Within the larger ABC superfamily of ATPases, ABCF family members eEF3 in Saccharomyces cerevisiae and EttA in Escherichia coli have been found to function as ribosomal translation factors. Several other ABCFs including biochemically characterized VgaA, LsaA and MsrE confer resistance to antibiotics that target the peptidyl transferase center and exit tunnel of the ribosome. However, the diversity of ABCF subfamilies, the relationships among subfamilies and the evolution of antibiotic resistance (ARE) factors from other ABCFs have not been explored. To address this, we analyzed the presence of ABCFs and their domain architectures in 4505 genomes across the tree of life. We find 45 distinct subfamilies of ABCFs that are widespread across bacterial and eukaryotic phyla, suggesting that they were present in the last common ancestor of both. Surprisingly, currently known ARE ABCFs are not confined to a distinct lineage of the ABCF family tree, suggesting that ARE can readily evolve from other ABCF functions. Our data suggest that there are a number of previously unidentified ARE ABCFs in antibiotic producers and important human pathogens. We also find that ATPase-deficient mutants of all four E. coli ABCFs (EttA, YbiT, YheS and Uup) inhibit protein synthesis, indicative of their ribosomal function, and demonstrate a genetic interaction of ABCFs Uup and YheS with translational GTPase BipA involved in assembly of the 50S ribosome subunit. Finally, we show that the ribosome-binding resistance factor VmlR from Bacillus subtilis is localized to the cytoplasm, ruling out a role in antibiotic efflux.

New strategies are continuously sought for the treatment of skin and wound infections due to increased problems with non-healing wounds. Electrospun nanofiber mats with antibacterial agents as drug delivery systems provide opportunities for the eradication of bacterial infections as well as wound healing. Antibacterial activities of such mats are directly linked with their drug release behavior. Traditional pharmacopoeial drug release testing settings are not always suitable for analyzing the release behavior of fiber mats intended for the local drug delivery. We tested and compared different drug release model systems for the previously characterized electrospun chloramphenicol (CAM)-loaded nanofiber (polycaprolactone (PCL)) and microfiber (PCL in combination with polyethylene oxide) mats with different drug release profiles. Drug release into buffer solution and hydrogel was investigated and drug concentration was determined using either high-performance liquid chromatography, ultraviolet-visible spectrophotometry, or ultraviolet (UV) imaging. The CAM release and its antibacterial effects in disc diffusion assay were assessed by bacterial bioreporters. All tested model systems enabled to study the drug release from electrospun mats. It was found that the release into buffer solution showed larger differences in the drug release rate between differently designed mats compared to the hydrogel release tests. The UV imaging method provided an insight into the interactions with an agarose hydrogel mimicking wound tissue, thus giving us information about early drug release from the mat. Bacterial bioreporters showed clear correlations between the drug release into gel and antibacterial activity of the electrospun CAM-loaded mats.

We developed a reporter system based on simultaneous expression of two fluorescent proteins: GFP as a reporter of the capacity of protein synthesis and mutated mScarlet-I as a reporter of translational errors. Because of the unique stop codons or frameshift mutations introduced into the mScarlet-I gene, red fluorescence was produced only after a mistranslation event. These reporters allowed us to estimate mistranslation at a single cell level using either flow cytometry or fluorescence microscopy. We found that laboratory strains of Escherichia coli are more prone to mistranslation compared to the clinical isolates. As relevant for uropathogenic E. coli, growth in human urine elevated translational frameshifting compared to standard laboratory media, whereas different standard media had a small effect on translational fidelity. Antibiotic-induced mistranslation was studied by using amikacin (aminoglycoside family) and azithromycin (macrolide family). Bactericidal amikacin induced preferably stop-codon readthrough at a moderate level. Bacteriostatic azithromycin on the other hand induced both frameshifting and stop-codon readthrough at much higher level. Single cell analysis revealed that fluorescent reporter-protein signal can be lost due to leakage from a fraction of bacteria in the presence of antibiotics, demonstrating the complexity of the antimicrobial activity.

Invasive salmonellosis caused by Salmonella enterica subspecies affects millions of people every year, mostly children from low-income countries, and is associated with a high mortality rate. Azithromycin is used to treat invasive salmonellosis resistant to first-line drugs despite conflicting effective concentrations in vitro and achievable serum concentrations in vivo . As resistance levels to azithromycin are rising, we demonstrate that nanomotion technology, which is based on measuring changes in bacterial nanoscale movements, can be used for rapid phenotypic testing of Salmonella ’s susceptibility to azithromycin. Additionally, the use of nanomotion enabled the detection of the bactericidal effect. Nanomotion changes under various culture conditions correlated with susceptibility measured by minimum inhibitory concenctration (MIC) determination, colony-forming unit (CFU) counting, and fluorescent reporter-based estimates of intrabacterial azithromycin accumulation. Environmental conditions, both during azithromycin treatment and throughout the recovery period, significantly affect the antibacterial response to azithromycin. Azithromycin susceptibility in Salmonella is detectable after only 2 h of treatment. This reflects the quick action of the antibiotic, which could be one of the contributing factors behind the clinical efficacy of azithromycin for Salmonella treatment. Our study underscores the critical role of assay conditions, which greatly influenced both azithromycin efficacy and the test results. IMPORTANCE: Azithromycin is used as a last-resort antibiotic to treat life-threatening infections caused by Salmonella enterica , a high-priority pathogen according to the World Health Organization. Resistance levels to azithromycin are increasing, highlighting the need for rapid susceptibility testing. In this study, we demonstrate that nanomotion technology can detect azithromycin susceptibility in Salmonella , suggesting its potential use for rapid resistance detection in clinical settings and its future use with azithromycin. Additionally, the study shows that nanomotion technology can be used for susceptibility and postantibiotic effect testing for various pathogens and antibacterials, including those generally regarded as bacteriostatic.

Guanosine tetra- and pentaphosphate ((p)ppGpp) are one of the key players in the stress response of bacteria. Accumulation of these alarmones activates the stringent response, usually triggered by different nutritional stresses. For Pseudomonas putida, there is only limited data available on the importance of the stringent response in stress situations. Also, in recent years, different specific phage defence systems have received much attention, but little is known about the involvement of stringent response in phage infection. Here, we show that P. putida PaW85 (p)ppGpp0 is prototrophic and tolerates chemical stress well. However, in the stationary phase P. putida cells deprived of (p)ppGpp have impaired membrane integrity. In addition, we conducted a large-scale screening of stringent response effects on phage infections using the CEPEST phage collection. We tested 67 phages of 22 different species and revealed that the lack of (p)ppGpp has opposing effects on phage infection with nearly half of the tested phages showing higher infection efficiency on the (p)ppGpp0 cells, whereas the other half shows reduced infection. We show that the differences in phage infection efficiency for phages Aura and Amme-3 are not caused by adsorption rate differences, but alterations in downstream steps of the infection cycle—prolonged latent period in the absence of (p)ppGpp or unproductive infection in the presence of (p)ppGpp. Altogether, results indicate that the role of stringent response in phage infection is highly diverse, and over half of the times the presence of (p)ppGpp facilitates phage infections rather than protects the cells.

Azithromycin is used to treat invasive salmonellosis, despite conflicting effective concentrations in vitro and in vivo . Resistance of Salmonella enterica to azithromycin is increasing. We demonstrate that nanomotion technology can be used for rapid phenotypic testing of Salmonella ’s susceptibility to azithromycin. Nanomotion changes under various culture conditions correlated with susceptibility measured by MIC determination, CFU counting, and fluorescent reporter-based estimates of intrabacterial azithromycin accumulation.