BIRMINGHAM, Ala. – Increases in multidrug-resistance in the bacteria Streptococcus pneumoniae have made it the fourth-leading cause of death associated with antibiotic resistance.
In a study in PLOS Biology, researchers report a new target to fight against pneumonia due to infections by this opportunistic lung pathogen — interference with the bacteria’s fermentation metabolism. This may offer a novel therapeutic option in the urgent need to discover new strategies to combat drug-resistant S. pneumoniae.
In a proof of principle, University of Alabama at Birmingham researchers showed that giving an existing drug — one already approved by the United States Food and Drug Administration to treat methanol poisoning – in combination with the antibiotic erythromycin significantly reduced disease in mice infected with a virulent, multidrug-resistant S. pneumoniae. The combination therapy reduced bacterial burden in the lungs by 95 percent, and bacterial burdens in the spleen and heart by 100- and 700-fold, respectively. The FDA-approved drug alone, or erythromycin alone, had no effect.
Fomepizole, the FDA-approved drug, disrupts activity of the enzyme alcohol dehydrogenase in the bacteria. The mice were infected intratracheally with the multidrug-resistant clinical isolate S. pneumoniae serotype 35B strain 162–5678, which has high resistance to erythromycin. Notably, the S. pneumoniae 35B serotype has been reported as an emerging multidrug-resistant serotype in clinical settings. Eighteen hours after infection, the mice were given a single injection of erythromycin, with or without fomepizole.
“Fomepizole, or other drugs that inhibit bacterial metabolism, have potential to dramatically increase the efficacy of erythromycin and other antibiotics, respectively, in vivo,” said Carlos Orihuela, Ph.D., professor and interim chair of the UAB Department of Microbiology.
A broad foundation of basic research preceded this proof-of-principle experiment.
S. pneumoniae relies on fermentation and glycolysis to produce energy. During fermentation, pyruvate is converted to lactate, acetate and ethanol, and NADH is oxidized to regenerate NAD+, which is needed for glycolysis. Accordingly, maintenance of an available NAD+ pool, necessary for redox balance, is vital for sustained energy production, bacterial growth and survival.
Orihuela and UAB colleagues made S. pneumoniae mutants in five enzymes involved in fermentation and NAD+ production, and they found, in general, that the mutants had impaired metabolism. Two of the mutants, one for lactate dehydrogenase and one for alcohol dehydrogenase, had stark decreases in intracellular pool of ATP, the energy molecule of living cells. The other three mutants had significant, but more modest, decreases.
NAD+/NADH redox imbalances in the mutants generally interfered with production of S. pneumoniae virulence factors and colonization in the mouse nasopharynx. Some of the mutations influenced susceptibility to antibiotics, as tested with three antibiotics, including erythromycin, that interfere with protein synthesis, two antibiotics that disrupt cell wall synthesis and one antibiotic that targets DNA transcription.
Researchers found that treating a wildtype S. pneumoniae, which did not have mutations in alcohol dehydrogenase or the other enzymes, with fomepizole alone caused redox imbalances. In vitro tests showed that treatment of S. pneumoniae with fomepizole enhanced the susceptibility to antibiotics, including fourfold decreases in the minimal inhibitory concentrations of the antibiotics erythromycin and gentamicin.
“We also evaluated whether fomepizole treatment impacted the antibiotic susceptibility of other anaerobic gram-positive bacteria, including other streptococcal pathogens, including Streptococcus pyogenes, Streptococcus agalactiae and Enterococcus faecium, to erythromycin or gentamicin,” Orihuela said. “We observed from twofold to eightfold decreased minimal inhibitory concentration with fomepizole in most cases, including E. faecium.”
“Our results indicate that the blocking of NAD+ regeneration pathways during infection is a way to increase antibiotic susceptibility in drug-resistant gram-positive anaerobic pathogens,” Orihuela said. “This has clinical potential with regard to microbial eradication and treatment of disseminated infection.”
Globally, more than 3 million individuals are hospitalized due to pneumococcal disease annually, and hundreds of thousands die as a result.
Co-authors with Orihuela in the study, “Targeting NAD+ regeneration enhances antibiotic susceptibility of Streptococcus pneumoniae during invasive disease,” are Hansol Im, Madison L. Pearson, Eriel Martinez, Xiuhong Song, Katherine L. Kruckow and Rachel M. Andrews, UAB Department of Microbiology; and Kyle H. Cichos and Elie S. Ghanem, UAB Department of Orthopaedic Surgery.
Support came from National Institutes of Health grants AI114800, AI148368, AI156898, AI172796 and HL129948.
At UAB, Microbiology and Orthopaedic Surgery are departments in the Marnix E. Heersink School of Medicine.