Development and evaluation of a core genome multilocus sequence typing scheme for Paenibacillus larvae, the deadly American foulbrood pathogen of honeybees.

Paenibacillus larvae is the causative agent of the fatal American foulbrood disease in honeybees (Apis mellifera). Strain identification is vital for preventing the spread of the disease. To date, the most accessible and robust scheme to identify strains is the multilocus sequence typing (MLST) method. However, this approach has limited resolution, especially for epidemiological studies. As the cost of whole-genome sequencing has decreased and as it becomes increasingly available to most laboratories, an extended MLST based on the core genome (cgMLST) presents a valuable tool for high-resolution investigations. In this study, we present a standardized, robust cgMLST scheme for P. larvae typing using whole-genome sequencing. A total of 333 genomes were used to identify, validate and evaluate 2419 core genes. The cgMLST allowed fine-scale differentiation between samples that had the same profile using traditional MLST and allowed for the characterization of strains impossible by MLST. The scheme was successfully used to trace a localized Swedish outbreak, where a cluster of 38 isolates was linked to a country-wide beekeeping operation. cgMLST greatly enhances the power of a traditional typing scheme, while preserving the same stability and standardization for sharing results and methods across different laboratories.

Honeybee gut microbiota dysbiosis in pesticide/parasite co-exposures is mainly induced by Nosema ceranae.

Honeybees ensure a key ecosystem service by pollinating many agricultural crops and wild plants. However, in the past few decades, managed bee colonies have been declining in Europe and North America. Researchers have emphasized both parasites and pesticides as the most important factors. Infection by the parasite Nosema ceranae and exposure to pesticides can contribute to gut dysbiosis, impacting the honeybee physiology. Here, we examined and quantified the effects of N. ceranae, the neonicotinoid thiamethoxam, the phenylpyrazole fipronil and the carboxamide boscalid, alone and in combination, on the honeybee gut microbiota. Chronic exposures to fipronil and thiamethoxam alone or combined with N. ceranae infection significantly decreased honeybee survival whereas the fungicide boscalid had no effect on uninfected bees. Interestingly, increased mortality was observed in N. ceranae-infected bees after exposure to boscalid, with synergistic negative effects. Regarding gut microbiota composition, co-exposure to the parasite and each pesticide led to decreased abundance of Alphaproteobacteria, and increased abundance of Gammaproteobacteria. The parasite also induced an increase of bacterial alpha-diversity (species richness). Our findings demonstrated that exposure of honeybees to N. ceranae and/or pesticides play a significant role in colony health and is associated with the establishment of a dysbiotic gut microbiota.

Impact of the microsporidian Nosema ceranae on the gut epithelium renewal of the honeybee, Apis mellifera.

The invasive microsporidian species, Nosema ceranae, causes nosemosis in honeybees and is suspected to be involved in Western honeybee (Apis mellifera) declines worldwide. The midgut of honeybees is the site of infection; the microsporidium can disturb the functioning of this organ and, thus, the bee physiology. Host defense against pathogens is not limited to resistance (i.e. the immune response) but also involves resilience. This process implies that the host can tolerate and repair damage inflicted by the infection- by the pathogen itself or by an excessive host immune response. Enterocyte damage caused by N. ceranae can be compensated by proliferation of intestinal stem cells (ISCs) that are under the control of multiple pathways. In the present study, we investigated the impact of N. ceranae on honeybee epithelium renewal by following the mitotic index of midgut stem cells during a 22-day N. ceranae infection. Fluorescence in situ hybridization (FISH) and immunostaining experiments were performed to follow the parasite proliferation/progression in the intestinal tissue, especially in the ISCs as they are key cells for the midgut homeostasis. We also monitored the transcriptomic profile of 7 genes coding for key proteins involved in pathways implicated in the gut epithelium renewal and homeostasis. We have shown for the first time that N. ceranae can negatively alter the gut epithelium renewal rate and disrupt some signaling pathways involved in the gut homeostasis. This alteration is correlated to a reduced longevity of N. ceranae-infected honeybees and we can assume that honeybee susceptibility to N. ceranae could be due to an impaired ability to repair gut damage.

Effect of subtherapeutic and therapeutic sulfamethazine concentrations on transcribed genes and translated proteins involved in Microbacterium sp. C448 resistance and degradation.

Microbacterium sp. C448, isolated from a soil regularly exposed to sulfamethazine (SMZ), can use various sulphonamide antibiotics as the sole carbon source for growth. The basis for the regulation of genes encoding the sulphonamide metabolism pathway, the dihydropteroate synthase sulphonamide target (folP), and the sulphonamide resistance (sul1) genes is unknown in this organism. In the present study, the response of the transcriptome and proteome of Microbacterium sp. C448 following exposure to subtherapeutic (33 µM) or therapeutic (832 µM) SMZ concentrations was evaluated. Therapeutic concentration induced the highest sad expression and Sad production, consistent with the activity of SMZ degradation observed in cellulo. Following complete SMZ degradation, Sad production tended to return to the basal level observed prior to SMZ exposure. Transcriptomic and proteomic kinetics were concomitant for the resistance genes and proteins. The abundance of Sul1 protein, 100-fold more abundant than FolP protein, did not change in response to SMZ exposure. Moreover, non-targeted analyses highlighted the increase of a deaminase RidA and a putative sulphate exporter expression and production. These two novel factors involved in the 4-aminophenol metabolite degradation and the export of sulphate residues formed during SMZ degradation, respectively, provided new insights into the Microbacterium sp. C448 SMZ detoxification process.

Toxicity of the Pesticides Imidacloprid, Difenoconazole and Glyphosate Alone and in Binary and Ternary Mixtures to Winter Honey Bees: Effects on Survival and Antioxidative Defenses.

To explain losses of bees that could occur after the winter season, we studied the effects of the insecticide imidacloprid, the herbicide glyphosate and the fungicide difenoconazole, alone and in binary and ternary mixtures, on winter honey bees orally exposed to food containing these pesticides at concentrations of 0, 0.01, 0.1, 1 and 10 µg/L. Attention was focused on bee survival, food consumption and oxidative stress. The effects on oxidative stress were assessed by determining the activity of enzymes involved in antioxidant defenses (superoxide dismutase, catalase, glutathione-S-transferase, glutathione reductase, glutathione peroxidase and glucose-6-phosphate dehydrogenase) in the head, abdomen and midgut; oxidative damage reflected by both lipid peroxidation and protein carbonylation was also evaluated. In general, no significant effect on food consumption was observed. Pesticide mixtures were more toxic than individual substances, and the highest mortalities were induced at intermediate doses of 0.1 and 1 µg/L. The toxicity was not always linked to the exposure level and the number of substances in the mixtures. Mixtures did not systematically induce synergistic effects, as antagonism, subadditivity and additivity were also observed. The tested pesticides, alone and in mixtures, triggered important, systemic oxidative stress that could largely explain pesticide toxicity to honey bees.

Contrasting Effects of Environmental Concentrations of Sulfonamides on Microbial Heterotrophic Activities in Freshwater Sediments.

The sulfonamide antibiotics sulfamethoxazole (SMX) and sulfamethazine (SMZ) are regularly detected in surface sediments of contaminated hydrosystems, with maximum concentrations that can reach tens of µg kg-1 in stream and river sediments. Little is known about the resulting effects on the exposed benthic organisms. Here we investigated the functional response of stream sediment microbial communities exposed for 4 weeks to two levels of environmentally relevant concentrations of SMX and SMZ, tested individually. To this end, we developed a laboratory channel experiment where natural stream sediments were immersed in water contaminated with nominal environmental concentrations of 500 and 5,000 ng L-1 of SMX or SMZ, causing their accumulation in surface sediments. The mean maximum concentrations measured in the sediment (about 2.1 µg SMX kg-1 dw and 4.5 µg SMZ kg-1 dw) were consistent with those reported in contaminated rivers. The resulting chronic exposure had various effects on the functional potential of the sediment microbial communities, according to the substance (SMX or SMZ), the type of treatment (high or low) and the measured activity, with a strong influence of temporal dynamics. Whereas the SMZ treatments resulted in only transient effects on the five microbial activities investigated, we observed a significant stimulation of the ß-glucosidase activity over the 28 days in the communities exposed to the high concentration of SMX. Together with the stimulation of aerobic respiration at low SMX concentrations and the reduced concentration observed in the last days, our results suggest a potential biodegradation of sulfonamides by microbial communities from sediments. Given the key functional role of surface sediment microbial communities in streams and rivers, our findings suggest that the frequently reported contamination of sediments by sulfonamides is likely to affect biogeochemical cycles, with possible impact on ecosystem functioning.

Antibiotrophy: Key Function for Antibiotic-Resistant Bacteria to Colonize Soils-Case of Sulfamethazine-Degrading Microbacterium sp. C448.

Chronic and repeated exposure of environmental bacterial communities to anthropogenic antibiotics have recently driven some antibiotic-resistant bacteria to acquire catabolic functions, enabling them to use antibiotics as nutritive sources (antibiotrophy). Antibiotrophy might confer a selective advantage facilitating the implantation and dispersion of antibiotrophs in contaminated environments. A microcosm experiment was conducted to test this hypothesis in an agroecosystem context. The sulfonamide-degrading and resistant bacterium Microbacterium sp. C448 was inoculated in four different soil types with and without added sulfamethazine and/or swine manure. After 1 month of incubation, Microbacterium sp. (and its antibiotrophic gene sadA) was detected only in the sulfamethazine-treated soils, suggesting a low competitiveness of the strain without antibiotic selection pressure. In the absence of manure and despite the presence of Microbacterium sp. C448, only one of the four sulfamethazine-treated soils exhibited mineralization capacities, which were low (inferior to 5.5 ± 0.3%). By contrast, manure addition significantly enhanced sulfamethazine mineralization in all the soil types (at least double, comprised between 5.6 ± 0.7% and 19.5 ± 1.2%). These results, which confirm that the presence of functional genes does not necessarily ensure functionality, suggest that sulfamethazine does not necessarily confer a selective advantage on the degrading strain as a nutritional source. 16S rDNA sequencing analyses strongly suggest that sulfamethazine released trophic niches by biocidal action. Accordingly, manure-originating bacteria and/or Microbacterium sp. C448 could gain access to low-competition or competition-free ecological niches. However, simultaneous inputs of manure and of the strain could induce competition detrimental for Microbacterium sp. C448, forcing it to use sulfamethazine as a nutritional source. Altogether, these results suggest that the antibiotrophic strain studied can modulate its sulfamethazine-degrading function depending on microbial competition and resource accessibility, to become established in an agricultural soil. Most importantly, this work highlights an increased dispersal potential of antibiotrophs in antibiotic-polluted environments, as antibiotics can not only release existing trophic niches but also form new ones.

Effects of the gut parasite Nosema ceranae on honey bee physiology and behavior.

The common and widespread parasite Nosema ceranae is considered a major threat to the Western honey bee at both the individual and colony levels. Several studies demonstrated that infection by this parasite may affect physiology, behavior, and survival of honey bees. N. ceranae infection impairs midgut integrity and alters the energy demand in honey bees. The infection can also significantly suppress the bee immune response and modify pheromone production in worker and queen honey bees leading to precocious foraging. However, the presence of N. ceranae is not systematically associated with colony weakening and honey bee mortality. This variability depends upon parasite or host genetics, nutrition, climate or interactions with other stressors such as environmental contaminants or other parasites.

Disruption of oxidative balance in the gut of the western honeybee Apis mellifera exposed to the intracellular parasite Nosema ceranae and to the insecticide fipronil.

The causes underlying the increased mortality of honeybee colonies remain unclear and may involve multiple stressors acting together, including both pathogens and pesticides. Previous studies suggested that infection by the gut parasite Nosema ceranae combined with chronic exposure to sublethal doses of the insecticide fipronil generated an increase in oxidative stress in the midgut of honeybees. To explore the impact of these two stressors on oxidative balance, we experimentally infected bees with N. ceranae and/or chronically exposed to fipronil at low doses for 22 days, and we measured soluble reactive oxygen species (ROS) and ROS damage by quantifying both protein and lipid oxidation in the midgut. Our results revealed a disruption of the oxidative balance, with a decrease in both the amount of ROS and ROS damage in the presence of the parasite alone. However, protein oxidation was significantly increased in the N. ceranae/fipronil combination, revealing an increase in oxidative damage and suggesting higher fipronil toxicity in infected bees. Furthermore, our results highlighted a temporal order in the appearance of oxidation events in the intestinal cells and revealed that all samples tended to undergo protein oxidation during ageing, regardless of treatment.