Rapid antimicrobial susceptibility testing and ß-lactam-induced cell morphology changes of Gram-negative biological threat pathogens by optical screening.

BACKGROUND: For Yersinia pestis, Burkholderia pseudomallei, and Burkholderia mallei, conventional broth microdilution (BMD) is considered the gold standard for antimicrobial susceptibility testing (AST) and, depending on the species, requires an incubation period of 16-20 h, or 24-48 h according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. After a diagnosis of plague, melioidosis or glanders during an outbreak or after an exposure event, the timely distribution of appropriate antibiotics for treatment or post-exposure prophylaxis of affected populations could reduce mortality rates. RESULTS: Herein, we developed and evaluated a rapid, automated susceptibility test for these Gram-negative bacterial pathogens based on time-lapse imaging of cells incubating in BMD microtitre drug panels using an optical screening instrument (oCelloScope). In real-time, the instrument screened each inoculated well containing broth with various concentrations of antibiotics published by CLSI for primary testing: ciprofloxacin (CIP), doxycycline (DOX) and gentamicin (GEN) for Y. pestis; imipenem (IPM), ceftazidime (CAZ) and DOX for B. mallei; and IPM, DOX, CAZ, amoxicillin-clavulanic acid (AMC) and trimethoprim-sulfamethoxazole (SXT) for B. pseudomallei. Based on automated growth kinetic data, the time required to accurately determine susceptibility decreased by ≥70% for Y. pestis and ≥ 50% for B. mallei and B. pseudomallei compared to the times required for conventional BMD testing. Susceptibility to GEN, IPM and DOX could be determined in as early as three to six hours. In the presence of CAZ, susceptibility based on instrument-derived growth values could not be determined for the majority of B. pseudomallei and B. mallei strains tested. Time-lapse video imaging of these cultures revealed that the formation of filaments in the presence of this cephalosporin at inhibitory concentrations was detected as growth. Other ß-lactam-induced cell morphology changes, such as the formation of spheroplasts and rapid cell lysis, were also observed and appear to be strain- and antibiotic concentration-dependent. CONCLUSIONS: A rapid, functional AST was developed and real-time video footage captured ß-lactam-induced morphologies of wild-type B. mallei and B. pseudomallei strains in broth. Optical screening reduced the time to results required for AST of three Gram-negative biothreat pathogens using clinically relevant, first-line antibiotics compared to conventional BMD.

Burkholderia mallei cluster 1 type VI secretion mutants exhibit growth and actin polymerization defects in RAW 264.7 murine macrophages.

Burkholderia mallei is a facultative intracellular pathogen that causes severe disease in animals and humans. Recent studies have shown that the cluster 1 type VI secretion system (T6SS-1) expressed by this organism is essential for survival in a hamster model of glanders. To better understand the role of T6SS-1 in the pathogenesis of disease, studies were initiated to examine the interactions of B. mallei tssE mutants with RAW 264.7 murine macrophages. Results obtained by utilizing modified gentamicin protection assays indicated that although the tssE mutants were able to survive within RAW 264.7 cells, significant growth defects were observed in comparison to controls. In addition, analysis of infected monolayers by differential interference contrast and fluorescence microscopy demonstrated that the tssE mutants lacked the ability to induce multinucleated giant cell formation. Via the use of fluorescence microscopy, tssE mutants were shown to undergo escape from lysosome-associated membrane protein 1-positive vacuoles. Curiously, however, following entry into the cytosol, the mutants exhibited actin polymerization defects resulting in inefficient intra- and intercellular spread characteristics. Importantly, all mutant phenotypes observed in this study could be restored by complementation. Based upon these findings, it appears that T6SS-1 plays a critical role in growth and actin-based motility following uptake of B. mallei by RAW 264.7 cells.

Burkholderia mallei cellular interactions in a respiratory cell model.

Burkholderia mallei is a facultative intracellular pathogen that survives and replicates in phagocytic cell lines. The bacterial burden recovered from naïve BALB/c mice infected by intranasal delivery indicated that B. mallei persists in the lower respiratory system. To address whether B. mallei invades respiratory non-professional phagocytes, this study utilized A549 and LA-4 respiratory epithelial cells and demonstrated that B. mallei possesses the capacity to adhere poorly to, but not to invade, these cells. Furthermore, it was found that B. mallei was taken up by the murine alveolar macrophage cell line MH-S following serum coating, an attribute suggestive of complement- or Fc receptor-mediated uptake. Invasion/intracellular survival assays of B. mallei-infected MH-S cells demonstrated decreased intracellular survival, whilst a type III secretion system effector bopA mutant strain survived longer than the wild-type. Evaluation of the potential mechanism(s) responsible for efficient clearing of intracellular organisms demonstrated comparable levels of caspase-3 in both the wild-type and bopA mutant with characteristics consistent with apoptosis of infected MH-S cells. Furthermore, challenge of BALB/c mice with the bopA mutant by the intranasal route resulted in increased survival. Overall, these data suggest that B. mallei induces apoptotic cell death, whilst the BopA effector protein participates in intracellular survival.

An allelic exchange system for compliant genetic manipulation of the select agents Burkholderia pseudomallei and Burkholderia mallei.

Burkholderia pseudomallei and B. mallei are Gram-negative bacterial pathogens that cause melioidosis in humans and glanders in horses, respectively. Both bacteria are classified as category B select agents in the United States. Due to strict select-agent regulations, the number of antibiotic selection markers approved for use in these bacteria is greatly limited. Approved markers for B. pseudomallei include genes encoding resistance to kanamycin (Km), gentamicin (Gm), and zeocin (Zeo); however, wild type B. pseudomallei is intrinsically resistant to these antibiotics. Selection markers for B. mallei are limited to Km and Zeo resistance genes. Additionally, there are few well developed counter-selection markers for use in Burkholderia. The use of SacB as a counter-selection method has been of limited success due to the presence of endogenous sacBC genes in the genomes of B. pseudomallei and B. mallei. These impediments have greatly hampered the genetic manipulation of B. pseudomallei and B. mallei and currently few reliable tools for the genetic manipulation of Burkholderia exist. To expand the repertoire of genetic tools for use in Burkholderia, we developed the suicide plasmid pMo130, which allows for the compliant genetic manipulation of the select agents B. pseudomallei and B. mallei using allelic exchange. pMo130 harbors an aphA gene which allows for Km selection, the reporter gene xylE, which allows for reliable visual detection of Burkholderia transformants, and carries a modified sacB gene that allows for the resolution of co-integrants. We employed this system to generate multiple unmarked and in-frame mutants in B. pseudomallei, and one mutant in B. mallei. This vector significantly expands the number of available tools that are select-agent compliant for the genetic manipulation of B. pseudomallei and B. mallei.