Burkholderia pseudomallei, a facultative intracellular pathogen, causes severe infections and is inherently refractory to many antibiotics. Previous studies from our group have shown that interferon gamma (IFN-␥) interacts synergistically with the antibiotic ceftazidime to kill bacteria in infected macrophages. The present study aimed to identify the underlying mechanism of that interaction. We first showed that blocking reactive oxygen species (ROS) pathways reversed IFN-␥-and ceftazidime-mediated killing, which led to our hypothesis that IFN-␥-induced ROS interacted with ceftazidime to synergistically kill Burkholderia bacteria. Consistent with this hypothesis, we also observed that buthionine sulfoximine (BSO), another inducer of ROS, could substitute for IFN-␥ to similarly potentiate the effect of ceftazidime on intracellular killing. Next, we observed that IFN-␥ induced ROS-mediated killing of intracellular but not extracellular bacteria. On the other hand, ceftazidime effectively reduced extracellular bacteria but was not capable of intracellular killing when applied at 10 g/ml. We investigated the exact role of IFN-␥-induced ROS responses on intracellular bacteria and notably observed a lack of actin polymerization associated with Burkholderia bacteria in IFN-␥-treated macrophages, which led to our finding that IFN-␥-induced ROS blocks vacuolar escape. Based on these results, we propose a model in which synergistically reduced bacterial burden is achieved primarily through separate and compartmentalized killing: intracellular killing by IFN-␥-induced ROS responses and extracellular killing by ceftazidime. Our findings suggest a means of enhancing antibiotic activity against Burkholderia bacteria through combination with drugs that induce ROS pathways or otherwise target intracellular spread and/or replication of bacteria.

This competitive drawing game, much like the “All Play” round of the popular board game Pictionary™, is a fast-paced, energy-rich review game, proven to keep students entertained, engaged, and learning. One representative from each of two competing teams simultaneously draw the same secret microbiology term, while their teammates frantically guess. The first team to correctly guess the word or phrase, wins a point. Unlike traditional Pictionary rules where only one team draws at a time, pitting teams against each other seems to increase the energy and engagement of student drawers and guessers, and allows every student to participate in each round of the game. Drawing microbiology terms not only helps strengthen associations between form and function, a crucial theme in microbiology, but also forces students to think differently and creatively about how to convey scientific terms through pictures. In addition, the game encourages simple recall of terms and definitions from past chapters. After one team has correctly guessed the secret term, all students participate in answering rapid-fire review questions related to the term. The result is a fun and stimulating review session of microbiology. In an anonymous survey administered after the game, about 94% of students found the activity to be an “effective” or “very effective” strategy to review previously covered material from the course. Out of those who participated in the survey, 100% rated the drawing game as a fun classroom activity, and 98% would recommend it for future semesters.

Burkholderia pseudomallei is a facultative intracellular pathogen and the causative agent of melioidosis, a potentially life-threatening disease endemic in Southeast Asia and Northern Australia. Treatment of melioidosis is a long and costly process and the pathogen is inherently resistant to several classes of antibiotics, therefore there is a need for new treatments that can help combat the pathogen. Previous work has shown that the combination of interferon-gamma, an immune system activator, and the antibiotic ceftazidime synergistically reduced the bacterial burden of RAW 264.7 macrophages that had been infected with either B. pseudomallei or Burkholderia thailandensis. The mechanism of the interaction was found to be partially dependent on interferon-gamma-induced production of reactive oxygen species inside the macrophages. To further confirm the role of reactive oxygen species in the effectiveness of the combination treatment, we investigated the impact of the antioxidant and reactive oxygen species scavenger, seleno-L-methionine, on intracellular and extracellular bacterial burden of the infected macrophages. In a dose-dependent manner, high concentrations of seleno-L-methionine (1000 μM) were protective towards infected macrophages, resulting in a reduction of bacteria, on its own, that exceeded the reduction caused by the antibiotic alone and rivaled the effect of ceftazidime and interferon-gamma combined. Seleno-L-methionine treatment also resulted in improved viability of infected macrophages compared to untreated controls. We show that the protective effect of seleno-L-methionine was partly due to its inhibition of bacterial growth. In summary, our study shows a role for high dose seleno-L-methionine to protect and treat macrophages infected with B. thailandensis.

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Burkholderia pseudomallei is a Gram-negative bacillus and facultative intracellular pathogen. It causes the disease melioidosis, which is a potentially fatal human disease found throughout the world but particularly in Southeast Asia and Northern Australia. B. pseudomallei is inherently antibiotic resistant and therefore new therapies are needed to combat this pathogen. Previous studies with the related organism Burkholderia thailandensis have shown that the antibiotic ceftazidime does not eliminate all bacteria in an in vitro macrophage model, and the remaining bacteria could still pose a health threat to a potential host. Due to their survival in the presence of antibiotics, we hypothesized that the remaining bacteria were one of two types of antibiotic tolerant cells: classically antibiotic resistant cells or persister cells. To test our hypothesis we isolated the bacteria that had survived ceftazidime treatment in the macrophage infection model and performed additional in vitro experiments to show that the surviving bacteria are neither antibiotic resistant nor persister cells. Instead, they are still susceptible to high doses (200 μg/ml) of the antibiotic over a period of 48 hours (p<0.001). We believe the bacteria survive exposure to the antibiotic during the macrophage infection because of their ability to move between intracellular and extracellular compartments, thus avoiding the antibiotic and its deadly effects. Our results provide evidence to suggest that intracellular pathogens, through movement between intracellular and extracellular compartments, may be protected from the effects of antibiotics in similar macrophage infection models.