NO-Stressed Y. pseudotuberculosis Has Decreased Cell Division Rates in the Mouse Spleen.

Fluorescence dilution approaches can detect bacterial cell division events and can detect if there are differential rates of cell division across individual cells within a population. This approach typically involves inducing expression of a fluorescent protein and then tracking partitioning of fluorescence into daughter cells. However, fluorescence can be diluted very quickly within a rapidly replicating population, such as pathogenic bacterial populations replicating within host tissues. To overcome this limitation, we have generated two revTetR reporter constructs, where either mCherry or yellow fluorescent protein (YFP) is constitutively expressed and repressed by addition of tetracyclines, resulting in fluorescence dilution within defined time frames. We show that fluorescent signals are diluted in replicating populations and that signal accumulates in growth-inhibited populations, including during nitric oxide (NO) exposure. Furthermore, we show that tetracyclines can be delivered to the mouse spleen during Yersinia pseudotuberculosis infection and defined a drug concentration that results in even exposure of cells to tetracyclines. We then used this system to visualize bacterial cell division within defined time frames postinfection. revTetR-mCherry allowed us to detect slow-growing cells in response to NO in culture; however, this strain had a growth defect within mouse tissues, which complicated results. To address this issue, we constructed revTetR-YFP using the less toxic YFP and showed that heightened NO exposure correlated with heightened YFP signal, indicating decreased cell division rates within this subpopulation in vivo. This revTetR reporter will provide a critical tool for future studies to identify and isolate slowly replicating bacterial subpopulations from host tissues.

Dynamic PET-facilitated modeling and high-dose rifampin regimens for Staphylococcus aureus orthopedic implant-associated infections.

Staphylococcus aureus is a major human pathogen causing serious implant­associated infections. Combination treatment with rifampin (10 to 15 mg/kg per day), which has dose-dependent activity, is recommended to treat S. aureus orthopedic implant­associated infections. Rifampin, however, has limited bone penetration. Here, dynamic 11C-rifampin positron emission tomography (PET) performed in prospectively enrolled patients with confirmed S. aureus bone infection (n = 3) or without orthopedic infection (n = 12) demonstrated bone/plasma area under the concentration-time curve ratio of 0.14 (interquartile range, 0.09 to 0.19), exposures lower than previously thought. PET-based pharmacokinetic modeling predicted rifampin concentration-time profiles in bone and facilitated studies in a mouse model of S. aureus orthopedic implant infection. Administration of high-dose rifampin (human equipotent to 35 mg/kg per day) substantially increased bone concentrations (2 mg/liter versus <0.2 mg/liter with standard dosing) in mice and achieved higher bacterial killing and biofilm disruption. Treatment for 4 weeks with high-dose rifampin and vancomycin was noninferior to the recommended 6-week treatment of standard-dose rifampin with vancomycin in mice (risk difference, −6.7% favoring high-dose rifampin regimen). High-dose rifampin treatment ameliorated antimicrobial resistance (0% versus 38%; P = 0.04) and mitigated adverse bone remodeling (P < 0.01). Last, whole-genome sequencing demonstrated that administration of high-dose rifampin in mice reduced selection of bacterial mutations conferring rifampin resistance (rpoB) and mutations in genes potentially linked to persistence. These data suggest that administration of high-dose rifampin is necessary to achieve optimal bone concentrations, which could shorten and improve treatments for S. aureus orthopedic implant infections.

Subpopulations of Stressed Yersinia pseudotuberculosis Preferentially Survive Doxycycline Treatment within Host Tissues.

Severe systemic bacterial infections result in colonization of deep tissues, which can be very difficult to eliminate with antibiotics. It remains unclear if this is because antibiotics are not reaching inhibitory concentrations within tissues, if subsets of bacteria are less susceptible to antibiotics, or if both contribute to limited treatment efficacy. To detect exposure to doxycycline (Dox) present in deep tissues following treatment, we generated a fluorescent transcriptional reporter derived from the tet operon to specifically detect intracellular tetracycline exposure at the single bacterial cell level. Dox exposure was detected in the spleen 2 h after intraperitoneal injection, and by 4 h postinjection, this treatment resulted in a significant decrease in viable Yersinia pseudotuberculosis bacteria in the spleen. Nitric oxide-stressed bacteria preferentially survived treatment, suggesting that stress was sufficient to alter Dox susceptibility. Many bacteria (∼10%) survived a single dose of Dox, and the antibiotic accumulated at the periphery of microcolonies to growth inhibitory concentrations until 48 h posttreatment. After this time point, antibiotic concentrations decreased and bacterial growth resumed. Dox-treated mice eventually succumbed to the infection, albeit with significantly prolonged survival relative to that of untreated mice. These results indicate that Dox delivery by intraperitoneal injection results in rapid diffusion of inhibitory concentrations of antibiotic into the spleen, but stressed cells preferentially survive drug treatment, and bacterial growth resumes once drug concentrations decrease. This fluorescent reporter strategy for antibiotic detection could easily be modified to detect the concentration of additional antimicrobial compounds within host tissues following drug administration.IMPORTANCE Bacterial infections are very difficult to treat when bacteria spread into the bloodstream and begin to replicate within deep tissues, such as the spleen. Subsets of bacteria can survive antibiotic treatment, but it remains unclear if this survival is because of limited drug diffusion into tissues, or if there are changes within the bacteria, promoting survival of some bacterial cells. Here, we have developed a fluorescent reporter to detect doxycycline (Dox) diffusion into host tissues, and we show that Dox impacts the bacterial population within hours of administration and inhibits bacterial growth for 48 h. However, bacterial growth resumes when antibiotic concentrations decrease. Subsets of bacteria, stressed by the host response to infection, survive Dox treatment at a higher rate. These results provide critical information about the dynamics that occur within deep tissues following antibiotic administration and suggest that subsets of bacteria are predisposed to survive inhibitory concentrations of antibiotic before exposure.