Isolation of bacterial microbiota associated to honey bees and evaluation of potential biocontrol agents of Varroa destructor.

The honey bee parasitic mite Varroa destructor is one of the main causes of depopulation of bee colonies. Bacterial symbionts associated to honey bees are known to produce a variety of bioactive molecules that have been suggested to play a protective role against honey bee pathogens. We hypothesised that among these bacteria, those colonising the external body of honey bees, and therefore able to survive and reproduce in the hive environment outside the insect gut, may be good candidate biocontrol agents to be tested against V. destructor. The aim of this study was to isolate bacterial species from healthy honey bees and dead varroa mites and to evaluate the potential miticidal effect of their spent medium containing both bacterial metabolites and viable cells, with the final objective of finding a long-lasting solution for mite control. 61 bacterial strains belonging to the Firmicutes, Proteobacteria and Actinobacteria phyla were isolated from the surface of foragers, nurse bees and larvae collected in 10 different apiaries. The most common species was Lactobacillus kunkeei (62%). Growth capability of a selection of isolates was observed at 30 and 34 °C with 1% and 20% glucose and fructose. Laboratory bioassays were conducted by spraying mites with six-day-grown bacterial cultures containing 107 cfu/ml of four strains of L. kunkeei, Bacillus thuringiensis, Bifidobacterium asteroides and an Acetobacteraceae bacterium. The effect of each strain on varroa survival was tested independently. The first three caused 95-100% mortality of mites in 3 days, indicating a potential role as natural antagonists towards varroa. The mediation of pH of the bacterial cultures did not appear to be determinant in mite inhibition, suggesting the involvement of other modes of action against varroa. The exploitation of honey bee microbiota for controlling one of the major threats for honey bee health may be a promising approach deserving further investigation.

Secondary Metabolites Produced by Honey Bee-Associated Bacteria for Apiary Health: Potential Activity of Platynecine.

Secondary metabolites of bacteria associated with honey bees were evaluated as part of an investigation on their potentiality for apiary health. Low molecular weight compounds released into culture filtrates by the four bacterial isolates taken from surface of healthy honey bees were analyzed using time-of-flight mass spectrometry. Only one low molecular weight compound was found in the culture filtrate of each isolate. Bacillus thuringiensis, Bifidobacterium asteroides and Acetobacteraceae bacterium, released into culture filtrates platynecine, a pyrrolizidine alkaloid of plant origin, which, until now, had never been reported as produced by bacteria. Lactobacillus kunkeei produced a 3,5-dinitropyridine, of unknown biological action never associated so far to bacteria. The highest relative concentration of platynecine was detected in B. thuringiensis (100%), B. asteroides and A. bacterium showed a concentration above 50% and below 25% that concentration. An in vitro assay on the potential for controlling the parasitic mite Varroa destructor by the culture filtrates of the three platynecine-producing bacteria was performed. Varroa mite mortality was proportional to the platynecine relative concentration into culture filtrate. Although miticidal activity of B. thuringiensis might be associated to other toxic proteins produced by this species, B. asteroides toxicity toward V. destructor along with the other findings of this study support the hypothesis that platynecine plays a direct or an indirect role in controlling varroa. Findings of this study suggest that secondary metabolites released by honey bee-associated bacteria represent a source of natural compounds to be considered in the challenge for counteracting the colony decline.

Impact of Ag and Al2O3 nanoparticles on soil organisms: in vitro and soil experiments.

In vitro analyses were conducted to assess the impact of Al2O3 and Ag nanoparticles on two common soil bacteria, Bacillus cereus and Pseudomonas stutzeri. Al2O3 nanoparticles did not show significant toxicity at any dose or time assayed, whereas exposure to 5 mg L(-1) Ag nanoparticles for 48 h caused bactericidal effects. Moreover, alterations at the morphological level were observed by transmission electron microscopy (TEM); Ag but not Al2O3 nanoparticles evoked the entrance of B. cereus cells in an early sporulation stage and both nanoparticles penetrated P. stutzeri cells. At the molecular level, a dramatic increase (8.2-fold) in katB gene expression was found in P. stutzeri following Al2O3 nanoparticles exposure, indicative of an oxidative stress-defence system enhancement in this bacterium. In the microcosm experiment, using two different natural soils, Al2O3 or Ag nanoparticles did not affect the Caenorhabditis elegans toxicity endpoints growth, survival, or reproduction. However, differences in microbial phylogenetic compositions were detected by fluorescence in situ hybridization (FISH). The use of katB- and pykA-based sequences showed that the microbial transcriptional response to nanoparticle exposure decreased, suggesting a decrease in cellular activity. These changes were attributable to both the nanoparticles treatment and soil characteristics, highlighting the importance of considering the soil matrix on a case by case basis.

Transcriptional and proteomic stress responses of a soil bacterium Bacillus cereus to nanosized zero-valent iron (nZVI) particles.

Nanosized zero valent iron (nZVI) is emerging as an option for treating contaminated soil and groundwater even though the potentially toxic impact exerted by nZVI on soil microorganisms remains uncertain. In this work, we focus on nanotoxicological studies performed in vitro using commercial nZVI and one common soil bacterium (Bacillus cereus). Results showed a negative impact of nZVI on B. cereus growth capability, consistent with the entrance of cells in an early sporulation stage, observed by TEM. Despite no changes at the transcriptional level are detected in genes of particular relevance in cellular activity (narG, nirS, pykA, gyrA and katB), the proteomic approach used highlights differentially expressed proteins in B. cereus under nZVI exposure. We demonstrate that proteins involved in oxidative stress-response and tricarboxilic acid cycle (TCA) modulation are overexpressed; moreover proteins involved in motility and wall biosynthesis are repressed. Our results enable to detect a molecular-level response as early warning signal, providing new insight into first line defense response of a soil bacterium after nZVI exposure.