e Tigecycline is a translational inhibitor with efficacy against a wide range of pathogens. Using experimental evolution, we adapted Acinetobacter baumannii, Enterococcus faecium, Escherichia coli, and Staphylococcus aureus to growth in elevated tigecycline concentrations. At the end of adaptation, 35 out of 47 replicate populations had clones with a mutation in rpsJ, the gene that encodes the ribosomal S10 protein. To validate the role of mutations in rpsJ in conferring tigecycline resistance, we showed that mutation of rpsJ alone in Enterococcus faecalis was sufficient to increase the tigecycline MIC to the clinical breakpoint of 0.5 g/ml. Importantly, we also report the first identification of rpsJ mutations associated with decreased tigecycline susceptibility in A. baumannii, E. coli, and S. aureus. The identified S10 mutations across both Gram-positive and -negative species cluster in the vertex of an extended loop that is located near the tigecycline-binding pocket within the 16S rRNA. These data indicate that S10 is a general target of tigecycline adaptation and a relevant marker for detecting reduced susceptibility in both Gram-positive and -negative pathogens. Tigecycline is an FDA-approved antibiotic used to treat complicated skin and skin structure infections (cSSSI), complicated intra-abdominal infections (cIAI), and community-associated bacterial pneumonia (CABP). Increasingly, tigecycline and other drugs of last resort, such as vancomycin and daptomycin, are used "off label" to combat infections arising from multidrug-resistant pathogens (1). As infections with multidrug-resistant pathogens increase, they pose a major threat to public health, with at least two million infections and 23,000 deaths annually in the United States (2). With increased usage, tigecycline resistance will inevitably develop. By understanding how drug resistance emerges, we can be more proactive in identifying the spread of alleles associated with nonsusceptibility or even move toward a preemptive strategy to block the emergence of resistance through new classes of "antievolution" drugs that could work with current antibiotics.The antibiotic tigecycline (TGC) has good in vitro activity against a broad spectrum of multidrug-resistant pathogens and is often used as part of a combination regimen in severe infections caused by multidrug-resistant isolates (3). TGC is a semisynthetic drug belonging to the tetracycline-derived glycylcycline family. Like tetracycline, TGC inhibits the elongation step of translation by binding reversibly to the 16S rRNA and blocking the entry of tRNAs into the A site. The therapeutic success of TGC is due, in part, to its high affinity for the ribosome, which is about 20-fold higher than that of tetracycline (4). TGC has a bulky t-butylglycylamido modification at the 9 position that has been proposed to limit the activity of common tetracycline-resistance mechanisms, such as ribosomal protection (tetM, tetO, etc.) and efflux pumps (tetK, tetA, etc.) (3).Despite the promising in vitro activity of TGC...

The evolution of hypermutators in response to antibiotic treatment in both clinical and laboratory settings provides a unique context for the study of adaptive evolution. With increased mutation rates, the number of hitchhiker mutations within an evolving hypermutator population is remarkably high and presents substantial challenges in determining which mutations are adaptive. Intriguingly however, hypermutators also provide an opportunity to explore deeply the accessible evolutionary trajectories that lead to increased organism fitness, in this case the evolution of antibiotic resistance to the clinically relevant antibiotic tigecycline by the hospital pathogen Acinetobacter baumannii. Using a continuous culture system, AB210M, a clinically derived strain of A. baumannii, was evolved to tigecycline resistance. Analysis of the adapted populations showed that nearly all the successful lineages became hypermutators via movement of a mobile element to inactivate mutS. In addition, metagenomic analysis of population samples revealed another 896 mutations that occurred at a frequency greater than 5% in the population, while 38 phenotypically distinct individual colonies harbored a total of 1712 mutations. These mutations were scattered throughout the genome and affected ~40% of the coding sequences. The most highly mutated gene was adeS, a known tigecycline-resistance gene; however, adeS was not solely responsible for the high level of TGC resistance. Sixteen other genes stood out as potentially relevant to increased resistance. The five most prominent candidate genes (adeS, rpsJ, rrf, msbA, and gna) consistently re-emerged in subsequent replicate population studies suggesting they are likely to play a role in adaptation to tigecycline. Interestingly, the repeated evolution of a hypermutator phenotype in response to antibiotic stress illustrates not only a highly adaptive strategy to resistance, but also a remarkably efficient survey of successful evolutionary trajectories.

Cell-free protein synthesis (CFPS) platforms, once primarily a research tool to produce difficult to express proteins, are increasingly being pursued by the synthetic biology community for applications including biomanufacturing, rapid screening systems, and field-ready sensors. While consistency within individual studies is apparent in the literature, challenges with reproducing results between laboratories, or even between individuals within a laboratory, are discussed openly by practitioners. As the field continues to grow and move toward applications, a quantitative understanding of expected variability for CFPS and the relative contribution of underlying sources will become increasingly important. Here we offer the first quantitative assessment of interlaboratory variability in CFPS. Three laboratories implemented a single CFPS protocol and performed a series of exchanges, both of material and personnel, designed to quantify relative contributions to variability associated with the site, operator, cell extract preparation, and supplemental reagent preparation. We found that materials prepared at each laboratory, exchanged pairwise, and tested at each site resulted in 40.3% coefficient of variation compared to 7.64% for a single operator across days using a single set of materials. Reagent preparations contributed significantly to observed variability; extract preparations, however, surprisingly did not explain any of the observed variability, even when prepared in different laboratories by different operators. Subsequent exchanges showed that both the site and the operator each contributed to observed interlaboratory variability. In addition to providing the first quantitative assessment of interlaboratory variability in CFPS, these results establish a baseline for individual operator variability across days that can be used as an initial benchmark for community-driven standardization efforts. We anticipate that our results will narrow future avenues of investigation to develop best practices that will ultimately drive down interlaboratory variability, accelerating research progress and informing the suitability of CFPS for real-world applications.

Horizontal gene transfer threatens the therapeutic success of antibiotics by facilitating the rapid dissemination of resistance alleles among bacterial species. The conjugative mobile element Tn916 provides an excellent context for examining the role of adaptive parasexuality as it carries the tetracycline-resistance allele tetM and has been identified in a wide range of pathogens. We have used a combination of experimental evolution and allelic frequency measurements to gain insights into the adaptive trajectories leading to tigecycline resistance in a hospital strain of Enterococcus faecalis and predict what mechanisms of resistance are most likely to appear in the clinical setting. Here, we show that antibiotic selection led to the near fixation of adaptive alleles that simultaneously altered TetM expression and produced remarkably increased levels of Tn916 horizontal gene transfer. In the absence of drug, approximately 1 in 120,000 of the nonadapted E. faecalis S613 cells had an excised copy of Tn916, whereas nearly 1 in 50 cells had an excised copy of Tn916 upon selection for resistance resulting in a more than 1,000-fold increase in conjugation rates. We also show that tigecycline, a translation inhibitor, selected for a mutation in the ribosomal S10 protein. Our results show the first example of mutations that concurrently confer resistance to an antibiotic and lead to constitutive conjugal-transfer of the resistance allele. Selection created a highly parasexual phenotype and high frequency of Tn916 jumping demonstrating how the use of antibiotics can lead directly to the proliferation of resistance in, and potentially among, pathogens.