Rapid antimicrobial susceptibility testing (AST) is urgently needed for treating infections with appropriate antibiotics and slowing down the emergence of antibiotic-resistant bacteria. Here, a phenotypic platform that rapidly produces AST results by femtosecond stimulated Raman scattering imaging of deuterium oxide (D 2 O) metabolism is reported. Metabolic incorporation of D 2 O into biomass in a single bacterium and the metabolic response to antibiotics are probed in as short as 10 min after culture in 70% D 2 O medium, the fastest among current technologies. Single-cell metabolism inactivation concentration (SC-MIC) is obtained in less than 2.5 h from colony to results. The SC-MIC results of 37 sets of bacterial isolate samples, which include 8 major bacterial species and 14 different antibiotics often encountered in clinic, are validated by standard minimal inhibitory concentration blindly measured via broth microdilution. Toward clinical translation, stimulated Raman scattering imaging of D 2 O metabolic incorporation and SC-MIC determination after 1 h antibiotic treatment and 30 min mixture of D 2 O and antibiotics incubation of bacteria in urine or whole blood is demonstrated.

The widespread use of antibiotics has significantly increased the number of resistant bacteria, which has also increased the urgency of rapid bacterial detection and profiling their antibiotic response. Current clinical methods for antibiotic susceptibility testing (AST) rely on culture and require at least 16 to 24 h to conduct. Therefore, there is an urgent need for a rapid method that can test the susceptibility of bacteria in a culture-free manner. Here we demonstrate a rapid AST method by monitoring the glucose metabolic activity of live bacteria at the single-cell level with hyperspectral stimulated Raman scattering (SRS) imaging. Using vancomycin-susceptible and -resistant enterococci E. faecalis as models, we demonstrate that the metabolic uptake of deuterated glucose in a single living bacterium can be quantitatively monitored via hyperspectral SRS imaging. Remarkably, the metabolic activity of susceptible bacteria responds differently to antibiotics from the resistant strain within only 0.5 h from the addition of antibiotics. Therefore, bacterial susceptibility and the minimum inhibitory concentration (MIC) of antibiotics can be determined within one cell cycle. Our metabolic imaging method is applicable to other bacteria species including E. coli, K. Pneumoniae, and S. aureus as well as different antibiotics, regardless of their mechanisms of inhibiting or killing bacteria.

Vancomycin-resistant enterococci (VRE) are the second leading cause of hospital-acquired infections (HAIs) attributed to a drug-resistant bacterium in the United States, and resistance to the frontline treatments is well documented. To combat VRE, we have repurposed the FDA-approved carbonic anhydrase drug acetazolamide to design potent antienterococcal agents. Through structure–activity relationship optimization we have arrived at two leads possessing improved potency against clinical VRE strains from MIC = 2 μg/mL (acetazolamide) to MIC = 0.007 μg/mL (22) and 1 μg/mL (26). Physicochemical properties were modified to design leads that have either high oral bioavailability to treat systemic infections or low intestinal permeability to treat VRE infections in the gastrointestinal tract. Our data suggest the intracellular targets for the molecules are putative α-carbonic and γ-carbonic anhydrases, and homology modeling and molecular dynamics simulations were performed. Together, this study presents potential anti-VRE therapeutic options to provide alternatives for problematic VRE infections.

Clostridium difficile is a deadly, opportunistic bacterial pathogen. In the last two decades, C. difficile infections (CDIs) have become a national concern due to the emergence of hypervirulent mutants armed with a higher capability of producing toxins and spores. This has resulted in an increased number of infections and death associated with CDI. The scarcity of anticlostridial drugs has led to unsatisfactory cure rates, elevated recurrence rates and permitted enhanced colonization with other drug-resistant pathogens (such as vancomycin-resistant enterococci), in afflicted patients. Therefore, both patients and physicians are facing an urgent need for more effective therapies to treat CDI. In an effort to find new anticlostridial drugs, we investigated auranofin, an FDA-approved oral antirheumatic drug which has recently been found to also possess antibacterial activity. Auranofin exhibited potent activity against C. difficile isolates inhibiting growth at a concentration of 1 μg/ml against 50% of all the tested isolates. Auranofin inhibited both toxin production and spore formation, a property that is lacking in both vancomycin and metronidazole (the primary agents used to treat CDI). Auranofin had a direct protective activity against C. difficile toxin-mediated inflammation and inhibited the growth of vancomycin-resistant enterococci. Overall, auranofin is a promising candidate that warrants further investigation as a treatment option for C. difficile infections.

No abstract

Multidrug-resistant enterococcal pathogens, especially vancomycin-resistant enterococci (VRE), are among the pathogens that require new antibiotic innovation. The colonization of the gut represents a major pathway by which VRE can cause infection and spread to other patients. In the current study, auranofin (FDA-approved rheumatoid arthritis drug) is evaluated for its potential use as a decolonizing agent for VRE. Auranofin was found to exert potent antimicrobial activity against a wide range of enterococcal clinical isolates with a minimum inhibitory concentration of 1 μg/mL. No resistant mutants could be developed against auranofin over the course of 14 passages. Auranofin was also found to exert potent anti-biofilm activity against VRE. Auranofin was superior to linezolid, the drug of choice for VRE infection treatment, in the in vivo mouse model. Auranofin significantly reduced the VRE burden in feces, cecum, and ileum contents after 8 days of treatment. Accordingly, this study provides valuable evidence that auranofin has significant promise as a novel gastrointestinal decolonizing agent for VRE.

Enterococci represent one of the microbial world’s most challenging enigmas. Colonization of the gastrointestinal tract (GIT) of high-risk/immunocompromised patients by enterococci exhibiting resistance to vancomycin (VRE) can lead to life-threating infections, including bloodstream infections and endocarditis. Decolonization of VRE from the GIT of high-risk patients represents an alternative method to suppress the risk of the infection. It could be considered as a preventative measure to protect against VRE infections in high-risk individuals. Though multiple agents (ramoplanin and bacitracin) have been evaluated clinically, no drugs are currently approved for use in VRE decolonization of the GIT. The present study evaluates ebselen, a clinical molecule, for use as a decolonizing agent against VRE. When evaluated against a broad array of enterococcal isolates in vitro, ebselen was found to be as potent as linezolid (minimum inhibitory concentration against 90% of clinical isolates tested was 2 μg/ml). Though VRE has a remarkable ability to develop resistance to antibacterial agents, no resistance to ebselen emerged after a clinical isolate of vancomycin-resistant E. faecium was serially-passaged with ebselen for 14 days. Against VRE biofilm, a virulence factor that enables the bacteria to colonize the gut, ebselen demonstrated the ability to both inhibit biofilm formation and disrupt mature biofilm. Furthermore, in a murine VRE colonization reduction model, ebselen proved as effective as ramoplanin in reducing the bacterial shedding and burden of VRE present in the fecal content (by > 99.99%), cecum, and ileum of mice. Based on the promising results obtained, ebselen warrants further investigation as a novel decolonizing agent to quell VRE infection.

A new series of phenylthiazoles with t-butyl lipophilic component was synthesized and their antibacterial activity against a panel of multidrug-resistant bacterial pathogens was evaluated. Five compounds demonstrated promising antibacterial activity against methicillin-resistant staphylococcal strains and several vancomycin-resistant staphylococcal and enterococcal species. Additionally, three derivatives 19, 23 and 26 exhibited rapid bactericidal activity, and remarkable ability to disrupt mature biofilm produced by MRSA USA300. More importantly, a resistant mutant to 19 couldn't be isolated after subjecting MRSA to sub-lethal doses for 14 days. Lastly, this new series of phenylthiazoles possesses an advantageous attribute over the first-generation compounds in their stability to hepatic metabolism, with a biological half-life of more than 9 h.