Paenibacillus larvae is the causative agent of American foulbrood (AFB), a disease affecting honey bee larvae. First-and secondinstar larvae become infected when they ingest food contaminated with P. larvae spores. The spores then germinate into vegetative cells that proliferate in the midgut of the honey bee. Although AFB affects honey bees only in the larval stage, P. larvae spores can be distributed throughout the hive. Because spore germination is critical for AFB establishment, we analyzed the requirements for P. larvae spore germination in vitro. We found that P. larvae spores germinated only in response to L-tyrosine plus uric acid under physiologic pH and temperature conditions. This suggests that the simultaneous presence of these signals is necessary for spore germination in vivo. Furthermore, the germination profiles of environmentally derived spores were identical to those of spores from a biochemically typed strain. Because L-tyrosine and uric acid are the only required germinants in vitro, we screened amino acid and purine analogs for their ability to act as antagonists of P. larvae spore germination. Indole and phenol, the side chains of tyrosine and tryptophan, strongly inhibited P. larvae spore germination. Methylation of the N-1 (but not the C-3) position of indole eliminated its ability to inhibit germination. Identification of the activators and inhibitors of P. larvae spore germination provides a basis for developing new tools to control AFB.A merican foulbrood (AFB) is a bacterial disease of honey bees that kills the developing larvae (1, 2). Paenibacillus larvae spores are the infectious agents for AFB, but it is the vegetative cells that cause disease (3, 4). In 2005, a survey of almond-pollinating bee colonies indicated that 4% of colonies had a significant AFB load (5). Once a beekeeping operation is contaminated, the bacterial spores are not easily removed (6). Although autoclaving and high concentrations of chemical disinfectants effectively kill the spores, these treatments are not viable for the beekeeping industry (7). Traditionally, Terramycin and other antibiotics have been used for the treatment and prevention of AFB. However, antibiotic treatment is ineffective in the spore stage of P. larvae, and overuse of the antibiotics leads to resistant strains (8, 9). Presently, the only accepted practice for controlling the spread of AFB is burning both the infected colonies and beekeeping equipment (3, 6).AFB occurs when first-or second-instar larvae (within 48 h after the egg hatches) ingest food that is contaminated with the P. larvae spores (10). Twelve hours after ingestion, P. larvae spores germinate and the new vegetative cells start to proliferate inside the larval gut (11). Several days postinfection, extreme bacteremia causes the death of the honey bee larvae (12-14). After the nutrient levels of the honey bees are depleted, P. larvae cells stop dividing and then sporulate. As a result, billions of spores are found in the dead remains of each bee larva (15, 16). Within the col...

Paenibacillus larvae, a Gram-positive bacterium, causes American foulbrood (AFB) in honey bee larvae (Apis mellifera Linnaeus [Hymenoptera: Apidae]). P. larvae spores exit dormancy in the gut of bee larvae, the germinated cells proliferate, and ultimately bacteremia kills the host. Hence, spore germination is a required step for establishing AFB disease. We previously found that P. larvae spores germinate in response to l-tyrosine plus uric acid in vitro. Additionally, we determined that indole and phenol blocked spore germination. In this work, we evaluated the antagonistic effect of 35 indole and phenol analogs and identified strong inhibitors of P. larvae spore germination in vitro. We further tested the most promising candidate, 5-chloroindole, and found that it significantly reduced bacterial proliferation. Finally, feeding artificial worker jelly containing anti-germination compounds to AFB-exposed larvae significantly decreased AFB infection in laboratory-reared honey bee larvae. Together, these results suggest that inhibitors of P. larvae spore germination could provide another method to control AFB.

Paenibacillus larvae endospores are the infectious particles of the honey bee brood disease, American Foulbrood. We demonstrate that our previously published protocol (Alvarado et al., 2013) consistently yields higher numbers and purer preparations of P. larvae endospores, than previously described protocols, regardless of the strain tested (B-3650, B-3554 or B-3685).

Ng KM, Ferreyra JA, Higginbottom SK et al. Microbiota-liberated host sugars facilitate post-antibiotic expansion of enteric pathogens. Nature 502(7469), 96-99 (2013). The human gut microbiota is a complex system of commensal microorganisms required for normal host physiology. Disruption of this protective barrier by antibiotics creates opportunities for enteric pathogens to establish infections. Although the correlation between the use of antibiotics and enteric infections have been known for some time, the specific signals that allow enteric pathogens to recognize a susceptible host have not been determined. In a recent article, Ng et al. demonstrated that the expansion of both Salmonella typhimurium and Clostridium difficile infections is enhanced by the availability of host-specific sugars liberated from the intestinal mucus by commensal bacteria. These results show how antibiotic removal of specific species from the gut microbiome allows symbiotic functions to be hijacked by pathogenic species.