Fructose and Trehalose Selectively Enhance In Vitro Sporulation of Paenibacillus larvae ERIC I and ERIC II Strains.

Maroš Laho,Juraj Majtán,Mária Šedivá,Jaroslav Klaudiny

doi:10.3390/microorganisms9020225

Abstract

Paenibacillus larvae is a Gram-positive bacterium, the spores of which are the causative agent of the most destructive brood disease of honeybees, American foulbrood (AFB). Obtaining viable spores of pathogen strains is requisite for different studies concerning AFB. The aim of this work was to investigate the effects of five saccharides that may naturally occur in higher amounts in bee larvae on in vitro sporulation of P. larvae. The effect of individual saccharides at different concentrations on spore yields of P. larvae strains of epidemiologically important ERIC genotypes was examined in Columbia sheep blood agar (CSA) and MYPGP agar media. It was found that fructose in ERIC I and trehalose in ERIC II strains at concentrations in the range of 0.5–2% represent new sporulation factors that significantly enhanced the yields of viable spores in both media, mostly in a concentration-dependent manner. The enhancements in spore yield were mainly caused by improvements of the germination ability of the spores produced. Glucose, maltose and sucrose at 1% or 0.5% concentrations also supported sporulation but to a lower extent and not in all strains and media. Based on the knowledge gained, a novel procedure was proposed for the preparation of viable P. larvae spores with supposed improved quality for AFB research.

Highlights

American foulbrood (AFB) causes large annual worldwide financial losses to beekeepers and farmers dependent on crop pollination
We demonstrated that fructose at 1–2% and trehalose at 0.5–2% concentrations in MYPGP or Columbia sheep blood agar (CSA) agar media significantly enhance the average yields of the viable heat-resistant spores of the ERIC I and ERIC II strains, respectively, in comparison with the yields achieved on the basal agar media themselves
The enhancements in spore yield with these saccharides were caused by similar processes to those we found for P. larvae sporulation

Summary

Introduction

American foulbrood (AFB) causes large annual worldwide financial losses to beekeepers and farmers dependent on crop pollination. The primary event in disease development in a colony is the infection of young larvae with Paenibacillus larvae spores, which contaminate larval food [1,2,3]. Ingested spores germinate in the larval midgut where the infection continues by massive proliferation of vegetative cells [4] associated with the production of substances that help bacteria to penetrate through the peritrophic matrix and midgut epithelium into the hemocoel (reviewed in [5,6]). The spores are transmitted by worker bees to other larvae and escalation of the infection in the colony leads to its collapse [3,7]. Spores are spread among colonies by bees and by beekeepers [8,9,10]. The use of antibiotics may be problematic because it can be connected with honey contamination [16,17] and their efficacy can impact the development of resistant strains of the pathogen [18,19,20]

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