It has become increasingly clear that low levels of antibiotics present in many environments can select for resistant bacteria, yet the evolutionary pathways for resistance development during exposure to low amounts of antibiotics remain poorly defined. Here we show that Salmonella enterica exposed to sub-MIC levels of streptomycin evolved high-level resistance via novel mechanisms that are different from those observed during lethal selections. During lethal selection only rpsL mutations are found, whereas at sub-MIC selection resistance is generated by several small-effect resistance mutations that combined confer high-level resistance via three different mechanisms: (i) alteration of the ribosomal RNA target (gidB mutations), (ii) reduction in aminoglycoside uptake (cyoB, nuoG, and trkH mutations), and (iii) induction of the aminoglycoside-modifying enzyme AadA (znuA mutations). These results demonstrate how the strength of the selective pressure influences evolutionary trajectories and that even weak selective pressures can cause evolution of high-level resistance.

BackgroundCoral reefs are colored by eukaryotic chromoproteins (CPs) that are homologous to green fluorescent protein. CPs differ from fluorescent proteins (FPs) by intensely absorbing visible light to give strong colors in ambient light. This endows CPs with certain advantages over FPs, such as instrument-free detection uncomplicated by ultra-violet light damage or background fluorescence, efficient Förster resonance energy transfer (FRET) quenching, and photoacoustic imaging. Thus, CPs have found utility as genetic markers and in teaching, and are attractive for potential cell biosensor applications in the field. Most near-term applications of CPs require expression in a different domain of life: bacteria. However, it is unclear which of the eukaryotic CP genes might be suitable and how best to assay them.ResultsHere, taking advantage of codon optimization programs in 12 cases, we engineered 14 CP sequences (meffRed, eforRed, asPink, spisPink, scOrange, fwYellow, amilGFP, amajLime, cjBlue, meffBlue, aeBlue, amilCP, tsPurple and gfasPurple) into a palette of Escherichia coli BioBrick plasmids. BioBricks comply with synthetic biology’s most widely used, simplified, cloning standard. Differences in color intensities, maturation times and fitness costs of expression were compared under the same conditions, and visible readout of gene expression was quantitated. A surprisingly large variation in cellular fitness costs was found, resulting in loss of color in some overnight liquid cultures of certain high-copy-plasmid-borne CPs, and cautioning the use of multiple CPs as markers in competition assays. We solved these two problems by integrating pairs of these genes into the chromosome and by engineering versions of the same CP with very different colors.ConclusionAvailability of 14 engineered CP genes compared in E. coli, together with chromosomal mutants suitable for competition assays, should simplify and expand CP study and applications. There was no single plasmid-borne CP that combined all of the most desirable features of intense color, fast maturation and low fitness cost, so this study should help direct future engineering efforts.Electronic supplementary materialThe online version of this article (10.1186/s13036-018-0100-0) contains supplementary material, which is available to authorized users.

Medical device-related biofilms are a major cause of hospital-acquired infections, especially chronic infections. Numerous diverse models to study surface-associated biofilms have been developed; however, their usability varies. Often, a simple method is desired without sacrificing throughput and biological relevance. Here, we present an in-house developed 3D-printed device (FlexiPeg) for biofilm growth, conceptually similar to the Calgary Biofilm device but aimed at increasing ease of use and versatility. Our device is modular with the lid and pegs as separate units, enabling flexible assembly with up- or down-scaling depending on the aims of the study. It also allows easy handling of individual pegs, especially when disruption of biofilm populations is needed for downstream analysis. The pegs can be printed in, or coated with, different materials to create surfaces relevant to the study of interest. We experimentally validated the use of the device by exploring the biofilms formed by clinical strains of Escherichia coli and Klebsiella pneumoniae, commonly associated with device-related infections. The biofilms were characterized by viable cell counts, biomass staining, and scanning electron microscopy (SEM) imaging. We evaluated the effects of different additive manufacturing technologies, 3D printing resins, and coatings with, for example, silicone, to mimic a medical device surface. The biofilms formed on our custom-made pegs could be clearly distinguished based on species or strain across all performed assays, and they corresponded well with observations made in other models and clinical settings, for example, on urinary catheters. Overall, our biofilm device is a robust, easy-to-use, and relevant assay, suitable for a wide range of applications in surface-associated biofilm studies, including materials testing, screening for biofilm formation capacity, and antibiotic susceptibility testing.

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Blood stream infection (BSI) is related to high mortality and morbidity. Early antimicrobial therapy is crucial in treating patients with BSI. The most common Gram-negative bacteria causing BSI is Escherichia coli. Targeted effective treatment of patients with BSI is only possible if it is based on antibiotic susceptibility testing (AST) data after blood culture positivity. However, there are very few methods available for rapid phenotypic AST and the fastest method takes 4 h. Here we analyzed the performance of a 30 min ultra-rapid method for AST of E. coli directly from positive blood cultures (BC). In total, 51 positive BC with E. coli were studied, and we evaluated the ultra-rapid method directly on positive BC as well as on E. coli colonies cultured on agar plates. The results obtained by the new method were compared with disk diffusion.The method provided accurate AST result in 30 min to Ciprofloxacin and Gentamicin for 92% and 84% of the positive BC samples, respectively. For E. coli isolates retrieved from agar plates, 86% and 96% of the AST results were accurate for Ciprofloxacin and Gentamicin, respectively, after 30 min of assay time. When time to result was modulated in-silico from 30 to 60 minutes for the agar plate samples, accuracy of AST results went up to 92% for Ciprofloxacin and to 100% for Gentamicin.The present study shows that the method is reliable and delivers ultra-rapid AST data in 30 minutes directly from positive BC and as well as from agar plates.