A non-invasive intratracheal inoculation method for the study of pulmonary melioidosis

Abstract

Pulmonary melioidosis, a disease manifestation caused by the bacterium Burkholderia pseudomallei, has been studied using aerosols or intranasal (IN) inoculation in small animal models. Both have inherent disadvantages which may not accurately model primary pulmonary melioidosis in humans. Intratracheal inoculation (IT) by direct visualization of the tracheal opening offers an alternative technique for infection that overcomes the disadvantages of aerosol and IN challenge. In this study, we describe a method which requires relatively inexpensive equipment, little training, and is compliant with the operational constraints of a BSL3 laboratory. Results obtained using trypan blue demonstrated that an inoculum can be accurately delivered into the lungs of mice within a biosafety cabinet (BSC). Whole body imaging and histopathology confirmed that mice inoculated intratracheally with B. pseudomallei develop the primary focus of infection in the lungs, and not the nasal passages which can lead to invasion of the central nervous system and potential neurologic complications. Further, based on colony counts and bioluminescent imaging, dissemination to secondary organs occurred as expected. Taken together, this intratracheal method of inoculation fulfills four goals: (1) to accurately deliver B. pseudomallei into the lungs of the animal model, (2) to avoid potentially confounding complications due to primary infections at sites other than the lung, (3) to maintain normal organ dissemination, and (4) to be BSL3 compliant.

Characterization of the role of the Burkholderia pseudomallei type 3 secretion system using in vivo imaging.

“…Both our laboratory and others have previously shown that the nasal cavity represents the predominant site of B. pseudomallei colonization in intranasally inoculated mice [73,93], whereas direct lung instillation of bacteria can abrogate nasal cavity colonization and the associated central nervous system involvement [78,99]. We have further characterized that lung-specific instillation of B.…”

“…The over-representation of these symptoms in mice have led us, and others, to investigate alternatives to the standard approaches of inoculating mice with B. pseudomallei through the nares. A recently developed intratracheal model of respiratory melioidosis succeeded in abrogating CNS infections, suggesting that URT colonization is directly responsible for the high levels of meningitis reported in the murine model [99,105]. Our current studies focused on advancing these findings by identifying whether URT infection in the mouse impacts the overall course of disease and ask whether these impacts might influence both basic and translational studies of respiratory melioidosis.…”

“…The IMIT inoculation method we developed is distinct from other non-invasive intratracheal instillation methods, including those used previously for B. pseudomallei instillation [99]. IMIT inoculation is a two-step process in which mouse intubation is followed by instillation of bacteria via a long blunt needle, and the approach facilitates an intermediate confirmation of correct catheter placement into the trachea (rather than the esophagus), and is therefore not prone to user error associated with unintentional misinoculation of the GI tract [106].…”

“…Opportunistic URT colonization by B. pseudomallei is associated with high rates of meningitis in murine respiratory models [93,101], and avoidance of bacterial deposition on the nasal mucosa by intratracheal instillation is associated with reduced CNS involvement [99]. We decided to investigate whether the URT colonization typical of current respiratory melioidosis models impacts the moribund disease presentation.…”

“…A recent study investigating an intratracheal instillation of B. pseudomallei directly into the lungs successfully demonstrated that avoidance of initial URT colonization could limit both CNS involvement and late stage URT colonization [99].…”