In the absence of antibiotic-mediated selection, sensitive bacteria are expected to displace their resistant counterparts if resistance genes are costly. However, many resistance genes persist for long periods in the absence of antibiotics. Horizontal gene transfer (primarily conjugation) could explain this persistence, but it has been suggested that very high conjugation rates would be required. Here, we show that common conjugal plasmids, even when costly, are indeed transferred at sufficiently high rates to be maintained in the absence of antibiotics in Escherichia coli. The notion is applicable to nine plasmids from six major incompatibility groups and mixed populations carrying multiple plasmids. These results suggest that reducing antibiotic use alone is likely insufficient for reversing resistance. Therefore, combining conjugation inhibition and promoting plasmid loss would be an effective strategy to limit conjugation-assisted persistence of antibiotic resistance.

It is generally assumed that antibiotics can promote horizontal gene transfer (HGT). However, because of a variety of confounding factors that complicate the interpretation of previous studies, the mechanisms by which antibiotics modulate HGT remain poorly understood. In particular, it is unclear whether antibiotics directly regulate the efficiency of HGT, serve as a selection force to modulate population dynamics after HGT has occurred, or both. Here, we address this question by quantifying conjugation dynamics in the presence and absence of antibiotic-mediated selection. Surprisingly, we find that sub-lethal concentrations of antibiotics from the most widely used classes do not significantly increase the conjugation efficiency. Instead, our modeling and experimental results demonstrate that conjugation dynamics are dictated by antibiotic-mediated selection, which can both promote and suppress conjugation dynamics. Our findings suggest that the contribution of antibiotics to the promotion of HGT may have been overestimated. These findings have implications for designing effective antibiotic treatment protocols and for assessing the risks of antibiotic use.

Bacteria have developed resistance against every antibiotic at an alarming rate, considering the timescale at which new antibiotics are developed. Thus, there is a critical need to use antibiotics more effectively, extend the shelf life of existing antibiotics, and minimize their side effects. This requires understanding the mechanisms underlying bacterial drug responses. Past studies have focused on survival in the presence of antibiotics by individual cells, as genetic mutants or persisters. In contrast, a population of bacterial cells can collectively survive antibiotic treatments lethal to individual cells. This tolerance can arise by diverse mechanisms, including resistance-conferring enzyme production, titration-mediated bistable growth inhibition, swarming, and inter-population interactions. These strategies can enable rapid population recovery after antibiotic treatment, and provide a time window for otherwise susceptible bacteria to acquire inheritable genetic resistance. Here, we emphasize the potential for targeting collective antibiotic tolerance behaviors as an antibacterial treatment strategy.

A body of evidence has shown that the control of E2F transcription factor activity is critical for determining cell cycle entry and cell proliferation. However, an understanding of the precise determinants of this control, including the role of other cell-cycle regulatory activities, has not been clearly defined. Here, recognizing that the contributions of individual regulatory components could be masked by heterogeneity in populations of cells, we model the potential roles of individual components together with the use of an integrated system to follow E2F dynamics at the single-cell level and in real time. These analyses reveal that crossing a threshold amplitude of E2F accumulation determines cell cycle commitment. Importantly, we find that Myc is critical in modulating the amplitude, whereas cyclin D/E activities have little effect on amplitude but do contribute to the modulation of duration of E2F activation, thereby affecting the pace of cell cycle progression.

The efficacy of many antibiotics decreases with increasing bacterial density, a phenomenon called the ‘inoculum effect' (IE). This study reveals that, for ribosome-targeting antibiotics, IE is due to bistable inhibition of bacterial growth, which reduces the efficacy of certain treatment frequencies.

Adeno-associated virus (AAV)-based gene therapies can restore endogenous factor VIII (FVIII) expression in hemophilia A (HA). AAV vectors typically utilize a B-domain deleted FVIII transgene, such as human FVIII-SQ in valoctocogene roxaparvovec (AAV5-FVIII-SQ). Surprisingly, the activity of transgene-produced FVIII-SQ was between 1.3 and 2.0 times higher in one-stage clot (OS) than chromogenic-substrate (CS) assays, while recombinant FVIII-SQ products have lower OS than CS activity. Transgene-produced and recombinant FVIII-SQ showed comparable specific activity (IU/mg) in the CS assay, demonstrating that the diverging activities arise in the OS assay. Higher OS activity for transgene-produced FVIII-SQ was observed across various assays kits and clinical laboratories, suggesting intrinsic molecular features as a potential root cause. Further experiments in two participants showed that transgene-produced FVIII-SQ accelerated early factor Xa and thrombin formation, which may explain the higher OS activity based on a kinetic bias between OS and CS assay readout times. Despite the faster onset of coagulation, global thrombin levels were unaffected. A correlation with joint bleeds suggested that both OS and CS assay remained clinically meaningful to distinguish hemophilic from non-hemophilic FVIII activity levels. During clinical development, the CS activity was chosen as a surrogate endpoint to conservatively assess hemostatic efficacy and enable comparison with recombinant FVIII-SQ products. Relevant trials are registered on Clinicaltrials.gov (NCT02576795 and NCT03370913).

Fig. 2. (A)Interaction web of top-down and bottom-up effects in the eelgrass study system. The top predator is the sea otter (E. lutris), the mesopredators are crabs (Cancer spp. and Pugettia producta), the epiphyte mesograzers are primarily an isopod (I. resecata) and a sea slug (P. taylori), and algal epiphyte competitors of eelgrass primarily consist of chain-forming diatoms, and the red alga Smithora naiadum. Solid arrows indicate direct effects, dashed arrows indicate indirect effects, and the plus and minus symbols indicate positive and/or negative effects on trophic guilds and eelgrass condition. C, competitive interaction; T, trophic interaction. (Original artwork by A. C. Hughes.) (B-E) Survey results testing for the effects of sea otter density on eelgrass bed community properties (Tables S2 and S3). Elkhorn Slough (sea otters present and high nutrients) eelgrass beds (n = 4) are coded in red, and the Tomales Bay reference site (no sea otters, low nutrients) beds (n = 4) are coded in blue. (B) Crab biomass and size structure of two species of Cancer crabs; (C) grazer biomass per shoot and large grazer density; (D) algal epiphyte loading; and (E) aboveground and belowground eelgrass biomass. DW, dry weight; FW, fresh weight.

The postantibiotic effect (PAE) refers to the temporary suppression of bacterial growth following transient antibiotic treatment. This effect has been observed for decades for a wide variety of antibiotics and microbial species. However, despite empirical observations, a mechanistic understanding of this phenomenon is lacking. Using a combination of modeling and quantitative experiments, we show that the PAE can be explained by the temporal dynamics of drug detoxification in individual cells after an antibiotic is removed from the extracellular environment. These dynamics are dictated by both the export of the antibiotic and the intracellular titration of the antibiotic by its target. This mechanism is generally applicable for antibiotics with different modes of action. We further show that efflux inhibition is effective against certain antibiotic motifs, which may help explain mixed cotreatment success.