Skip to main content
SpringerLink
Log in

Comparative analysis of Paenibacillus larvae genotypes isolated in Connecticut

  • Original Paper
  • Published:
Archives of Microbiology Aims and scope Submit manuscript

Abstract

Ninety-six strains of Paenibacillus larvae, causative agent of American foulbrood in honey bee (Apis mellifera) larvae, collected from Connecticut, USA (CT), honey bees, and 12 P. larvae strains not from CT, were genotyped via ERIC-PCR and XbaI-RFLP analysis. All CT-isolates, five strains isolated in South America, three strains from North America (not CT), and one strain isolated in Australia grouped into the ERIC I genotype. Three P. larvae formerly subsp. pulvifaciens strains grouped into ERIC III and IV genotypes. XbaI-RFLP genotyping showed three genotypes within the CT-isolates, and two were identified as XbaI-RFLP Type I and III. The third XbaI-RFLP genotype (Type Ib) represented one of four new XbaI-RFLP genotypes identified. Comparison of genotype results for the P. larvae strains tested was used to develop a correlation between ERIC-PCR genotyping and XbaI-RFLP genotyping. Sixteen CT-isolates were tetracycline-resistant and demonstrated PCR amplification using oligonucleotide primers for tetL. All 16 isolates grouped within XbaI-RFLP Type Ib, suggesting limited introduction of a tetracycline-resistant strain into CT.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  • Alippi AM, Aguilar OM (1998a) Unique DNA fingerprint patterns of Paenibacillus larvae subsp. larvae strains. J Apic Res 37:273–280

    CAS  Google Scholar 

  • Alippi AM, Aguilar OM (1998b) Characterization of isolates of Paenibacillus larvae subsp. larvae from diverse geographical origin by the polymerase chain reaction and BOX primers. J Invertebr Pathol 72:21–27

    Article  CAS  PubMed  Google Scholar 

  • Alippi AM, López AC, Aguilar OM (2002) Differentiation of Paenibacillus larvae subsp. larvae, the cause of American foulbrood of honeybees, by using PCR and restriction fragment analysis of genes encoding 16S rRNA. Appl Environ Microbiol 68:3655–3660

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Alippi A, Reynaldi FJ, López AC, De Giusti MR, Aguilar M (2004) Molecular epidemiology of Paenibacillus larvae larvae and incidence of American foulbrood in Argentinean honeys from Buenos Aires province. J Apic Res 43:135–143

    CAS  Google Scholar 

  • Alippi AM, López AC, Reynaldi FJ, Grasso DH, Aguilar OM (2007) Evidence for plasmid-mediated tetracycline resistance in Paenibacillus larvae, the causal agent of American Foulbrood (AFB) disease in honeybees. Vet Microbiol 125:290–303

    Article  CAS  PubMed  Google Scholar 

  • Antúnez K, Piccini C, Castro-Sowinski S, Rosado AS, Seldin L, Zunino P (2007) Phenotypic and genotypic characterization of Paenibacillus larvae isolates. Vet Microbiol 124:178–183

    Article  PubMed  Google Scholar 

  • Ashiralieva A, Genersch E (2006) Reclassification, genotypes and virulence of Paenibacillus larvae, the etiological agent of American foulbrood in honeybees—a review. Apidologie 37:411–420

    Article  Google Scholar 

  • de Graaf DC, Alippi AM, Antúnez K, Aronstein KA, Budge G, De Koker D, De Smet L, Dingman DW, Evans JD, Foster LJ, Fünfhaus A, Garcia-Gonzalez E, Gregorc A, Human H, Murray KD, Nguyen BK, Poppinga L, Spivak M, vanEngelsdorp D, Wilkins S, Genersch E (2013) Standard methods for American foulbrood research. J Apic Res 52(1). http://dx.doi.org/10.3896/IBRA.1.52.1.11

  • Di Pinto A, Novello L, Terio V, Tantillo G (2011) ERIC-PCR genotyping of Paenibacillus larvae in southern Italian honey and brood combs. Curr Microbiol 63:416–419

    Article  PubMed  Google Scholar 

  • Dingman DW (2012) Paenibacillus larvae 16S-23S rDNA intergenic transcribed spacer (ITS) regions: DNA fingerprinting and characterization. J Invertebr Pathol 110:352–358

    Article  CAS  PubMed  Google Scholar 

  • Dingman DW, Stahly DP (1983) Medium promoting sporulation of Bacillus larvae and metabolism of medium components. Appl Environ Microbiol 46:860–869

    CAS  PubMed Central  PubMed  Google Scholar 

  • Forsgren E, Stevanovic J, Fries I (2008) Variability in germination and in temperature and storage resistance among Paenibacillus larvae genotypes. Vet Microbiol 129:342–349

    Article  CAS  PubMed  Google Scholar 

  • Gautom RK (1997) Rapid pulsed-field gel electrophoresis protocol for typing of Escherichia coli O157:H7 and other gram-negative organisms in 1 day. J Clin Microbiol 35:2977–2980

    CAS  PubMed Central  PubMed  Google Scholar 

  • 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 34:195–206

    Article  CAS  Google Scholar 

  • Genersch E, Ashiralieva A, Fries I (2005) Strain-and genotype-specific differences in virulence of Paenibacillus larvae subsp. larvae, a bacterial pathogen causing American foulbrood disease in honeybees. Appl Environ Microbiol 71:7551–7555

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Genersch E, Forsgren E, Pentikäinen J, Ashiralieva A, Rauch S, Kilwinski J, Fries I (2006) Reclassification of Paenibacillus larvae subsp. pulvifaciens and Paenibacillus larvae subsp. larvae as Paenibacillus larvae without subspecies differentiation. Int J Syst Evol Microbiol 56:501–511

    Article  CAS  PubMed  Google Scholar 

  • Gordon RE, Haynes WC, Pang CH (1973) The genus Bacillus. Agriculture handbook no. 427. Agricultural Research Service, US Dept Agriculture, Washington

    Google Scholar 

  • Hitchcock JD, Stoner A, Wilson WT, Menapace DM (1979) Pathogenicity of Bacillus pulvifaciens to honey bee larvae of various ages (Hymenoptera: Apidae). J Kansas Entomol Soc 52:238–246

    Google Scholar 

  • Johnson JR, Clabots C (2000) Improved repetitive-element PCR fingerprinting of Salmonella enterica with the use of extremely elevated annealing temperatures. Clin Diagn Lab Immunol 7:258–264

    CAS  PubMed Central  PubMed  Google Scholar 

  • Katznelson H (1950) Bacillus pulvifaciens (n. sp.), an organism associated with powdery scale of honeybee larvae. J Bacteriol 59:153–155

    CAS  PubMed Central  PubMed  Google Scholar 

  • Morrissey BJ, Helgason T, Poppinga L, Fünfhaus A, Genersch E, Budge GE (2014) Biogeography of Paenibacillus larvae, the causative agent of American foulbrood, using a new MLST scheme. Environ Microbiol. doi:10.1111/1462-2920.12625

    PubMed Central  Google Scholar 

  • Murray KD, Aronstein KA (2006) Oxytetracycline-resistance in the honey bee pathogen Paenibacillus larvae is encoded on novel plasmid pMA67. J Apic Res 45:207–214

    Article  CAS  Google Scholar 

  • Neuendorf S, Hedtke K, Tangen G, Genersch E (2004) Biochemical characterization of different genotypes of Paenibacillus larvae subsp. larvae, a honey bee bacterial pathogen. Microbiology 150:2381–2390

    Article  CAS  PubMed  Google Scholar 

  • Ng LK, Martin I, Alfa M, Mulvey M (2001) Multiplex PCR for the detection of tetracycline resistant genes. Mol Cell Probes 15:209–215

    Article  CAS  PubMed  Google Scholar 

  • Peters M, Kilwinski J, Beringhoff A, Reckling D, Genersch E (2006) American foulbrood of the honey bee: occurrence and distribution of different genotypes of Paenibacillus larvae in the administrative district of Arnsberg (North Rhine-Westphalia). J Vet Med B 53:100–104

    Article  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  • 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 170:291–297

    Article  PubMed  Google Scholar 

  • Steinkraus KH, Morse RA (1996) Media for the detection of Bacillus larvae spores in honey. Acta Biotechnol 16:57–64

    Article  CAS  Google Scholar 

  • Tyler KD, Wang G, Tyler SD, Johnson WM (1997) Factors affecting reliability and reproducibility of amplification-based DNA fingerprinting of representative bacterial pathogens. J Clin Microbiol 35:339–346

    CAS  PubMed Central  PubMed  Google Scholar 

  • Versalovic J, Schneider M, De Bruijn FJ, Lupski JR (1994) Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Methods Mol Cell Biol 5:25–40

    CAS  Google Scholar 

  • Wu XY, Chin J, Ghalayini A, Hornitzky M (2005) Pulsed-field gel electrophoresis typing and oxytetracycline sensitivity of Paenibacillus larvae subsp. larvae isolates of Australian origin and those recovered from honey imported from Argentina. J Apic Res 44:87–92

    CAS  Google Scholar 

Download references

Acknowledgments

I thank S.M. Douglas for helpful suggestions in the preparation of this manuscript and Regan Huntley for technical assistance. I also thank A. Alippi for kindly providing bacterial strains for use in this investigation. This research was supported by Federal Hatch fund USDA CONH00250.

Author information

Authors and Affiliations

  1. Department of Entomology, The Connecticut Agricultural Experiment Station, 123 Huntington Street, P. O. Box 1106, New Haven, CT, 06504, USA

    Douglas W. Dingman

Authors
  1. Douglas W. Dingman
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Douglas W. Dingman.

Additional information

Communicated by Erko Stackebrandt.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Dingman, D.W. Comparative analysis of Paenibacillus larvae genotypes isolated in Connecticut. Arch Microbiol 197, 789–795 (2015). https://doi.org/10.1007/s00203-015-1113-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00203-015-1113-4

Keywords

Search

Navigation