Sporulation and Germination of Paenibacillus larvae Cells.

Endospores are metabolically dormant cells formed by a variety of Gram-positive bacteria within the phylum Firmicutes in response to nutrient limiting or otherwise unfavorable growth conditions. American foulbrood disease (AFB) is a serious disease of honeybees that is caused by the introduction of Paenibacillus larvae endospores into a honeybee colony. Progression to fulminant disease and eventual collapse of the colony requires multiple rounds of endospore germination, vegetative replication, endospore formation, and subsequent spread within the colony. This unit includes protocols for the in vitro sporulation and germination of P. larvae to assist investigators in the study of these processes. © 2018 by John Wiley & Sons, Inc.

Growth and Laboratory Maintenance of Paenibacillus larvae.

Paenibacillus larvae is a Gram-positive, spore-forming bacterium and the causative agent of American foulbrood disease (AFB), a highly contagious, fatal disease affecting managed honeybee (Apis mellifera) colonies. As the etiological agent of American foulbrood disease, P. larvae is the most economically significant bacterial pathogen infecting honeybees. This unit includes protocols for the in vitro growth and laboratory maintenance of P. larvae. © 2018 by John Wiley & Sons, Inc.

The orange spotted cockroach (Blaptica dubia, Serville 1839) is a permissive experimental host for Francisella tularensis.

Francisella tularensis is a zoonotic bacterial pathogen that causes severe disease in a wide range of host animals, including humans. Well-developed murine models of F. tularensis pathogenesis are available, but they do not meet the needs of all investigators. However, researchers are increasingly turning to insect host systems as a cost-effective alternative that allows greater increased experimental throughput without the regulatory requirements associated with the use of mammals in biomedical research. Unfortunately, the utility of previously-described insect hosts is limited because of temperature restriction, short lifespans, and concerns about the immunological status of insects mass-produced for other purposes. Here, we present a novel host species, the orange spotted (OS) cockroach (Blaptica dubia), that overcomes these limitations and is readily infected by F. tularensis. Intrahemocoel inoculation was accomplished using standard laboratory equipment and lethality was directly proportional to the number of bacteria injected. Progression of infection differed in insects housed at low and high temperatures and F. tularensis mutants lacking key virulence components were attenuated in OS cockroaches. Finally, antibiotics were delivered to infected OS cockroaches by systemic injection and controlled feeding; in the latter case, protection correlated with oral bioavailability in mammals. Collectively, these results demonstrate that this new host system provides investigators with a new tool capable of interrogating F. tularensis virulence and immune evasion in situations where mammalian models are not available or appropriate, such as undirected screens of large mutant libraries.

Laboratory culture and maintenance of Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is a ubiquitous soil bacterium that is increasingly recognized as an emerging nosocomial pathogen. This unit includes protocols for the in vitro growth and maintenance of S. maltophilia.