran NA (2014) Variation in gut microbial communities and its association with pathogen infection in wild bumble bees (Bombus). ISME J 8(12):2369–2379CrossRefGoogle Scholar

Copyright information

© Sociedade Brasileira de Microbiologia 2019

Authors and Affiliations

  • Vicente Daniel Moreno Andrade
    • 1
  • José Luis Hernández Flores
    • 2
  • Miguel Angel Ramos López
    • 1
  • Andrés Cruz Hernández
    • 3
  • Sergio Romero Gómez
    • 1
  • Rosa Paulina Medina Calvillo
    • 1
  • Ana Gabriel Estrada Martínez
    • 1
  • Juan Caballero Pérez
    • 1
  • Iván Arvizu Hernández
    • 1
  • Erika Álvarez Hidalgo
    • 1
  • Claudia Álvarado Osuna
    • 4
  • George H. Jones
    • 5
  • Juan Campos Guillén
    • 1
    Email author
  1. 1.Facultad de QuímicaUniversidad Autónoma de QuerétaroSantiago de QuerétaroMéxico
  2. 2.Laboratorio de Bioseguridad y Análisis de Riesgo, Departamento de Ingeniería GenéticaCentro de Investigación y de Estudios Avanzados del IPNIrapuatoMéxico
  3. 3.Escuela de AgronomíaUniversidad De La Salle BajíoLeónMéxico
  4. 4.CIATEJZapopanMéxico
  5. 5.Department of BiologyEmory UniversityAtlantaUSA

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