Solvent Extraction of Klebsiella pneumoniae Bacteriophage Lysates with 1-Dodecanol Results in Endotoxin Reduction with Low Risk of Solvent Contamination.

Antimicrobial resistance in pathogenic bacteria is increasing worldwide. One solution to this crisis is bacteriophage therapy, a treatment that harnesses naturally occurring bacterial viruses to invade and lyse antimicrobial resistant bacterial hosts. In Gram-negative hosts, a by-product of bacteriophage production is bacterial endotoxin, which can cause serious immune reactions in vivo. Purification methods using organic solvent extraction can remove endotoxin in bacteriophage lysates. In this study, we investigate a method for removal of endotoxin from 16 high-titer Klebsiella pneumoniae lysates by extraction with 1-dodecanol, 1-octanol, dodecane, or decane. In these experiments, treatment with either 1-dodecanol or 1-octanol resulted in removal of 104-105 endotoxin units/mL. Recovery of bacteriophage in lysates treated with dodecanol without dialysis was >90%, and residual dodecanol was low (10-1500 ppm). Overall these results suggest that organic solvent extraction using 1-dodecanol is effective at removing bacterial endotoxin, maintaining bacteriophage titer, and reducing solvent contamination in 16 K. pneumoniae bacteriophage lysates.

Genetic Determinants of Intrinsic Antibiotic Tolerance in Mycobacterium avium.

The Mycobacterium avium complex (MAC) is one of the most prevalent causes of nontuberculous mycobacteria pulmonary infection in the United States, and yet it remains understudied. Current MAC treatment requires more than a year of intermittent to daily combination antibiotic therapy, depending on disease severity. In order to shorten and simplify curative regimens, it is important to identify the innate bacterial factors contributing to reduced antibiotic susceptibility, namely, antibiotic tolerance genes. In this study, we performed a genome-wide transposon screen to elucidate M. avium genes that play a role in the bacterium's tolerance to first- and second-line antibiotics. We identified a total of 193 unique M. avium mutants with significantly altered susceptibility to at least one of the four clinically used antibiotics we tested, including two mutants (in DFS55_00905 and DFS55_12730) with panhypersusceptibility. The products of the antibiotic tolerance genes we have identified may represent novel targets for future drug development studies aimed at shortening the duration of therapy for MAC infections. IMPORTANCE The prolonged treatment required to eradicate Mycobacterium avium complex (MAC) infection is likely due to the presence of subpopulations of antibiotic-tolerant bacteria with reduced susceptibility to currently available drugs. However, little is known about the genes and pathways responsible for antibiotic tolerance in MAC. In this study, we performed a forward genetic screen to identify M. avium antibiotic tolerance genes, whose products may represent attractive targets for the development of novel adjunctive drugs capable of shortening the curative treatment for MAC infections.

Mechanisms of Antibiotic Tolerance in Mycobacterium avium Complex: Lessons From Related Mycobacteria.

Mycobacterium avium complex (MAC) species are the most commonly isolated nontuberculous mycobacteria to cause pulmonary infections worldwide. The lengthy and complicated therapy required to cure lung disease due to MAC is at least in part due to the phenomenon of antibiotic tolerance. In this review, we will define antibiotic tolerance and contrast it with persistence and antibiotic resistance. We will discuss physiologically relevant stress conditions that induce altered metabolism and antibiotic tolerance in mycobacteria. Next, we will review general molecular mechanisms underlying bacterial antibiotic tolerance, particularly those described for MAC and related mycobacteria, including Mycobacterium tuberculosis, with a focus on genes containing significant sequence homology in MAC. An improved understanding of antibiotic tolerance mechanisms can lay the foundation for novel approaches to target antibiotic-tolerant mycobacteria, with the goal of shortening the duration of curative treatment and improving survival in patients with MAC.