Drug-Driven Phenotypic Convergence Supports Rational Treatment Strategies of Chronic Infections.

Chronic Pseudomonas aeruginosa infections evade antibiotic therapy and are associated with mortality in cystic fibrosis (CF) patients. We find that in vitro resistance evolution of P. aeruginosa toward clinically relevant antibiotics leads to phenotypic convergence toward distinct states. These states are associated with collateral sensitivity toward several antibiotic classes and encoded by mutations in antibiotic resistance genes, including transcriptional regulator nfxB. Longitudinal analysis of isolates from CF patients reveals similar and defined phenotypic states, which are associated with extinction of specific sub-lineages in patients. In-depth investigation of chronic P. aeruginosa populations in a CF patient during antibiotic therapy revealed dramatic genotypic and phenotypic convergence. Notably, fluoroquinolone-resistant subpopulations harboring nfxB mutations were eradicated by antibiotic therapy as predicted by our in vitro data. This study supports the hypothesis that antibiotic treatment of chronic infections can be optimized by targeting phenotypic states associated with specific mutations to improve treatment success in chronic infections.

Metabolic specialization drives reduced pathogenicity in Pseudomonas aeruginosa isolates from cystic fibrosis patients.

Metabolism provides the foundation for all cellular functions. During persistent infections, in adapted pathogenic bacteria metabolism functions radically differently compared with more naïve strains. Whether this is simply a necessary accommodation to the persistence phenotype or if metabolism plays a direct role in achieving persistence in the host is still unclear. Here, we characterize a convergent shift in metabolic function(s) linked with the persistence phenotype during Pseudomonas aeruginosa colonization in the airways of people with cystic fibrosis. We show that clinically relevant mutations in the key metabolic enzyme, pyruvate dehydrogenase, lead to a host-specialized metabolism together with a lower virulence and immune response recruitment. These changes in infection phenotype are mediated by impaired type III secretion system activity and by secretion of the antioxidant metabolite, pyruvate, respectively. Our results show how metabolic adaptations directly impinge on persistence and pathogenicity in this organism.

Adaptive Evolution of Pseudomonas aeruginosa in Human Airways Shows Phenotypic Convergence Despite Diverse Patterns of Genomic Changes.

Selective forces in the environment drive bacterial adaptation to novel niches, choosing the fitter variants in the population. However, in dynamic and changing environments, the evolutionary processes controlling bacterial adaptation are difficult to monitor. Here, we follow 9 people with cystic fibrosis chronically infected with Pseudomonas aeruginosa, as a proxy for bacterial adaptation. We identify and describe the bacterial changes and evolution occurring between 15 and 35 yr of within-host evolution. We combine whole-genome sequencing, RNA sequencing, and metabolomics and compare the evolutionary trajectories directed by the adaptation of 4 different P. aeruginosa lineages to the lung. Our data suggest divergent evolution at the genomic level for most of the genes, with signs of convergent evolution with respect to the acquisition of mutations in regulatory genes, which drive the transcriptional and metabolomic program at late time of evolution. Metabolomics further confirmed convergent adaptive phenotypic evolution as documented by the reduction of the quorum-sensing molecules acyl-homoserine lactone, phenazines, and rhamnolipids (except for quinolones). The modulation of the quorum-sensing repertoire suggests that similar selective forces characterize at late times of evolution independent of the patient. Collectively, our data suggest that similar environments and similar P. aeruginosa populations in the patients at prolonged time of infection are associated with an overall reduction of virulence-associated features and phenotypic convergence.

Role of bacterial multidrug efflux pumps during infection.

Multidrug efflux pumps are protein complexes located in the cell envelope that enable bacteria to expel, not only antibiotics, but also a wide array of molecules relevant for infection. Hence, they are important players in microbial pathogenesis. On the one hand, efflux pumps can extrude exogenous compounds, including host-produced antimicrobial molecules. Through this extrusion, pathogens can resist antimicrobial agents and evade host defenses. On the other hand, efflux pumps also have a role in the extrusion of endogenous compounds, such as bacterial intercommunication signaling molecules, virulence factors or metabolites. Therefore, efflux pumps are involved in the modulation of bacterial behavior and virulence, as well as in the maintenance of the bacterial homeostasis under different stresses found within the host. This review delves into the multifaceted roles that efflux pumps have, shedding light on their impact on bacterial virulence and their contribution to bacterial infection. These observations suggest that strategies targeting bacterial efflux pumps could both reinvigorate the efficacy of existing antibiotics and modulate the bacterial pathogenicity to the host. Thus, a comprehensive understanding of bacterial efflux pumps can be pivotal for the development of new effective strategies for the management of infectious diseases.

Bacterial persisters in long-term infection: Emergence and fitness in a complex host environment.

Despite intensive antibiotic treatment, Pseudomonas aeruginosa often persists in the airways of cystic fibrosis (CF) patients for decades, and can do so without antibiotic resistance development. Using high-throughput screening assays of bacterial survival after treatment with high concentrations of ciprofloxacin, we have determined the prevalence of persisters in a large patient cohort using 460 longitudinal isolates of P. aeruginosa from 39 CF patients. Isolates were classed as high persister variants (Hip) if they regrew following antibiotic treatment in at least 75% of the experimental replicates. Strain genomic data, isolate phenotyping, and patient treatment records were integrated in a lineage-based analysis of persister formation and clinical impact. In total, 19% of the isolates were classified as Hip and Hip emergence increased over lineage colonization time within 22 Hip+ patients. Most Hip+ lineages produced multiple Hip isolates, but few Hip+ lineages were dominated by Hip. While we observed no strong signal of adaptive genetic convergence within Hip isolates, they generally emerged in parallel or following the development of ciprofloxacin resistance and slowed growth. Transient lineages were majority Hip-, while strains that persisted over a clinically diagnosed 'eradication' period were majority Hip+. Patients received indistinguishable treatment regimens before Hip emergence, but Hip+ patients overall were treated significantly more than Hip- patients, signaling repeated treatment failure. When subjected to in vivo-similar antibiotic dosing, a Hip isolate survived better than a non-Hip in a structured biofilm environment. In sum, the Hip phenotype appears to substantially contribute to long-term establishment of a lineage in the CF lung environment. Our results argue against the existence of a single dominant molecular mechanism underlying bacterial antibiotic persistence. We instead show that many routes, both phenotypic and genetic, are available for persister formation and consequent increases in strain fitness and treatment failure in CF airways.

Structure of Pseudomonas aeruginosa ribosomes from an aminoglycoside-resistant clinical isolate.

Resistance to antibiotics has become a major threat to modern medicine. The ribosome plays a fundamental role in cell vitality by the translation of the genetic code into proteins; hence, it is a major target for clinically useful antibiotics. We report here the cryo-electron microscopy structures of the ribosome of a pathogenic aminoglycoside (AG)-resistant Pseudomonas aeruginosa strain, as well as of a nonresistance strain isolated from a cystic fibrosis patient. The structural studies disclosed defective ribosome complex formation due to a conformational change of rRNA helix H69, an essential intersubunit bridge, and a secondary binding site of the AGs. In addition, a stable conformation of nucleotides A1486 and A1487, pointing into helix h44, is created compared to a non-AG-bound ribosome. We suggest that altering the conformations of ribosomal protein uL6 and rRNA helix H69, which interact with initiation-factor IF2, interferes with proper protein synthesis initiation.

Omics-based tracking of Pseudomonas aeruginosa persistence in "eradicated" cystic fibrosis patients.

Whenever Pseudomonas aeruginosa is cultured from cystic fibrosis (CF) patient airways, the primary goal is eradication by antibiotic therapy. Success is defined by ≥6 months of negative bacterial airway cultures. However, we suspect that P. aeruginosa persists in airways without clinical detection for long periods.Out of 298 P. aeruginosa-infected Copenhagen CF patients, we identified 80 with complete P. aeruginosa monitoring records and measured their maximum P. aeruginosa-free eradication periods (MEP). Isolates from 72 patients were whole-genome sequenced (n=567) and clone typed. Select isolate relatedness was examined through phylogenetic analysis and phenotypic multivariate modelling.69 (86%) patients exhibited eradication in the monitoring period (2002-2018). Sequenced isolates bridged the MEP of 42 patients, and the same clone type persisted over the MEP in 18 (43%) patients. Patients with failed eradication were on average treated more intensively with antibiotics, but this may be linked to their more severe pre-MEP infection trajectories. Of the 42 patients, 26 also had sinus surgery; the majority (n=15) showed MEPs adjacent to surgery, and only five had persisting clone types. Importantly, combined phylogenetic-phenomic evaluation suggests that persisting clone types are a result of re-emergence of the same strain rather than re-infection from the environment, and similar relatedness is exhibited by paired lower and upper airway samples and in transmission cases.In conclusion, nearly half of CF patients with supposed eradication may not truly be cleared of their original bacteria according to omics-based monitoring. This distinct cohort that is persistently infected would probably benefit from tailored antibiotic therapy.

Bacterial Cell Cultures in a Lab-on-a-Disc: A Simple and Versatile Tool for Quantification of Antibiotic Treatment Efficacy.

Pathogenic bacterial biofilms can be life-threatening, greatly decrease patient's quality of life, and are a substantial burden on the healthcare system. Current methods for evaluation of antibacterial treatments in clinics and in vitro systems used in drug development and screening either do not facilitate biofilm formation or are cumbersome to operate, need large reagent volumes, and are costly, limiting their usability. To address these issues, this work presents the development of a robust in vitro cell culture platform compatible with confocal microscopy. The platform shaped as a compact disc facilitates long-term bacterial culture without external pumps and tubing and can be operated for several days without additional liquid handling. As an example, Pseudomonas aeruginosa biofilm is grown from single cells, and it is shown that (1) the platform delivers reproducible and reliable results; (2) growth is dependent on flow rate and growth medium composition; and (3) efficacy of antibiotic treatment depends on the formed biofilm. This platform enables biofilm growth, quantification, and treatment as in a conventional flow setup while decreasing the application barrier of lab-on-chip systems. It provides an easy-to-use, affordable option for end users working with cell culturing in relation to, e.g., diagnostics and drug screening.

Nanograss sensor for selective detection of Pseudomonas aeruginosa by pyocyanin identification in airway samples.

Pyocyanin is a virulence factor solely produced by the pathogen Pseudomonas aeruginosa. Pyocyanin is also a redox active molecule that can be directly detected by electrochemical sensing. A nanograss (NG) based sensor for sensitive quantification of pyocyanin in sputum samples from cystic fibrosis (CF) patients is presented here. The NG sensors were custom made in a cleanroom environment by etching nanograss topography on the electrode surface followed by depositing 200 nm gold. The NG sensors were utilized for amperometric quantification of pyocyanin in spiked hypertonic saline samples, resulting in a linear calibration curve with a R2 value of 0.9901 and a limit of detection of 172 nM. The NG sensors were applied in a small pilot test on five airway samples from five CF patients. The NG sensor was capable of identifying P. aeruginosa in the airway samples in 60 s without any sample pretreatment.

Electrochemical Detection of Pyocyanin as a Biomarker for Pseudomonas aeruginosa: A Focused Review.

Pseudomonas aeruginosa (PA) is a pathogen that is recognized for its advanced antibiotic resistance and its association with serious diseases such as ventilator-associated pneumonia and cystic fibrosis. The ability to rapidly detect the presence of pathogenic bacteria in patient samples is crucial for the immediate eradication of the infection. Pyocyanin is one of PA's virulence factors used to establish infections. Pyocyanin promotes virulence by interfering in numerous cellular functions in host cells due to its redox-activity. Fortunately, the redox-active nature of pyocyanin makes it ideal for detection with simple electrochemical techniques without sample pretreatment or sensor functionalization. The previous decade has seen an increased interest in the electrochemical detection of pyocyanin either as an indicator of the presence of PA in samples or as a tool for quantifying PA virulence. This review provides the first overview of the advances in electrochemical detection of pyocyanin and offers an input regarding the future directions in the field.

Oxidative stress under low oxygen conditions triggers hyperflagellation and motility in the Antarctic bacterium Pseudomonas extremaustralis.

Reactive oxygen species and nitrogen species (ROS and RNS), produced in a wide range of physiological process even under low oxygen availability, are among the main stressors found in the environment. Strategies developed to combat them constitute key features in bacterial adaptability and survival. Pseudomonas extremaustralis is a metabolic versatile and stress resistant Antarctic bacterium, able to grow under different oxygen conditions. The present work explores the effect of oxidative stress under low oxygen conditions in P. extremaustralis, by combining RNA deep sequencing analysis and physiological studies. Cells grown under microaerobiosis exhibited more oxidative damage in macromolecules and lower survival rates than under aerobiosis. RNA-seq analysis showed an up-regulation of genes related with oxidative stress response, flagella, chemotaxis and biofilm formation while chaperones and cytochromes were down-regulated. Microaerobic cultures exposed to H2O2 also displayed a hyper-flagellated phenotype coupled with a high motility behavior. Moreover, cells that were subjected to oxidative stress presented increased biofilm formation. Altogether, our results suggest that a higher motile behavior and augmented capacity to form biofilm structures could work in addition to well-known antioxidant enzymes and non-enzymatic ROS scavenging mechanisms to cope with oxidative stress at low oxygen tensions.

Microaerophilic alkane degradation in Pseudomonas extremaustralis: a transcriptomic and physiological approach.

Diesel fuel is one of the most important sources of hydrocarbon contamination worldwide. Its composition consists of a complex mixture of n-alkanes, branched alkanes and aromatic compounds. Hydrocarbon degradation in Pseudomonas species has been mostly studied under aerobic conditions; however, a dynamic spectrum of oxygen availability can be found in the environment. Pseudomonas extremaustralis, an Antarctic bacterium isolated from a pristine environment, is able to degrade diesel fuel and presents a wide microaerophilic metabolism. In this work RNA-deep sequence experiments were analyzed comparing the expression profile in aerobic and microaerophilic cultures. Interestingly, genes involved in alkane degradation, including alkB, were over-expressed in micro-aerobiosis in absence of hydrocarbon compounds. In minimal media supplemented with diesel fuel, n-alkanes degradation (C13-C19) after 7 days was observed under low oxygen conditions but not in aerobiosis. In-silico analysis of the alkB promoter zone showed a putative binding sequence for the anaerobic global regulator, Anr. Our results indicate that some diesel fuel components can be utilized as sole carbon source under microaerophilic conditions for cell maintenance or slow growth in a Pseudomonas species and this metabolism could represent an adaptive advantage in polluted environments.

Long-term social dynamics drive loss of function in pathogenic bacteria.

Laboratory experiments show that social interactions between bacterial cells can drive evolutionary change at the population level, but significant challenges limit attempts to assess the relevance of these findings to natural populations, where selection pressures are unknown. We have increasingly sophisticated methods for monitoring phenotypic and genotypic dynamics in bacteria causing infectious disease, but in contrast, we lack evidence-based adaptive explanations for those changes. Evolutionary change during infection is often interpreted as host adaptation, but this assumption neglects to consider social dynamics shown to drive evolutionary change in vitro. We provide evidence to show that long-term behavioral dynamics observed in a pathogen are driven by selection to outcompete neighboring conspecific cells through social interactions. We find that Pseudomonas aeruginosa bacteria, causing lung infections in patients with cystic fibrosis, lose cooperative iron acquisition by siderophore production during infection. This loss could be caused by changes in iron availability in the lung, but surprisingly, we find that cells retain the ability to take up siderophores produced by conspecifics, even after they have lost the ability to synthesize siderophores. Only when cooperative producers are lost from the population is the receptor for uptake lost. This finding highlights the potential pitfalls of interpreting loss of function in pathogenic bacterial populations as evidence for trait redundancy in the host environment. More generally, we provide an example of how sequence analysis can be used to generate testable hypotheses about selection driving long-term phenotypic changes of pathogenic bacteria in situ.

Global transcriptional response of solvent-sensitive and solvent-tolerant Pseudomonas putida strains exposed to toluene.

Pseudomonas putida strains are generally recognized as solvent tolerant, exhibiting varied sensitivity to organic solvents. Pan-genome analysis has revealed that 30% of genes belong to the core-genome of Pseudomonas. Accessory and unique genes confer high degree of adaptability and capabilities for the degradation and synthesis of a wide range of chemicals. For the use of these microbes in bioremediation and biocatalysis, it is critical to understand the mechanisms underlying these phenotypic differences. In this study, RNA-seq analysis compared the short- and long-term responses of the toluene-sensitive KT2440 strain and the highly tolerant DOT-T1E strain. The sensitive strain activates a larger number of genes in a higher magnitude than DOT-T1E. This is expected because KT2440 bears one toluene tolerant pump, while DOT-T1E encodes three of these pumps. Both strains activate membrane modifications to reduce toluene membrane permeability. The KT2440 strain activates the TCA cycle to generate energy, while avoiding energy-intensive processes such as flagellar biosynthesis. This suggests that KT2440 responds to toluene by focusing on survival mechanisms. The DOT-T1E strain activates toluene degradation pathways, using toluene as source of energy. Among the unique genes encoded by DOT-T1E is a 70 kb island composed of genes of unknown function induced in response to toluene.

Coexistence and within-host evolution of diversified lineages of hypermutable Pseudomonas aeruginosa in long-term cystic fibrosis infections.

The advent of high-throughput sequencing techniques has made it possible to follow the genomic evolution of pathogenic bacteria by comparing longitudinally collected bacteria sampled from human hosts. Such studies in the context of chronic airway infections by Pseudomonas aeruginosa in cystic fibrosis (CF) patients have indicated high bacterial population diversity. Such diversity may be driven by hypermutability resulting from DNA mismatch repair system (MRS) deficiency, a common trait evolved by P. aeruginosa strains in CF infections. No studies to date have utilized whole-genome sequencing to investigate within-host population diversity or long-term evolution of mutators in CF airways. We sequenced the genomes of 13 and 14 isolates of P. aeruginosa mutator populations from an Argentinian and a Danish CF patient, respectively. Our collection of isolates spanned 6 and 20 years of patient infection history, respectively. We sequenced 11 isolates from a single sample from each patient to allow in-depth analysis of population diversity. Each patient was infected by clonal populations of bacteria that were dominated by mutators. The in vivo mutation rate of the populations was ∼100 SNPs/year-∼40-fold higher than rates in normo-mutable populations. Comparison of the genomes of 11 isolates from the same sample showed extensive within-patient genomic diversification; the populations were composed of different sub-lineages that had coexisted for many years since the initial colonization of the patient. Analysis of the mutations identified genes that underwent convergent evolution across lineages and sub-lineages, suggesting that the genes were targeted by mutation to optimize pathogenic fitness. Parallel evolution was observed in reduction of overall catabolic capacity of the populations. These findings are useful for understanding the evolution of pathogen populations and identifying new targets for control of chronic infections.

Is genotyping of single isolates sufficient for population structure analysis of Pseudomonas aeruginosa in cystic fibrosis airways?

BACKGROUND: The primary cause of morbidity and mortality in cystic fibrosis (CF) patients is lung infection by Pseudomonas aeruginosa. Therefore much work has been done to understand the adaptation and evolution of P. aeruginosa in the CF lung. However, many of these studies have focused on longitudinally collected single isolates, and only few have included cross-sectional analyses of entire P. aeruginosa populations in sputum samples. To date only few studies have used the approach of metagenomic analysis for the purpose of investigating P. aeruginosa populations in CF airways. RESULTS: We analysed five metagenomes together with longitudinally collected single isolates from four recently chronically infected CF patients. With this approach we were able to link the clone type and the majority of SNP profiles of the single isolates to that of the metagenome(s) for each individual patient. CONCLUSION: Based on our analysis we find that when having access to comprehensive collections of longitudinal single isolates it is possible to rediscover the genotypes of the single isolates in the metagenomic samples. This suggests that information gained from genome sequencing of comprehensive collections of single isolates is satisfactory for many investigations of adaptation and evolution of P. aeruginosa to the CF airways.

Genome analysis of a transmissible lineage of pseudomonas aeruginosa reveals pathoadaptive mutations and distinct evolutionary paths of hypermutators.

Genome sequencing of bacterial pathogens has advanced our understanding of their evolution, epidemiology, and response to antibiotic therapy. However, we still have only a limited knowledge of the molecular changes in in vivo evolving bacterial populations in relation to long-term, chronic infections. For example, it remains unclear what genes are mutated to facilitate the establishment of long-term existence in the human host environment, and in which way acquisition of a hypermutator phenotype with enhanced rates of spontaneous mutations influences the evolutionary trajectory of the pathogen. Here we perform a retrospective study of the DK2 clone type of P. aeruginosa isolated from Danish patients suffering from cystic fibrosis (CF), and analyze the genomes of 55 bacterial isolates collected from 21 infected individuals over 38 years. Our phylogenetic analysis of 8,530 mutations in the DK2 genomes shows that the ancestral DK2 clone type spread among CF patients through several independent transmission events. Subsequent to transmission, sub-lineages evolved independently for years in separate hosts, creating a unique possibility to study parallel evolution and identification of genes targeted by mutations to optimize pathogen fitness (pathoadaptive mutations). These genes were related to antibiotic resistance, the cell envelope, or regulatory functions, and we find that the prevalence of pathoadaptive mutations correlates with evolutionary success of co-evolving sub-lineages. The long-term co-existence of both normal and hypermutator populations enabled comparative investigations of the mutation dynamics in homopolymeric sequences in which hypermutators are particularly prone to mutations. We find a positive exponential correlation between the length of the homopolymer and its likelihood to acquire mutations and identify two homopolymer-containing genes preferentially mutated in hypermutators. This homopolymer facilitated differential mutagenesis provides a novel genome-wide perspective on the different evolutionary trajectories of hypermutators, which may help explain their emergence in CF infections.

Evolutionary remodeling of global regulatory networks during long-term bacterial adaptation to human hosts.

The genetic basis of bacterial adaptation to a natural environment has been investigated in a highly successful Pseudomonas aeruginosa lineage (DK2) that evolved within the airways of patients with cystic fibrosis (CF) for more than 35 y. During evolution in the CF airways, the DK2 lineage underwent substantial phenotypic changes, which correlated with temporal fixation of specific mutations in the genes mucA (frame-shift), algT (substitution), rpoN (substitution), lasR (deletion), and rpoD (in-frame deletion), all encoding regulators of large gene networks. To clarify the consequences of these genetic changes, we moved the specific mutations, alone and in combination, to the genome of the reference strain PAO1. The phenotypes of the engineered PAO1 derivatives showed striking similarities with phenotypes observed among the DK2 isolates. The phenotypes observed in the DK2 isolates and PAO1 mutants were the results of individual, additive and epistatic effects of the regulatory mutations. The mutations fixed in the σ factor encoding genes algT, rpoN, and rpoD caused minor changes in σ factor activity, resulting in remodeling of the regulatory networks to facilitate generation of unexpected phenotypes. Our results suggest that adaptation to a highly selective environment, such as the CF airways, is a highly dynamic and complex process, which involves continuous optimization of existing regulatory networks to match the fluctuations in the environment.

Fast Selective Detection of Pyocyanin Using Cyclic Voltammetry.

Pyocyanin is a virulence factor uniquely produced by the pathogen Pseudomonas aeruginosa. The fast and selective detection of pyocyanin in clinical samples can reveal important information about the presence of this microorganism in patients. Electrochemical sensing of the redox-active pyocyanin is a route to directly quantify pyocyanin in real time and in situ in hospitals and clinics. The selective quantification of pyocyanin is, however, limited by other redox-active compounds existing in human fluids and by other metabolites produced by pathogenic bacteria. Here we present a direct selective method to detect pyocyanin in a complex electroactive environment using commercially available electrodes. It is shown that cyclic voltammetry measurements between -1.0 V to 1.0 V reveal a potential detection window of pyocyanin of 0.58-0.82 V that is unaffected by other redox-active interferents. The linear quantification of pyocyanin has an R² value of 0.991 across the clinically relevant concentration range of 2-100 µM. The proposed method was tested on human saliva showing a standard deviation of 2.5% ± 1% (n = 5) from the known added pyocyanin concentration to the samples. This inexpensive procedure is suggested for clinical use in monitoring the presence and state of P. aeruginosa infection in patients.

Differential transcriptional response to antibiotics by Pseudomonas putida†DOT-T1E.

Multi-drug resistant bacteria are a major threat to humanity, especially because the current battery of known antibiotics is not sufficient to combat infections produced by these microbes. Therefore, the study of how current antibiotics act and how bacteria defend themselves against antibiotics is of critical importance. Pseudomonas putida DOT-T1E exhibits an impressive array of RND efflux pumps, which confer this microorganism high resistance to organic solvents and antibiotics that would kill most other microorganisms. We have chosen DOT-T1E as a model microbe to study the microbial responses to a wide battery of antibiotics (chloramphenicol, rifampicin, tetracycline, ciprofloxacin, ampicillin, kanamycin, spectinomycin and gentamicin). Ribonucleic acid sequencing (RNA)-seq analyses revealed that each antibiotic provokes a unique transcriptional response profile in DOT-T1E. While many of the genes identified were related to known antibiotic targets, others were unrelated or encoded hypothetical proteins. These results indicate that our knowledge of antibiotic resistance mechanisms is still partial. We also identified 138 new small RNAs (sRNAs) in DOT-T1E, dramatically adding to the 16 that have been previously described. Importantly, our results reveal that a correlation exists between the expression of messenger RNA and sRNA, indicating that some of these sRNAs are likely involved in fine tuning the expression of antibiotic resistance genes. Taken together, these findings open new frontiers in the fight against multi-drug resistant bacteria and point to the potential use of sRNAs as novel antimicrobial targets.