Background: Recent scientific reports on the use of high dose tigecycline monotherapy as a “drug of last resort” warrant further research into the use of this regimen for the treatment of severe multidrug-resistant, Gram-negative bacterial infections. In the current study, the therapeutic efficacy of tigecycline monotherapy was investigated and compared to meropenem monotherapy in a newly developed rat model of fatal lobar pneumonia–septicemia. Methods: A Klebsiella pneumoniae producing extended-spectrum β-lactamase (ESBL) and an isogenic variant producing K. pneumoniae carbapenemase (KPC) were used in the study. Both strains were tested for their in vitro antibiotic susceptibility and used to induce pneumonia–septicemia in rats, which was characterized using disease progression parameters. Therapy with tigecycline or meropenem was initiated at the moment that rats suffered from progressive infection and was administered 12-hourly over 10 days. The pharmacokinetics of meropenem were determined in infected rats. Results: In rats with ESBL pneumonia–septicemia, the minimum dosage of meropenem achieving survival of all rats was 25 mg/kg/day. However, in rats with KPC pneumonia–septicemia, this meropenem dosage was unsuccessful. In contrast, all rats with KPC pneumonia–septicemia were successfully cured by administration of high-dose tigecycline monotherapy of 25 mg/kg/day . Conclusions: The current study supports recent literature recommending high-dose tigecycline as a last resort regimen for the treatment of severe multidrug-resistant bacterial infections. The use of ESBL- and KPC-producing K. pneumoniae strains in the current rat model of pneumonia–septicemia enables further investigation, helping provide supporting data for follow-up clinical trials in patients suffering from severe multidrug-resistant bacterial respiratory infections. Klebsiella pneumoniae carbapenemase (KPC)—can no longer be effectively treated with β-lactam antibiotics and tend to be susceptible only to “drugs of last resort” [Recent data from the U.S. indicate that Gram-negative bacteria are responsible for more than 30% of hospital-acquired infections . A compl resort” ,3.Tigecycline (an antibiotic belonging to the class of glycylcyclines) is one such drug of last resort, although resistance to tigecycline has been reported . FurtherK. pneumoniae strain or its isogenic KPC-positive variant. The model was used to investigate the therapeutic efficacy of tigecycline as monotherapy as compared to meropenem monotherapy, a potent carbapenem antibiotic that serves as the conventional treatment for multidrug-resistant, Gram-negative bacterial infections [To investigate this issue further, in rats we developed a model of bilateral pneumonia–septicemia, in which we were able to compare the therapeutic efficacy of individual antibiotics at similar conditions of severity, duration of infection, and host defense. This novel pneumonia–septicemia model was based on a previously established rat model of unilateral pneumonia–septicemia (in which one lung was left uninfected), which was intended for antimicrobial pharmacokinetic and pharmacodynamic studies . Furtherfections . K. pneumoniae American Type Culture Collection (ATCC) 43816™ (capsular serotype 2) was used as a parent strain to generate the isogenic ESBL-producing variant K. pneumoniae ESBL EMC2003 (referred to in this study as K. pneumoniae ESBL) and the isogenic KPC-producing variant K. pneumoniae KPC EMC2014 (referred to in this study as K. pneumoniae KPC) via bacterial conjugation with an ESBL-producing or KPC-producing clinical isolate and subsequent selection on antibiotic agar plates. The stability of both plasmid-containing strains was assessed through five consecutive passages in Mueller–Hinton II broth . The virulence of the bacterial strains was maintained by rat lung passage every 12 months. ATCC Quality Control strains K. pneumoniae ATCC 13883™ (WT), K. pneumoniae ATCC 700603™ (ESBL), and K. pneumoniae ATCC BAA1705™ (KPC) were used as reference strains.K. pneumoniae isolates [Due to the complex relationship between antibiotic resistance and virulence factors, it is important to report the genotypic data of bacterial strains used in microbiological studies ,14. Polyisolates . To furtisolates .The antibiotics used were amikacin hydrate, ceftazidime hydrate, ciprofloxacin hydrochloride monohydrate, colistin sulfate salt, gentamicin sulfate, meropenem trihydrate, norfloxacin, tigecycline, tobramycin sulfate salt, trimethoprim, and sulfamethoxazole . For co-trimoxazole, the ratio of trimethoprim/sulfamethoxazole was 1:19. Meropenem was combined with cilastatin sodium  (1:1) when used in vivo to inhibit the function of dehydropeptidase enzyme, which is a known catalyst of meropenem in rats . The anesthetics used were medetomidine hydrochloride , fentanyl citrate , and midazolam hydrochloride , which were administered together intraperitoneally at 0.5 mg/kg, 50 μg/kg, and 5 mg/kg, respectively. Antagonists were atipamezole hydrochloride , naloxone hydrochloride dihydrate (Hameln Pharma Plus GmbH), and flumazenil , which were administered together intraperitoneally at 1.5 mg/kg, 0.7 mg/kg, and 0.3 mg/kg, respectively.®2 system and AST-N344 Gram-Negative Susceptibility Cards .The antimicrobial susceptibility of bacterial strains was assessed by determination of the minimum inhibitory concentration (MIC) using the broth microdilution method following EUCAST guidelines , which rK. pneumoniae for meropenem is 0.125 mg/L and for tigecycline was 1 mg/L, but has recently been increased to 2 mg/L. Samples were centrifuged at 12,500× g for 5 min to avoid drug carry-over, serially 10-fold diluted, and subcultured on Mueller Hinton II agar plates  for colony-forming unit (CFU) counts after 24 h at 37 °C. At 24 h, changes in antibiotic susceptibility were determined via MIC assay using subsamples of 1 mL to prepare bacterial inocula.The bacterial killing capacity of meropenem and tigecycline was investigated using the time–kill kinetics (TKK) assay, as previously described . Two-fol2 exposure when humane endpoints were reached or at termination of experiments. Specified pathogen-free (SPF) male strain RP–AEur–RijHsd albino rats bred at the Laboratory Animals Center of Erasmus University Medical Center Rotterdam (Erasmus MC) were used. Rats  were housed individually in ventilated cages and were given food and water ad libitum. Rats were randomly allocated to experimental groups once they reached the appropriate age and body weight. Group sizes were based on estimates of the hazard ratio. Euthanasia was applied by COAnimals were maintained and handled in accordance with the Guidelines for Accommodation and Care of Animals . All animal procedures were performed in accordance with the Dutch Animal Experimentation Act BWBR0003081), which meets the requirements of the European Union Animal Directive (2010/63/EU). Experimental procedures were approved by the Institutional Animal Care and Use Committee of the Erasmus MC. The current study was designed to abide by the “three Rs” principles of animal research  wherever possible  and was 81, which100) inocula: 2 × 106K. pneumoniae ESBL or 6 × 107K. pneumoniae KPC. An implantable programmable temperature transponder (IPTT)-300  was implanted subcutaneously. Infected rats were monitored 12-hourly for 11 days to assess the disease progression and pre-defined humane endpoints by changes in body temperature and body weight, as well as by external symptoms of disease, including ungroomed appearance, pallor, nose bleeding, lack of reactivity, inactivity, instability, or abnormal breathing of rats. Rat survival was based on rats reaching humane endpoints, at which point euthanasia was applied by CO2 exposure. After dissection, bacteria isolated from lungs and blood were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry  to rule out co-infection, a pre-established exclusion criterium. Acute bilateral pneumonia–septicemia in rats (referred to in this study as ESBL pneumonia–septicemia or KPC pneumonia–septicemia) was established in groups of 11 rats using suspensions of washed bacteria in the logarithmic phase of growth. After intubation and cannulation of the trachea under anesthesia, rats were held in a vertical position and the lungs were inoculated with 60 µL PBS containing the respective absolute lethal dose . The five lung lobes were collected separately in 2 mL PBS each and homogenized using the gentleMACS™ Octo Dissociator . Blood and lung homogenates, either undiluted or in 10-fold dilutions, were subcultured on Mueller–Hinton II agar plates  for CFU counts after 24 h at 37 °C. In blood plasma samples, the alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) levels were assessed for hepatic function, and the creatinine and blood urea nitrogen (BUN) levels were assessed for renal function. Histopathological examination of the infected lungs at 24 h after infection was performed in 2 rats with ESBL pneumonia–septicemia and 2 rats with KPC pneumonia–septicemia. In sacrificed rats, in situ lungs were fixated with 10% formalin under constant pressure to re-expand the lungs. Segments of the left lung were dehydrated in ethanol and toluol, and finally embedded in paraffin. Paraffin-embedded tissues were cut into 4-µm sections, from which one in every 15 cuts was used for hematoxylin–eosin (HE) staining and one for Gram staining.The early phase of infection was characterized at 24 h and 48 h after initiation of infection by viable counts of K. pneumoniae ESBL strain [The therapeutic response to meropenem was determined in rats with ESBL pneumonia–septicemia and rats with KPC pneumonia–septicemia. The response to tigecycline was only investigated in rats with KPC pneumonia–septicemia, as this had already been investigated in a previous unilateral pneumonia–septicemia rat model caused by the identical L strain . TreatmeL strain . Disease1/2) of the meropenem elimination process were calculated using a PKSolver 2.0 one-compartment model of extravascular administration with time delay to account for absorption [ƒT > MIC) and (2) the highest unbound meropenem concentration reached in blood plasma (ƒCmax), were calculated based on unbound meropenem concentrations.Meropenem plasma concentrations were determined in 3 rats with ESBL pneumonia–septicemia. Rats received 3 doses of 25 mg/kg/day meropenem 12-hourly, starting at 24 h after initiation of infection. Blood obtained by tail vein puncture was collected in ethylenediaminetetraacetic acid (EDTA) tubes . Meropenem concentrations were determined by high-performance liquid chromatography-mass spectrometry (HPLC-MS) . The lowsorption . The V asorption . As onlyKaplan–Meier rat survival curves were generated, and statistical differences in rat survival rates were calculated using the log rank test using Prism 5.01 .K. pneumoniae ESBL and K. pneumoniae KPC strains. ESBL and KPC resistance genes were detected via PCR, as expected in the respective strains , and can be considered representative of the tigecycline susceptibility of wild-type K. pneumoniae populations—albeit on the high end of the wild-type distribution [The antimicrobial susceptibility of C values . The isois study . Howeverribution . ®2 data  rats with ESBL pneumonia–septicemia and in 4/6 (66%) rats with KPC pneumonia–septicemia. Changes in liver and kidney function in terms of ALAT, ASAT, creatinine, and BUN were not found. Histopathological examination of the infected lungs at 24 h after initiation of infection revealed distinctive alveolar pathology, as shown for KPC pneumonia–septicemia (Rats were inoculated with bacteria . As a copticemia . SimilarK. pneumoniae strain (p = 0.4662). In both cases, the body temperatures  did not reach the respective meropenem MIC (16 mg/L), resulting in a ƒT > MIC of 0 h.Meropenem plasma concentrations were determined to investigate the steady-state pharmacokinetic profile of meropenem at MED in rats with ESBL pneumonia–septicemia. The plasma concentration–time curve indicated a one-compartment deposition . The corƒT > MIC of 0 h. This treatment resulted in survival of only 1/12 rats with KPC pneumonia–septicemia (p = 0.5728). The failure of meropenem treatment was also reflected in the body temperatures and the body weight values.Rats with KPC pneumonia–septicemia were treated with 25 mg/kg/day meropenem 12-hourly for 10 days, which correlates to a pticemia , which wK. pneumoniae ESBL strain [3K. pneumoniae and an upper limit of 1.9 × 104K. pneumoniae. None of the isolated bacteria showed a change in susceptibility to tigecycline.Next, the therapeutic response to tigecycline treatment of 25 mg/kg/day 12-hourly for 10 days was investigated in rats with KPC pneumonia–septicemia. This dosage was the MED of tigecycline found in a previous unilateral pneumonia–septicemia rat model caused by an identical L strain . The 25 K. pneumoniae. This bacterial species is becoming increasingly resistant to currently available antibiotics [K. pneumoniae, showing that the cumulative fractional response rate of >90% was only achievable with the high-dose tigecycline regimen [Antimicrobial resistance continues to spread worldwide and is often encountered in clinically relevant organisms such as ibiotics ,34, withibiotics . Additioibiotics , and a nibiotics ,38,39,40ibiotics . Furtheribiotics ,42,43. Tibiotics ,45,46,47ibiotics , and by  regimen . Taken tK. pneumoniae with meropenem as a comparator drug. This model is representative of a particularly severe multidrug-resistant infection in human patients [Complicating this debate is the fact that tigecycline is frequently applied in the treatment of infections caused by multidrug-resistant bacteria as part of combination therapy rather than monotherapy . Based opatients ,51,52 anpatients ,10.K. pneumoniae ESBL unilateral pneumonia–septicemia model, this tigecycline dosage was the successful MED, generating 100% survival of rats and normalization of body temperature. In the same study, the efficacy of tigecycline was found to be dose-dependent and was correlated to the ratio of the area under the plasma concentration–time curve (AUC) to the MIC (AUC/MIC). The AUC during a 24-h period (AUC0–24h) for this tigecycline dosage in rats with was similar to the AUC0–24h of high-dose tigecycline treatments in human patients (a loading dose of 300 mg tigecycline followed by 150 mg tigecycline every 12 h) [K. pneumoniae ESBL with a tigecycline MIC of 1 mg/L, the AUC/MIC was 34.29 mg·h/L at a tigecycline dosage of 25 mg/kg/day, which was the MED of tigecycline for survival of all rats. As the tigecycline MIC of K. pneumoniae KPC is 2 mg/L, the extrapolated AUC/MIC ratio for KPC pneumonia–septicemia at a tigecycline dosage of 25 mg/kg/day is calculated to be 17.15 mg·h/L. Despite this MED having a lower AUC/MIC ratio in rats with bilateral KPC pneumonia–septicemia, the tigecycline treatment at 25 mg/kg/day 12-hourly for 10 days remained successful. Unlike the meropenem treatment, this treatment did not result in normalization of body weight, which might be explained by either the persistence of low bacterial numbers in the lungs of the rats at the end of treatment or by potential gastro-intestinal side effects of tigecycline.Treatment with tigecycline at 25 mg/kg/day 12-hourly for 10 days resulted in 100% survival of rats with KPC pneumonia–septicemia and normalization of body temperature. In our previous study using the ry 12 h) . For pneK. pneumoniae ESBL for >5 h, achieving the bactericidal target of carbapenem regimens in human patients, which is a ƒT > MIC of approximately 40% of the dosing interval [Treatment with meropenem in rats with ESBL pneumonia–septicemia showed a clear dose–response relationship. At the MED of 25 mg/kg/day, meropenem resulted in 100% rat survival, stabilization of body weight, body temperature returning to the circadian rhythm, and effected bacterial elimination from the lung tissue at the end of treatment. Meropenem concentrations in rat plasma remained above the MIC of interval . In terminterval .K. pneumoniae ESBL and K. pneumoniae KPC. This difference in susceptibility resulted in different ƒT > MIC values for the two strains, with unbound meropenem concentrations in rat plasma remaining above the MIC of K. pneumoniae ESBL for >5 h, whereas the ƒCmax (<14 mg/L) did not reach the meropenem MIC of K. pneumoniae KPC (16 mg/L), resulting in a ƒT > MIC of 0 h. The respective therapeutic success or failure of this meropenem dose in both infection models can be explained as a consequence of the time-dependent bactericidal activity of meropenem, which was shown in the in vitro TKK data in the present study, as well as in other in vivo pharmacokinetic and pharmacodynamic studies [The MED of meropenem in rats with ESBL pneumonia–septicemia was not successful in rats with KPC pneumonia–septicemia, which is in line with the difference in susceptibility of  studies .K. pneumoniae bacteria, resulting in rapid mortality when left untreated [K. pneumoniae ESBL (1 mg/L) and K. pneumoniae KPC (2 mg/L) are considered resistant, these MICs remain within the wild-type distribution of tigecycline MICs for K. pneumoniae [K. pneumoniae strains. Future research can improve on the generalizability of these results by investigating high-dosage tigecycline regimens in the treatment of infection models caused by other K. pneumoniae strains or different Gram-negative bacterial species. Another interesting avenue for further research with the current pneumonia–septicemia model would be the investigation of high-dose meropenem regimens [The pneumonia–septicemia model established in the present study is representative of a particularly severe human infection with ntreated ,51,52. Feumoniae ,28. In veumoniae , demonstregimens  or high-regimens .K. pneumoniae. Based on the therapeutic tigecycline dose observed in this publication, the limited susceptibility to tigecycline of both strains, and current clinical guidelines, the in vivo data reported in the present study further supports recent literature on the applicability of high-dosage tigecycline as a treatment of “last resort” in patients for severe multidrug-resistant K. pneumoniae infections. The present work involves a clinically relevant animal model for multidrug-resistant K. pneumoniae infections that facilitates the future investigation of antibiotics and novel therapeutic approaches to pneumonia–septicemia.The data obtained in the current study provide evidence that high-dosage tigecycline monotherapy is effective in a rat model representative of human pneumonia–septicemia caused by multidrug-resistant


Borrelia burgdorferi, is the most common tick-borne infectious disease in the United States. We examined gene expression patterns in the blood of individuals with early disseminated LD at the time of diagnosis (acute) and also at approximately 1 month and 6 months following antibiotic treatment. A distinct acute LD profile was observed that was sustained during early convalescence (1 month) but returned to control levels 6 months after treatment. Using a computer learning algorithm, we identified sets of 20 classifier genes that discriminate LD from other bacterial and viral infections. In addition, these novel LD biomarkers are highly accurate in distinguishing patients with acute LD from healthy subjects and in discriminating between individuals with active and resolved infection. This computational approach offers the potential for more accurate diagnosis of early disseminated Lyme disease. It may also allow improved monitoring of treatment efficacy and disease resolution.Lyme disease (LD), caused by  A bioinformatics approach was employed to identify transcriptome alterations in the peripheral blood mononuclear cells of well-characterized human subjects who were diagnosed with early disseminated Lyme disease (LD) based on stringent microbiological and clinical criteria. Transcriptomes were assessed at the time of presentation and also at approximately 1 month  and 6 months  after initiation of an appropriate antibiotic regimen. Comparative transcriptomics identified 335 transcripts, representing 233 unique genes, with significant alterations of at least 2-fold expression in acute- or convalescent-phase blood samples from LD subjects relative to healthy donors. Acute-phase blood samples from LD subjects had the largest number of differentially expressed transcripts . This transcriptional profile, which was dominated by interferon-regulated genes, was sustained during early convalescence. 6 months after antibiotic treatment the transcriptome of LD subjects was indistinguishable from that of healthy controls based on two separate methods of analysis. Return of the LD expression profile to levels found in control subjects was concordant with disease outcome; 82% of subjects with LD experienced at least one symptom at the baseline visit compared to 43% at the early convalescence time point and only a single patient (9%) at the 6-month convalescence time point. Using the random forest machine learning algorithm, we developed an efficient computational framework to identify sets of 20 classifier genes that discriminated LD from other bacterial and viral infections. These novel LD biomarkers not only differentiated subjects with acute disseminated LD from healthy controls with 96% accuracy but also distinguished between subjects with acute and resolved  disease with 97% accuracy. Borrelia burgdorferi, is the most common tick-borne infectious disease in the United States, with an average of >25,000 reported cases per year during the past decade and an estimated annual incidence possibly as high as 300,000 cases per year (25–Lyme disease (LD), a multisystem inflammatory disorder caused by per year . Diagnoser year 24. Treatmr year 257. If lefr year 25–10.B. burgdorferi is the mainstay of laboratory diagnosis of LD  disease with 97% accuracy.B. burgdorferi from blood. The only exception was a study subject who presented with facial palsy, a sign of disseminated infection, and who was seropositive by two-tier serologic testing. Serologic testing by a first-tier whole-cell sonicate enzyme-linked immunosorbent assay (ELISA) was conducted at each sample collection time. All EM subjects except one were either seropositive by ELISA at presentation or seroconverted during the course of the study. B. burgdorferi was cultivated from the blood of 29 subjects with EM , early convalescent LD , and late convalescent LD  with PBMCs from healthy donors (n = 21) using whole-genome oligonucleotide arrays. Principal-component analysis was performed using all samples. x axis) accounts for 37.7% of the variability in the data and, with few exceptions, clearly separates the healthy donor and late convalescent LD blood samples from the acute and early convalescent LD blood samples. No further separation of samples within each of these groups occurs when the second (y axis) or third (z axis) principal component is applied.To characterize the host response to P value of <0.05 and at least a 2-fold change in expression at any time point relative to the healthy donor group. A total of 335 DETs, representing 233 unique genes, were identified  were defined as those having a fied see . The 1-m10.1128/mBio.00047-20.1TABLE S1Table S1, DOCX file, 0.04 MB.List of transcripts differentially expressed in Lyme disease patient PBMCs during acute disease and convalescence. Download Copyright © 2020 Petzke et al.2020Petzke et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the B. burgdorferi infection among subjects. Consistent with the Venn diagrams, the profiles for acute LD and 1-month convalescent LD samples were found to be strikingly similar; however, the intensity of many of the transcripts was slightly reduced in the 1-month convalescent samples. Importantly, expression intensities for the 6-month convalescent LD samples showed greater variability in general, as was observed in healthy donors , wound healing (P = 3.28E–04), blood coagulation (P = 0.001), hemostasis (P = 0.001), and response to stress (P = 0.005). Cluster 3 featured genes involved in fatty acid catabolism , choline kinase activity (P = 2.72E–04), ethanolamine kinase activity (P = 2.72E–04), and intracellular lipid transport (P = 5.32E–04).DETs separated into five gene clusters . Signifitabolism . SignifiB. burgdorferiin vitro  are involved in innate immune cell functions, including pathogen recognition, phagocytosis, neutrophil activation, chemotaxis and cell migration, and inflammation. The most highly induced transcript encodes DEFA1/DEFA1B/DEFA3 , microbicidal proteins of neutrophil granules that effectively kill in vitro  (Table 2P = 4.98E–06), response to wounding (P = 1.21E–05), and cell migration (P = 2.92E–04).Cluster 2 contained 22 genes (26 probe sets) that, with three exceptions, were not significantly changed during acute or early convalescent LD but were significantly induced in the late convalescent LD (6 months) subjects. Cluster 5 consisted of transcripts for 50 genes that were significantly repressed in the majority of acute and early convalescent LD patients relative to healthy subjects. Significant GO terms for these genes included immune system process (http://www.interferome.org/interferome/home.jspx), a database of interferon (IFN)-regulated genes . In the first comparison, the capability of this model to correctly distinguish between subjects with acute LD and healthy controls was determined and the top 20 genes with the highest random forest importance levels were identified . HierarcAnother major limitation of most serologic diagnostic tests is the inability to distinguish between active and prior infection as circulating antibodies are present long after the pathogen is cleared. Application of the random forest algorithm to samples from LD subjects at baseline and at 6-month convalescence resulted in a separate distinct set of 20 classifier genes . HierarcStaphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, or influenza A virus (n = 10) of the random forest algorithm were run to identify the top 20 genes associated with each infectious agent that had the highest importance levels (B. burgdorferi), 95% (S. pneumoniae and S. aureus), and 94% (E. coli). Comparison of the 20-gene sets revealed that acute infections due to E. coli, S. aureus, and S. pneumoniae shared multiple classifiers; the greatest number (eight) of shared classifiers was between E. coli and S. aureus infections (B. burgdorferi (n = 15) and influenza A (n = 6)  from patients with acute infections caused by common bacterial and viral pathogens:  A virus . First, e levels . Using tfections . The gen (n = 6) . RemarkaIn this study, multiple approaches were used to identify a peripheral blood signature that would enable reliable detection of early disseminated LD at a time point when standard serologic testing may be suboptimally sensitive. A 20-gene classifier set that correctly distinguished subjects with acute LD from healthy donors with 96% accuracy, 100% sensitivity, and 90% specificity was identified. A second major limitation of antibody-based tests is the inability to differentiate between acute infection and resolved infection (after antibiotic treatment) due to specific circulating antibodies that may persist for years after the microbe has been eliminated. The identified 20-gene classifier set was able to discriminate acute LD from 6-month convalescent subjects with 97% accuracy, 100% sensitivity, and 90% specificity. Notably, gene expression changes corresponded to reported symptoms. The greatest number of genes with altered expression was present in the acute LD group; symptoms were reported by 82% of all acute LD subjects in this study and by 93% of the 28 subjects whose blood was analyzed for gene expression. In contrast, return of the gene expression profile to that observed in the healthy donors corresponded with resolution of symptoms: only one 6-month LD convalescent subject (9%) reported having any symptom. Thus, the identified classifier set has the potential for serving as a test for disease resolution.B. burgdorferi infection was applied to published microarray data sets for PBMCs collected from patients with acute infections caused by three common bacterial pathogens or by influenza A virus. Importantly, all 20 classifier genes for acute B. burgdorferi infection were completely unique and were not associated with any of these four pathogens. Therefore, the gene classifier sets described here not only demonstrated high sensitivity for acute LD relative to healthy donors and convalescent LD patients, but the 20-gene classifier set for acute LD distinguished B. burgdorferi infection from the other tested bacterial or viral infections with 100% specificity.The algorithm used to generate the classifier gene set for acute B. burgdorferi and influenza A infection were both characterized by an IFN-regulated signature, although the individual genes comprising each set were unique. IFI27  is the classifier gene for influenza A that has the highest random forest importance value. IFI27 has been described in a separate study as a novel single-gene biomarker in patient blood that was able to discriminate, with 88% diagnostic accuracy and 90% specificity, between influenza virus- and bacterium-associated respiratory infections  and/or positive blood culture for B. burgdorferi (74%); the remaining subject had facial palsy, a sign of disseminated LD. The inclusion criteria of Bouquet et al. were less stringent and consisted of a physician-documented EM of >5 cm with at least one concurrent nonspecific symptom . Cultivation of B. burgdorferi from any clinical samples was not reported, and only 43% of LD subjects had MEM. Of the 29 subjects with LD in the Bouquet et al. study, 8 did not seroconvert, and 1 was not tested. In addition to the enrollment criteria, the definition for altered gene expression differed between the studies; Bouquet et al. used a 1.5-fold change cutoff compared to the 2-fold change in the present study. Significantly, in the present study, random forest analysis was employed to build predictive models. Classifier gene sets that could separately distinguish healthy controls from patients with acute disseminated infection, and between such patients and those with resolved infection, were identified.Bouquet and colleagues also examined the transcriptional profile in PBMCs of LD patients with EM before antibiotic treatment, 3 weeks later, and then 6 months after the completion of antibiotic therapy . There iB. burgdorferi is still localized to the skin. Current studies are under way to test the sensitivity of the diagnostic biomarker set using samples from subjects with EM, but without evidence of dissemination. It should also be noted, that the use of published data sets rather than prospectively collected samples  skin lesions and/or the cultivation of B. burgdorferi from blood, as previously described . Healthy donors were recruited from NYMC staff, excluding members of the investigators’ laboratories, and met the following inclusion criteria: no history of LD, no receipt of a Lyme disease vaccine, no evidence of a current infectious disease, not pregnant, and no usage of an immunosuppressive medication. Patients were recruited from the Lyme Disease Diagnostic Center of NYMC during the summer seasons of 2005 to 2006 and 2010 to 2013. Blood samples were collected at the time of diagnosis (acute LD) and at approximately 1 and 6 months after the initiation of a recommended course of antibiotics . Serologescribed .Venous blood was collected directly into BD-Vacutainer CPT tubes . PBMCs were isolated by centrifugation, according to the manufacturer’s protocol, no later than 3 h after blood collection. PBMCs were washed with Hanks’ balanced salt solution without calcium, magnesium, or phenol red , and RNA was isolated immediately thereafter under RNase-free conditions using the PureScript total RNA isolation kit  or the Ambion ToTALLY RNA isolation kit , according to the manufacturers’ instructions. Contaminating DNA was removed using the DNA-free kit . RNA was eluted in 20 μl RNase/DNase-free water and stored at –80°C after the addition of 32 U of RNase inhibitor . RNA integrity was assessed by electrophoresis using an Agilent Bioanalyzer 2100  prior to cDNA synthesis for microarray hybridization. Samples having an RNA integrity number below 6 were excluded from further analysis.Between 5 and 20 ng of total RNA from each PBMC sample was used to generate high-fidelity cDNA using an Ovation RNA amplification system  according to the manufacturer’s protocol. The amplified cDNA was fragmented to 50 to 100 nucleotides, labeled with biotin, and hybridized to the Affymetrix GeneChip.HG-U219 high-density oligonucleotide array . After hybridization, the arrays were stained with streptavidin-phycoerythrin and washed in an Affymetrix fluidics module using standard Affymetrix protocols. The detection and quantitation of target hybridization was performed using a GeneArray Scanner 3000 (Affymetrix). All procedures were performed at the Bionomics Research and Technology Center, Rutgers University, Piscataway, NJ.P value of <0.05 and a fold change of at least 2 relative to the healthy donor group, were subjected to hierarchical clustering and principal-component analysis.Microarray data were analyzed using GeneSpring GX14.9 software . Raw expression values in CEL file format were normalized by robust multiarray analysis (RMA) and quantile normalization, filtered to include only those with intensity values above the 20th percentile, and baseline transformed to the median of all samples. Statistical analysis was performed using one-way analysis of variance with Benjamini-Hochberg multiple testing correction to reduce false positives . Differen = 28) versus healthy donors (n = 21) and acute LD versus 6-month convalescent LD (n = 10). In the first step, the distribution of the gene expression variance across all experimental groups was computed, and genes with variance at or above the 90th percentile were identified. This threshold is a parameter of the framework and can be appropriately set based on the variance distribution in a considered cohort of samples. In the second step, expression data containing the top 10% of variance in each experimental group were subjected to iterations (n = 50) of random forest analysis, a well-established machine learning algorithm  data repository under accession number


A meta-analysis was conducted to evaluate the efficacy and safety of topical treatments (including steroids and antibiotics) for adults with blepharokeratoconjunctivitis (BKC). Q-test and I2 statistic.  The following databases were searched for relevant randomised controlled trials (RCTs): China National Knowledge Infrastructure (CNKI), Web of Science, MEDLINE, PubMed, Embase, and Cochrane Central Register of Controlled Trials database (CENTRAL). Two reviewers selected studies and analyzed the risk of bias independently. The treatments were loteprednol 0.5%/tobramycin 0.3% (LE/T) and dexamethasone 0.1%/tobramycin 0.3% (DM/T). The efficacy outcome measures were change from baseline (CFB) in composite scores of ocular symptoms and signs; the CFB in the signs composite scores for blepharitis, conjunctivitis, and keratitis at each visit; the total ocular adverse event incidence (AEs); and the incidence of intraocular pressure (IOP) increase after treatment. Prepost mean differences (MDs) were compared for continuous outcome variables, and incidences were analyzed for dichotomous data. The pooled effect sizes were analyzed using 95% confidence intervals (CIs) in a fixed-effect model. Heterogeneity was evaluated using the P=0.21). The CFB in signs composite scores for blepharitis , conjunctivitis , and keratitis  was also similar with the two treatments. LE/T was a safer intervention than DM/T, with fewer overall adverse events  and significantly less elevation of intraocular pressure (IOP) .  The CFB to final visit in ocular symptoms and signs of BKC was not statistically different between the two treatments (95% CI, −0.33 to 1.50; MD = 0.58;  DM/T and LE/T are both effective treatments for BKC, but LE/T may be a safer intervention. Blepharokeratoconjunctivitis (BKC) is an inflammatory disorder of the eyelid margin with secondary conjunctivitis and keratopathy . The maiConventional treatments for BKC include nonsteroidal anti-inflammatory drugs (NSAIDs), antibiotics, and corticosteroids and are often prescribed in combination with artificial eye drops and eyelid hygiene, massage, or light therapy , 7. SeveTopical steroids have also proven useful in relieving ocular symptoms, but ineffective in eliminating harmful bacteria from eyelids . ClinicaClinicalTrials.gov) was also searched to look for any ongoing or possibly unpublished relevant trials. The PubMed, MEDLINE, CNKI, Web of Science, Embase, and CENTRAL search strategies are presented in Supplementary Materials.The following databases were searched for RCTs relevant to the review question: PubMed, China National Knowledge Infrastructure (CNKI), Web of Science, Embase, MEDLINE, and CENTRAL. The primary search strategy in PubMed used the relevant medical subject heading (MeSH) terms “blepharokeratoconjunctivitis” and “therapeutics.” The detailed electronic search strategy in PubMed was as follows including Boolean operators: (blepharokeratoconjunctivitis[Title]) OR Blepharitis-Associated Keratoconjunctivitis[Title]) OR BKC[Title]) OR blepharokeratitis[Title]) OR blepharoconjunctivitis[Title]) AND (Therapeutics[Title/Abstract]) OR Therapies[Title/Abstract]) OR Treatments[Title/Abstract]) OR Therapeutic[Title/Abstract]) OR Therapy[Title/Abstract]) OR Treatment[Title/Abstract]). The reference lists of selected articles were also checked. The search was conducted with no restrictions on dates and languages. The United States National Institutes of Health register of clinical trials (Two reviewers (LZ and YJS) selected studies independently using predetermined selection criteria see . The titStudies meeting the following criteria were included: (1) the study included patients who were at least 18 years old; (2) patients had a diagnosis of BKC prior to the treatments; (3) the presence of ocular signs was evidenced by slit-lamp examination; (4) patients had received topical ocular treatments; and (5) the study design was RCT.The patients had previously or concurrently taken oral or topical treatmentsThe data could not be included in statistical analysis due to incomplete reporting .Trials were excluded if:Two reviewers (LZ and YJS) selected the studies based on the criteria outlined in Risk of bias was accessed by two reviewers (LZ and YJS) independently based on the Cochrane Handbook criteria focusing on seven aspects as follows: allocation concealment, blinding of outcome assessors, incomplete outcome data, random sequence generation, participant and personnel blinding, selective reporting, and other sources of bias. Each included trial was critically appraised by the reviewers considering each of the above areas. Any unresolved disagreement between the two reviewers was resolved following review by author ZQP.Q-test and I2 statistic were used to assess heterogeneity, with values of 25%, 50%, and 75% indicating low, medium, and high levels, respectively. At I2 level greater than 50%, meta-analysis was conducted using the fixed-effects model, and at lower levels, the random-effects model. Statistical analyses in this study were conducted using Review Manager Software , and P values lower than 0.05 were considered significant.The primary outcome measure was CFB of the ocular symptoms and signs composite score at the final follow-up visit (day 15 ± 1). The secondary outcome measures were the CFB in composite score of ocular signs of blepharitis, conjunctivitis, and keratitis at each follow-up. The safety outcome measure was overall incidence of ocular AEs (including increased IOP). The weighted mean difference (WMD) of continuous data with 95% CI was determined using the inverse variance method. For dichotomous data, the risk ratio (RR) was calculated with 95% CI and the weighted summary RR was determined using the Mantel–Haenszel method. The The data searches yielded 587 studies  of whichCharacteristics of the included studies are summarized in Overall, risk of bias assessment found that the included studies were at low risk, and therefore, high quality , althougI2) was zero for most outcome measures, and a fixed-effects model was used for these. The RCTs tested effects of the two interventions by comparing the ocular signs and symptoms composite score between the start point and endpoints. The endpoints included the score at the second visit (day 3 ± 1), the third visit (day 7 ± 1), and the fourth visit (day 15 ± 1).The primary outcomes of the meta-analysis are presented in I2 = 0%; MD = 0.29) or the fourth follow-up visits . However, the LE/T group showed a higher improvement from baseline than the DM/T group at the third visit   .2 = 0%, MD = 0.13), the third , or the fourth follow-up visit   .2 = 0%, MD = −0.02), the third visit , or the fourth visit   .Statistically significant heterogeneity was found in the conjunctivitis composite score at the final visit (day 15 ± 1), so a random-effect model was used .2 = 0%, MD = 0.03), third , or fourth visits   .n = 27, 8.6%) were fewer than those in the DM/T group , with 0.52 risk ratio  . More patients treated with DM/T experienced a small (10 mmHg or less) increase in mean IOP than those treated with LE/T  (P=0.08)  . The poo=0.0002) . Similar(P=0.08) .BKC is an ocular disease that presents challenges to ophthalmologists in clinical practice. As its pathogenesis is not completely understood, clinical guidelines have not yet been provided. The purpose of BKC treatment is to modify meibomian gland function and the bacterial flora of the eyelid and conjunctiva and to reduce ocular inflammation . FortunaExisting systematic reviews on the safety and efﬁcacy of topical and systematic treatments for BKC in children found no high-quality evidence , 20. DesThe present analysis reveals no significant differences between LE/T and DM/T as treatments of BKC in terms of ocular signs and symptoms. These results are inconsistent with those reported by Rhee and Mah  in whichIncidences of nonocular treatment-emergent adverse events were comparable between treatments  in the current meta-analysis. The nonocular side-effects were mostly mild to moderate. Only one hypertension subject in the LE/T group and one with a headache in the DM/T group were considered severe. Fewer ocular adverse events occurred in the LE/T group (6/315) than in the DM/T group (14/316). Most ocular AEs were mild to moderate, but IOP was increased, with significantly higher frequency in the DM/T than LE/T group. In addition, changes in IOP found among Chinese patients were higher than those among Americans, according to these two RCTs, in agreement with previous findings . HoweverCorticosteroids may lead to multiple physiological changes involving the deposition of extracellular matrix material, the production of cross-linked actin fibers, inhibition of cell phagocytosis, and possibly to increased resistance to aqueous outflow and an elevation of IOP , 26. DueSome studies have shown that topical calcineurin inhibitors  have positive impacts on chronic blepharokeratoconjunctivitis treatments including longer remission periods than topical corticosteroids , 29, butBlepharitis is a common condition, and its treatment is an important clinical issue . For DemFinally, dry eye secondary to BKC should also be considered. Artificial eye drops are an important treatment option because of the evaporative drying that accompanies meibomian gland disfunction and changes of the ocular surface microenvironment .This review has some limitations. First, the number of clinical trials included is small. Only a few studies have focused on topical treatment of adult BKC. Second, this meta-analysis lacks subgroup analysis on different severities of BKC. Further high-quality randomised controlled trials with large stratified samples including different severity levels are warranted to determine the efficacy and limitations of the antibiotic plus steroid and other treatments for these different clinical categories of BKC.This meta-analysis evaluated the evidence on efficacy and safety of topical antibiotics and steroids for adults with BKC. The results showed that LE/T and DM/T are similarly effective in the treatment of BKC, and that LE/T is associated with better safety.


Pseudomonas aeruginosa as a model, we demonstrate that the self-produced antibiotic pyocyanin (PYO) activates defenses that confer collateral tolerance specifically to structurally similar synthetic clinical antibiotics. Non-PYO-producing opportunistic pathogens, such as members of the Burkholderia cepacia complex, likewise display elevated antibiotic tolerance when cocultured with PYO-producing strains. Furthermore, by widening the population bottleneck that occurs during antibiotic selection and promoting the establishment of a more diverse range of mutant lineages, PYO increases apparent rates of mutation to antibiotic resistance to a degree that can rival clinically relevant hypermutator strains. Together, these results reveal an overlooked mechanism by which opportunistic pathogens that produce natural toxins can dramatically modulate the efficacy of clinical antibiotics and the evolution of antibiotic resistance, both for themselves and other members of clinically relevant polymicrobial communities.Bacterial opportunistic human pathogens frequently exhibit intrinsic antibiotic tolerance and resistance, resulting in infections that can be nearly impossible to eradicate. We asked whether this recalcitrance could be driven by these organisms’ evolutionary history as environmental microbes that engage in chemical warfare. Using  This study shows that pyocyanin, a toxin secreted by the opportunistic pathogen Pseudomonas aeruginosa, induces defense responses that decrease the efficacy of structurally-similar clinical antibiotics and accelerate the evolution of antibiotic resistance, both in the producer and in other members of clinically-relevant polymicrobial communities. The emergence and spread of bacterial resistance to clinical antibiotics is a growing public health concern worldwide . MoreoveConsidering that most of the antibiotics used today are derived from microbially produced molecules, we hypothesized that molecular defenses that originally evolved to protect cells from a natural antibiotic in the environment might also promote tolerance and/or resistance to structurally or mechanistically similar clinical drugs. Indeed, several clinical antibiotic resistance genes are thought to have originated in nonpathogenic soil bacteria, but it has often been assumed that intermediate steps of horizontal gene transfer are necessary in order for such genes to be acquired by human pathogens . In thisPseudomonas aeruginosa, which is notorious for causing chronic lung infections in cystic fibrosis (CF) patients, as well as other types of infections in immunocompromised hosts [P. aeruginosa produces several redox-active, heterocyclic compounds known as phenazines [P. aeruginosa itself [P. aeruginosa is driven by cellular defenses that evolved to mitigate self-induced toxicity. We also tested whether phenazine production by P. aeruginosa could promote antibiotic tolerance in other clinically relevant opportunistic pathogens from the Burkholderia and Stenotrophomonas genera. Finally, we explored the ramifications of phenazine-induced tolerance for the evolution of heritable antibiotic resistance, both in P. aeruginosa and in a clinical isolate from the Burkholderia cepacia complex.One organism that is well suited to testing these hypotheses is the opportunistic pathogen ed hosts . P. aeruenazines . Phenazienazines , which ienazines ; (ii) acenazines ; (iii) penazines ; and  [We started by characterizing the defense mechanisms in (PYO) ,16. To dThe fitness effects of different transposon insertions largely aligned with what is thought to be the primary mode of PYO toxicity, which is the generation of reactive oxygen species (ROS) ,20. For P. aeruginosa with PYO in the presence of the broad-spectrum RND efflux inhibitor phenylalanine-arginine β-naphthylamide (PAβN), cell death was accelerated, confirming that efflux pumps are necessary for minimizing PYO toxicity  efflux system genes , which wtoxicity .P. aeruginosa to tolerate PYO toxicity might overlap more broadly with those that confer tolerance to fluoroquinolones. To address this question, we compared our dataset to a recent Tn-seq study that screened for genes that affect P. aeruginosa survival in the presence of the broad-spectrum fluoroquinolone ciprofloxacin [Mutations in the efflux system repressors identified in our Tn-seq screen are commonly found in clinical isolates that are resistant to synthetic fluoroquinolone antibiotics, as the efflux systems regulated by these repressors efficiently export this class of drugs ,23. We tfloxacin . Across ahpB  and katB  [mexEF-oprN and mexGHI-opmD [phz mutant that does not produce PYO, and Δphz treated with exogenous PYO  , but alsGHI-opmD ,16. We cnous PYO –S3 Figs.ycosides  Fig 2A)ahpB  rather than phenotypic resistance. Importantly, PYO also induced ciprofloxacin tolerance when P. aeruginosa was grown in synthetic cystic fibrosis sputum medium (SCFM)  , suggestP. aeruginosa and PYO-treated Δphz exhibited a shorter lag phase compared to non-PYO-treated Δphz following ciprofloxacin treatment . We verified that the transcription levels of mexGHI-opmD under arabinose induction were comparable to when PYO is present  as a proxy for the likelihood of evolving antibiotic resistance. We calculated this parameter using standard methods for fluctuation test analysis  by enhancing the growth of preexisting “partially resistant” mutants during exposure to the antibiotic; (B) by increasing the proportion of cells that survive and subsequently mutate to resistance while still in the presence of the antibiotic; or (C) by a combination of A and B. To distinguish between these scenarios, we implemented a 2-pronged approach. First, to explore the possibility of scenario A, we isolated and characterized several mutants from the fluctuation test plates containing ciprofloxacin. We regrew the isolates under nonselective conditions both with and without PYO treatment and calculated the percentage of colony-forming units (CFUs) that could subsequently be recovered on ciprofloxacin plates relative to nonselective plates, as a metric for each isolate’s level of resistance. We defined as “partially resistant” those isolates for which only a subset of the population could grow under the antibiotic selection without PYO treatment, as evidenced by lower CFU counts on antibiotic plates compared to nonselective plates. Second, to determine the relative likelihoods of scenario A and scenario B, we examined the fit of our fluctuation test data to different formulations of the theoretical Luria–Delbrück (LD) distribution. Specifically, we compared mathematical models that make different assumptions regarding whether mutants arise prior to or during the antibiotic selection.phz parent strain were below the level of detection on the ciprofloxacin plates even in the presence of PYO, confirming that PYO-induced defenses alone, in the absence of a resistance mutation, were insufficient to enable growth under the selection condition used for the fluctuation tests (5 CFU/mL) and short incubation time (18 hours) used for standard MIC assays can preclude detection of weak growth at a given antibiotic concentration. As a further validation of our assay for detection of partially resistant mutants, we also tested isolates with distinct colony morphologies that were not enriched on the PYO-containing antibiotic plates relative to PYO-free antibiotic plates in the original fluctuation tests. As expected, these mutants were fully resistant to ciprofloxacin at the original selection concentration  and increased occurrence of post-plating mutations resulting from elevated survival on the antibiotic plates (the proposed scenario B).That PYO increases μrt model   and clinical contexts  [P. aeruginosa would have evolved inducible defense mechanisms against PYO toxicity and that production of PYO by P. aeruginosa might therefore also increase tolerance and resistance to clinical antibiotics in these community members. To test this hypothesis, we focused on the genera Burkholderia and Stenotrophomonas, both of which are (i) soil-borne gram-negative opportunistic pathogens that are frequently refractory to clinical antibiotic treatments [P. aeruginosa, e.g., in CF patients [B. cepacia ATCC 25416; a non-CF clinical isolate of Stenotrophomonas, Stenotrophomonas maltophilia ATCC 13637; and several clinical isolates of the 3 most prevalent Burkholderia species found in CF patients [Burkholderia cenocepacia, Burkholderia multivorans, and Burkholderia gladioli  –54. We heatments –57; and patients . Specifipatients : BurkholS. maltophilia, which was sensitive to PYO ppGpp synthetase gene, which is known to affect antibiotic tolerance and resistance [B. multivorans CipR-7 possessed a point mutation in DNA gyrase A (S83R), along with a point mutation in a malto-oligosyltrehalose synthase. Given that DNA gyrase A is the target of ciprofloxacin and that the specific mutated residue is likely homologous to the T83 residue that was mutated in a study of fluoroquinolone-resistant mutants in B. cepacia [Whole-genome sequencing of the fluctuation test isolates revealed that ry genes . B. multsistance . Finally cepacia , it is iB. multivorans mutants we detected primarily arose prior to or during the antibiotic selection. In all cases, the distribution of mutants closely matched the Hamon and Ycart formulation of the theoretical LD distribution, suggesting that the detected mutants arose prior to the antibiotic exposure . In addition, unlike for P. aeruginosa, the mixed LD–Poisson distribution that allows for post-plating mutations was a poorer fit than the Hamon and Ycart model for all PYO-treated B. multivorans samples  and phenazine-1-carboxamide [P. aeruginosa is less tolerant to colistin than the Δphz strain. The mechanism of this synergistic lethality warrants further investigation. Our results highlight that identifying the cellular defenses induced by natural antibiotics, not only in the case of P. aeruginosa and phenazines, but also potentially other opportunistic pathogens and their endogenously produced natural antibiotics, is essential for accurately predicting clinical antibiotic efficacy.Interestingly, our finding that PYO does not increase tolerance to aminoglycosides , S4B Figboxamide , which aboxamide  and consboxamide . Phenaziboxamide ,17,69; tboxamide , but theboxamide . NotablyStaphylococcus aureus highlighted the important role that preexisting genetic diversity in the population can play in shaping the evolution of antibiotic resistance [B. multivorans CipR-7 strain, which had acquired a mutation in the cellular target of ciprofloxacin , yet still required exposure to PYO in order to grow at the ciprofloxacin concentration on which it was originally selected , 42.8 mM NaCl, 1 mM MgSO4, 9.35 mM NH4Cl, and a trace elements solution [4 with the other components was crucial for consistent PYO production by WT P. aeruginosa UCBPP-PA14 in GMM. Luria–Bertani (LB) Miller broth  and BBL cation-adjusted Mueller–Hinton (MH) II broth (BD Biosciences) were prepared according to the manufacturer’s instructions (notably with only a 10 minute autoclave step for MH medium), with the addition of 1.5% Bacto agar (BD Biosciences) to make solid media. SCFM composition was prepared as described previously [2SO4 and no nitrate. In addition, 3.6 μM FeSO4.7H2O and 0.3 mM N-acetyl-glucosamine were added [4.7H2O and N-acetyl-glucosamine solutions were prepared fresh each time or stored at −20°C, respectively, and added to SCFM on the day of use. For SCFM agar, a 2× solution of the medium components was prepared and added to a separately autoclaved 3% molten agar solution, for a final concentration of 1× SCFM and 1.5% agar.Different culture media were used for different experiments as indicated throughout the Methods. Succinate minimal medium (SMM) composition was 40 mM sodium succinate , 50 mM KHsolution . Glucoseeviously , with 1.re added . All comAntibiotics were prepared in concentrated stock solutions (100× or greater) and stored at −20°C. Ciprofloxacin was dissolved in 0.1 M or 20 mM HCl, while levofloxacin, gentamicin, tobramycin, and colistin were dissolved in sterile deionized water. PAβN dihydrochloride  was dissolved in sterile deionized water (50 mg/mL). PYO was synthesized and purified as previously described ,84 and dP. aeruginosa PA14 was used for all experiments unless otherwise noted. For a full list of strains made in this study, see P. aeruginosa PA14 backgrounds: (i) unmarked deletions, used for Tn-seq validation experiments; (ii) fluorescent strains for time-lapse microscopy experiments; and (iii) strains overexpressing one of the following 3 systems: mexGHI-opmD, ahpB, and katB. Established protocols were used for all these procedures [In this study, we use PA14 as an abbreviation for UCBPP-PA14. ocedures .P. aeruginosa PA14 genomic DNA (gDNA) and cleaned up using the Monarch PCR Purification kit  were used for Gibson assembly together with pMQ30 cut with SacI and HindIII. The assembled construct was then transformed into E. coli DH10B, with transformants being selected in LB with 20 μg/mL gentamicin. All correctly assembled plasmids were identified by colony PCR and verified by Sanger sequencing . Next, for the insertion of the constructs into P. aeruginosa PA14 genome, triparental conjugation was performed following Choi and Schweizer [P. aeruginosa PA14 Δphz background  with 100 μg/mL gentamicin following Choi and Schweizer [Briefly, for unmarked deletions, approximately 1-kb fragments immediately upstream and downstream of the target locus were cloned using Gibson assembly into the pMQ30 suicide vector ,87. Fragchweizer . All unms strain ), allowichweizer . FinallyrpsG gene were inserted in the attTn7 site of P. aeruginosa PA14 Δphz chromosome by tetraparental conjugation, followed with selection on VBMM with 100 μg/mL gentamicin [Fluorescent strains used in time-lapse microscopy were made using previously published plasmids ,89. Consntamicin .ara [mexGHI-opmD, ahpB, and katB) were all amplified by PCR. Next, using Gibson assembly, the targets were cloned downstream of Para in the pUC18T-miniTn7T-GmR vector, resulting in the 3 different overexpression constructs: Para:mexGHI-opmD, Para:ahpB, and Para:katB. The final constructs were introduced into the attTn7 of the P. aeruginosa PA14 Δphz background strain by tetraparental conjugation [Finally, overexpressing strains were made as previously described . The preara ) and thejugation .P. aeruginosa PA14 transposon library previously prepared [500) of 0.05 in 50 mL of SMM, and grown aerobically under shaking conditions (250 rpm) at 37°C for approximately 4 to 5 generations to an OD500 of 0.8–1. These growing conditions were used for all the stages of the experiment. After growth in SMM, each aliquot was considered an independent replicate. Cells from each replicate were pelleted, washed, and resuspended  in minimal phosphate buffer  with and without 100 μM PYO. Cells were then incubated for 26 hours under shaking conditions at 37°C. Therefore, the experiment consisted of 4 different samples that were later sequenced: (i) “R1 No PYO”; (ii) “R1 + PYO”; (iii) “R2 No PYO”; and (iv) “R2 + PYO.” After the incubation, cultures from all treatments were pelleted, washed again to remove PYO, and resuspended in fresh SMM. Immediately, an aliquot of each sample was diluted to a starting OD500 of approximately 0.05 in 25 mL SMM, followed by outgrowth for approximately 4 to 5 generations to an OD500 of 0.8–1. After outgrowth, 2.5 mL of each sample was pelleted and stored at −80°C.The Tn-seq experiment was performed following the design presented in prepared  were thaP. aeruginosa UCBPP-PA14 genome sequence using Bowtie 2 [2 transformed. Finally, the p-values for both replicates were combined using the Fisher combined probability test as done in Basta and colleagues [2 ratios of the 2 replicates are also shown. For the log2 ratios and p-values for all PA14 genes, see 2 ratios (fitness) for the selected genes were plotted using the geom_tile function from the ggplot2 package in R [gDNA was extracted from the pelleted samples using the DNeasy Blood & Tissue kit . All the steps for sequencing library preparation followed exactly the protocol used by Basta and colleagues , includiBowtie 2  and wereBowtie 2 , with noBowtie 2 ,92. UsinBowtie 2 ,92. Nextlleagues , and theage in R ,94.P. aeruginosa PAO1 strain background . Orthologs were determined using the “pseudomonas.com” database [To compare the results of this Tn-seq analysis with a previously published study  analyzinckground , the datdatabase .phzΔackAΔpta, ΔphzΔlptA, ΔphzΔmexS, and ΔphzΔdctBD) to the survival of the Δphz strain upon exposure to PYO. The experimental design was very similar to the one used in for the Tn-seq, with minor adaptations. An overnight culture (5 mL) of each strain was grown in SMM (40 mM succinate) from LB plates. Cells were washed and resuspended at an OD500 of 0.1 (or 0.25 for ΔphzΔdctBD) in the same medium to start the new cultures (5 mL), which were grown to OD500 approximately 0.8–1, pelleted, washed, and resuspended in the same minimal medium without succinate (no carbon source) at OD500 of 1. For each strain, the culture was split across 8 to 12 wells (150 μL cultures) in a 96-well plate, with 100 μM PYO added to half of the cultures. Moreover, 70 μL of mineral oil was added to the top of the wells to prevent evaporation. PI at 5 μM was also added to the cultures to monitor cell death [phz parent strain ; these “fitness” values were log2 transformed for plotting.To validate the Tn-seq results , experimll death . The plaP. aeruginosa for tolerance against PYO toxicity. From a Δphz overnight culture pre-grown in SMM (20 mM succinate), a new 7-mL culture was started in fresh SMM at an OD500 of 0.05 and was incubated for around 10 hours (enough to reach stationary phase). Cells were then pelleted, washed, and resuspended in MPB at an OD500 of 1 (10 mL of culture was prepared). The culture was then split into 4 different treatments: (i) no PYO, no PAβN; (ii) 100 μM PYO, no PAβN; (iii) no PYO, with PAβN (50 μg/mL); and (iv) 100 μM PYO, with PAβN. Each of the treatments were split across 12 wells containing 150 μL of culture + 70 μL of mineral oil in a 96-well plate. The plate was incubated at 37°C under shaking conditions using a BioTek Synergy 4 plate reader. Samples were serially diluted in MPB and plated for CFUs on LB agar after 12, 24, and 48 hours. Survival for treatments containing PYO was calculated based on the CFUs counted for the negative control without PYO  in GMM (10 mM glucose) per treatment. Three treatments were prepared: WT, Δphz (no PYO), and Δphz + 100 μM PYO, with 4 independent biological replicates for each. Each of the 4 individual cultures (replicates) were incubated for around 20 hours, reaching stationary phase, in 7-mL cultures (18 × 150 mm glass tubes). Each individual culture (replicate) was then split into a negative control (no antibiotic) or antibiotic treatment . After addition of the antibiotic from concentrated stocks, cultures were incubated for 4 hours, serially diluted in MPB and then plated for CFUs on LB agar. Unless stated otherwise, cells were not washed before plating. We observed that washing the cells did not make any difference in the outcome of the experiments. In addition, washing was not feasible for the tolerance assays using smaller volumes . The only 2 experiments where washing was performed are described below (“Tolerance assay with PAβN” and “Tolerance assay to measure the lag in CFUs appearance”). In these cases, cells were washed because (i) the ciprofloxacin concentrations were higher (10 μg/mL) and more likely to affect P. aeruginosa cells on the plate; and (ii) for the case of the PAβN experiment, we wanted to avoid having cells be in contact with the inhibitor while growing on the plate. Antibiotics were used at the concentrations mentioned in figure legends. Plates were incubated at RT, and CFUs were counted after 36 to 48 hours. Plates were always checked again after 7 days to count any late-arising CFUs. Importantly, for all tolerance experiments performed in this study , each experiment was repeated at least twice on different days, with similar results.For most antibiotic tolerance assays , the experimental design shown in P. aeruginosa mutants with partial resistance to ciprofloxacin  were grown with and without 20 mM arabinose for induction of the controlled systems and then exposed to ciprofloxacin the same way described above. To rule out any nonspecific interference of the inducer, negative controls with and without 20 mM arabinose using the parent Δphz strain (without the insertions in the attTn7 site) were also done. Adding arabinose to the Δphz strain did not impact tolerance levels . Next, cells were pelleted, washed, and resuspended into 2 new cultures, one with PYO (100 μM) and one without PYO, at an OD500 of 0.05 in GMM . Cultures were grown until OD500 = 0.5 (around 5 to 6 hours). Cells were then washed and resuspended in the same medium at an OD500 of 0.5, but without the nitrogen source . PYO was re-added after washes to the culture that was pre-grown with PYO. The cultures, one with and one without PYO, were then split into different treatments: negative control (no antibiotic), ciprofloxacin (0.5 μg/mL), levofloxacin (1 μg/mL), gentamicin (16 μg/mL), and tobramycin (4 μg/mL). Then, they were all transferred to wells in a 96-well plate . Cultures within wells contained 150 μL with an additional 70 μL of mineral oil on top to prevent evaporation. The depletion of nitrogen prevented growth in the negative control, which limited overestimation of the antibiotic killing effect . The plates were incubated for 4 hours at 37°C under shaking conditions (175 rpm) using a benchtop incubator . The 96-well plate was kept inside an airtight plastic container with several wet paper towels to maintain high humidity attached to the shaker. After incubation, cells were serially diluted and plated on LB agar for CFU counting  and the Δphz background control  was divided by the area covered by Δphz cells to obtain the relative “growth area ratios”. This was done for first and last time points. Three experiments were performed, with different fluorescent protein/strain combinations: (i) WT::mApple/Δphz::GFP  of the first and last time points were segmented using the “Auto Threshold” function and “Default” setting. The area of the segmented cells was then recorded using the “Analyze Particles” function in Fiji . This al n = 19, ; and  Δphz + 1 μM PYO; (iv) Δphz + 10 μM PYO; (v) Δphz + 100 μM PYO; and (vi) Δphz + 200 μM PYO. Cultures of WT or Δphz were grown overnight in GMM (20 mM glucose), then cells were washed and resuspended at an OD500 of 0.05 (3 replicates) in fresh GMM (5 mL in culture tubes). Different concentrations of PYO were added to Δphz cultures as mentioned, and all cultures were incubated for around 8.5 hours (until early stationary phase). This was enough time for WT to make PYO  WT PA14; (ii) Δat OD691 ). After phz, Δphz Para:mexGHI-opmD, Δphz Para:ahpB, and Δphz Para:katB) were incubated with and without 20 mM arabinose for artificial induction of the constructs. Cultures of the 4 strains were grown overnight in GMM (20 mM glucose), then cells were washed and resuspended at an OD500 of 0.05 (3 replicates) in the same medium (5 mL in culture tubes), with and without 20 mM arabinose . Cultures were incubated for around 8.5 hours, then pelleted, immediately frozen using liquid nitrogen, and stored at −80°C.Eight different treatments were prepared for this qRT-PCR experiment, in which each of the 4 tested strains  and the log2 fold change in expression are shown in phz samples without added PYO , and B. gladioli AU42104 were used in the growth experiments shown in 500 of 0.05 in the same medium. Cultures were then split, different concentrations of PYO were added , and moved to a 96-well plate . Cultures within wells contained 150 μL with an additional 70 μL of mineral oil on top to prevent evaporation. The plates were incubated at 37°C under shaking conditions using a BioTek Synergy 4 plate reader with OD500 measurements every 15 minutes for 24 hours to measure growth. Assays for tolerance to ciprofloxacin with or without exogenous PYO were performed for S. maltophilia (sensitive to PYO) and for 4 Burkholderia strains : B. cepacia, B. cenocepacia, B. multivorans 1, and B. multivorans AU18358 (B. multivorans 4). The experiments followed exactly what was done for P. aeruginosa .ruginosa , except vorans 1  and follP. aeruginosa impacts tolerance to ciprofloxacin in other species, coculture experiments were performed using membrane-separated 12-well tissue plate cultures containing 0.1 μm pore PET membranes (VWR Cat. No. 10769–226). Briefly, overnight cultures of the P. aeruginosa strain (WT/Δphz PA14 or PA 76–11) and the respective other species tested  were prepared in GMM (20 mM glucose) + AA. Cells were pelleted, washed, and resuspended to different ODs as follows: (i) for any P. aeruginosa–Burkholderia assay, P. aeruginosa starting OD500 = 0.05 and Burkholderia starting OD500 = 0.025; and (ii) for the P. aeruginosa–S. maltophilia assay, P. aeruginosa starting OD500 = 0.01 and S. maltophilia starting OD500 = 0.1. P. aeruginosa was cultured in the bottom part of the well (600 μL), while the other species was cultured in the upper part of the well (100 μL), as shown in B. cepacia and S. maltophilia were cultured either with WT or Δphz P. aeruginosa PA14 (with and without 100 μM PYO exogenously added).To test how PYO produced by B. cenocepacia and B. multivorans 1 were cultured either with PA 76–11 (a P. aeruginosa strain isolated from CF sputum that produced 50 to 100 μM PYO in these assays) or alone in the presence or absence of 100 μM PYO. For cases where Burkholderia was grown alone, the strain tested was grown in both the bottom and top parts of the membrane-separated wells. In all experiments, cocultures were grown for around 20 hours at 37°C under shaking conditions (175 rpm) using a benchtop incubator, followed by addition of ciprofloxacin  and incubation for 4 hours. The membrane-separated plates were kept inside an airtight plastic container with several wet paper towels to maintain high humidity attached to the shaker. For every coculture combination in the membrane-separated plate, 3 wells were used as a negative control (no antibiotic), and 3 wells were used for ciprofloxacin treatment; each well was considered an independent replicate. After incubation with ciprofloxacin, cells were serially diluted in MPB and plated for CFUs on LB. In most cases, only Burkholderia cells were plated  to test if PYO produced by PA14 WT increases tolerance in Burkholderia in this medium  in each well.The antibiotic concentrations used for selecting de novo antibiotic-resistant mutants in the fluctuation tests were chosen based on the results of a modified agar dilution MIC assay. Overnight cultures were grown for each strain in GMM (with 10 mM glucose) or GMM (10 mM glucose) + AA, respectively, then diluted to an ODe EUCAST  resistane EUCAST . EUCAST 5 CFUs/mL in a 2-fold dilution series of ciprofloxacin in MH broth, with or without 100 μM PYO. The dilution series were set up in a final volume of 100 μL per well in 96-well microtiter plates, with appropriate no-antibiotic and cell-free controls. Three biological replicates (independent overnight cultures or cell suspensions) were prepared for each tested strain. Following inoculation, the microtiter plates were sealed with a plastic film to prevent evaporation and incubated in a single layer at 37°C, without shaking. The wells were assessed for growth (turbidity) visible to the naked eye after 18 hours of incubation.Ciprofloxacin MICs for parent strains and isolated mutants from the fluctuation tests were determined according to standard clinical methods for broth microdilution assays . In brieP. aeruginosa are from experiments using the Δphz strain. We also performed fluctuation tests using the P. aeruginosa PA14 WT strain and performed phenotypic and genotypic characterization of partially resistant mutants detected in those experiments (see below); however, the effect of PYO on apparent mutation rates in WT was difficult to interpret due to inconsistent PYO production in the 96-well plates. For cultures that were grown with PYO (or arabinose in the case of strains with arabinose-inducible constructs), the PYO (or 20 mM arabinose) was added to the medium before inoculation. The cultures were inoculated with a 10−6 dilution of a single overnight culture  that had first been diluted to a standard OD500 of 1.0, corresponding to an initial cell density of approximately 2,000 to 2,500 CFUs/mL (400 to 500 cells/culture). Each treatment condition consisted of 44 such parallel cultures.For all tested strains and conditions, fluctuation tests were performed by inoculating 200-μL cultures in parallel in a flat-bottomed 96-well plate. All reported fluctuation test data for 500 of 0.4 to 0.7 for P. aeruginosa in GMM with 10 mM glucose or 0.9 to 1.2 for P. aeruginosa in SCFM or B. multivorans in GMM + AA). For plating during stationary phase, the cultures were incubated for 24 hours. The cultures were then plated by spotting 40 to 50 μL per culture into single wells of 24-well plates ; each well contained 1 mL of MH agar or SFCM agar plus an antibiotic, with or without 100 μM PYO (or 20 mM arabinose for strains with arabinose-inducible constructs). In the case of B. multivorans cultures that were spotted onto antibiotic plates containing 100 μM PYO, the cultures were first diluted 1:10 (if not pretreated with 100 μM PYO) or 1:100 (if pretreated with 100 μM PYO).The 96-well plates were placed inside an airtight plastic container with several wet paper towels to maintain high humidity, then incubated at 37°C with shaking at 250 rpm. For plating during log phase, the cultures were incubated until reaching approximately half-maximal density  or 40 to 48 hours for B. multivorans. Subsequently, colonies were counted under a stereoscope at the highest magnification for which the field of view still encompassed an entire well; occasionally, a well contained too many colonies to count , in whim, the expected number of mutations per culture. This is a maximum likelihood-based method for inferring mutation rates from fluctuation test colony counts, based on the classic LD distribution with a correction to account for the effects of partial plating  [m when partial plating is involved [app (apparent mutation rate per generation) from m, we divided m by the total number of cells per parallel culture [Mutation rates reported in the figures were calculated using the function newton.LD.plating from the R package rSalvador  to estim volume) . We chosinvolved ,100. To  culture , as estim and w (relative fitness of mutants compared to the parent strain in the nonselective pre-plating liquid growth medium) using the function GF.est from the R script available at http://ljk.imag.fr/membres/Bernard.Ycart/LD/ ; then, we used the function pLD from the same script to generate the theoretical distribution. For the mixed LD–Poisson and basic LD models, we wrote and used an R translation of the MATLAB code written by Lang and colleagues [https://github.com/AWMurrayLab/FluctuationTest_GregLang. The basic LD model used by Lang and colleagues [m to infer the mutation rate, not when comparing the fits of different models to the empirical cumulative distribution of the raw colony counts.To compare the fits of different formulations of the LD distribution to our data, we generated theoretical cumulative distributions using the parameter values estimated for our data. Specifically, for the Hamon and Ycart version of the LD model , we estilleagues ; the orilleagues  is equivapp from the Hamon and Ycart model [Plots of the empirical cumulative distributions of our data against the theoretical models showed that the Hamon and Ycart model was a visually good fit in all cases . Putative mutants that were not enriched by PYO were restreaked on ciprofloxacin agar plates without PYO. Frozen stocks of each restreaked, visually pure isolate were prepared by inoculating cultures with single colonies in 5 mL of liquid LB, incubating to stationary phase, mixing 1:1 with 50% glycerol, and storing at −80°C.We defined putative ciprofloxacin-resistant mutants as “enriched” by PYO in the fluctuation tests if colonies with a given morphology were at least 2 times more numerous on the PYO-containing ciprofloxacin plate than the respective non-PYO-containing ciprofloxacin plate derived from the same 200-μL culture. These putative mutants could be either from the PYO pretreated or non-PYO pretreated branches of the fluctuation test. Putative mutants that were seemingly enriched by PYO were restreaked for purity on PYO-containing agar plates at the same ciprofloxacin concentration on which they were selected in the fluctuation test (0.5 μg/mL for P. aeruginosa) or GMM + AA (for B. multivorans) were inoculated directly from the frozen stock to minimize the number of generations in which secondary mutations could be acquired. The cultures were grown to stationary phase overnight, then subcultured to an OD500 of 0.05 in 5 mL of fresh GMM (for P. aeruginosa) or GMM + AA (for B. multivorans), with or without 100 μM PYO. The new cultures were grown to mid-log phase, then serially diluted in GMM or GMM + AA (+/− 100 μM PYO as appropriate) and plated for CFUs (10 μL per dilution step) on (1) plain MH agar; (2) MH agar + ciprofloxacin; and (3) MH agar + ciprofloxacin + 100 μM PYO. The lowest plated dilution was 10−1, making the limit of detection approximately 1,000 CFUs/mL.The levels of ciprofloxacin resistance of selected isolates, as well as the parent strains, were assessed using a CFU recovery assay as follows. For each isolate, four 5 mL cultures in GMM  with default parameters [P. aeruginosa mutants were derived from the Δphz strain except for CipR-33 and CipR-40, which were derived from the WT strain. In the case of B. multivorans, several dozen putative mutations were identified that were common to all 3 sequenced putative mutants. We assumed that these represented assembly errors in the parent strain genome scaffold, but even if they were genuine mutations, these would not account for the phenotypic differences between the isolates; therefore, B. multivorans isolates were identified by retrieving the surrounding 200 bp from the parent genome scaffold and using the nucleotide BLAST tool on the MicroScope platform [B. multivorans ATCC 17616 genome.gDNA was isolated from selected putative mutants and the parent strains using the DNeasy Blood & Tissue kit (Qiagen). Library preparation and 2 × 150 bp paired-end Illumina sequencing was performed by the Microbial Genome Sequencing Center , with a minimum of 300-Mb sequencing output per sample (approximately 50 × coverage). Forward and reverse sequencing reads were concatenated into a single file for each isolate, and quality control was performed using Trimmomatic (version 0.39)  with therameters . This scplatform  to find P. aeruginosa Δphz and GMM + AA for B. multivorans 1. The cultures were prepared and incubated in the same manner as the fluctuation tests, except that 5 μM PI was added at the beginning of the experiment, and measurements for OD500 and PI fluorescence  were taken periodically using a Spark 10M plate reader . Importantly, PI stock concentration  was prepared in DMSO, and the final concentration of DMSO in the cultures did not exceed 0.1%. In addition, black 96-well plates with clear bottoms were used to minimize the effects of adjacent wells on fluorescence readings.To verify that PYO did not increase the population turnover rate  in our log-phase fluctuation tests prior to the antibiotic selection step, we performed growth curves in the presence of different concentrations of PYO, with the addition of PI as a fluorescent marker for cell death. The use of PI as a marker for PYO-induced cell death has previously been validated under similar conditions . The grot tests or 1-way analysis of variance (ANOVA) with post hoc Tukey honestly significant difference (HSD) test for multiple comparisons were used for tolerance assay data. The likelihood ratio test as implemented by the rSalvador function LRT.LD.plating was used to compare mutation rates, alongside the alternative criterion of nonoverlapping 84% confidence intervals as a proxy for the p < 0.05 threshold for statistical significance. The Mann–Whitney U test was used to compare the distributions of mutant frequencies. Welch unpaired t tests were used for comparisons of CFU recovery on ciprofloxacin plates under different PYO treatments. Benjamini–Hochberg corrections were used in all cases to control false discovery rates, except where Tukey HSD test was performed. For all antibiotic tolerance assays measured by CFUs, survival data were log10 transformed before statistical analyses.All statistical analyses were performed using R . Welch uS1 FigA) Normalized cDNA levels measured by qRT-PCR. cDNA measurements were normalized by levels of the housekeeping gene oprI (see (B) Fold change in expression upon PYO treatment, relative to the measurements in untreated Δphz. ahpB: alkyl hydroperoxide reductase B; katB: catalase B. Black horizontal lines mark the mean value for independent biological cultures (n = 3). The data underlying this figure can be found in Table G in (oprI see . (B) Fol(TIF)Click here for additional data file.S2 FigP. aeruginosa are shown. cDNA levels for each gene were measured by qRT-PCR during early stationary phase and normalized by the levels of the housekeeping gene oprI (see n = 3). The data underlying this figure can be found in Table H in The normalized cDNA levels for genes within operons coding for the 11 main RND efflux systems in oprI see . This da(TIF)Click here for additional data file.S3 FigP. aeruginosa are shown. These plots are derived from the normalized cDNA dataset shown in phz were used as the basis for calculation of changes of expression (shown as log2 fold change). Black horizontal lines mark the mean value for independent biological cultures (n = 3). The data underlying this figure can be found in Table H in The PYO-induced changes in expression for genes within operons coding for the 11 main RND efflux systems in (TIF)Click here for additional data file.S4 Fig(A) Experimental design for survival assay to measure tolerance to clinical antibiotics. In conditions with exogenous PYO, the PYO was added when cultures were inoculated. PYO itself was not lethal under these experimental conditions . (B) Tolerance levels of Δphz cells harvested in log phase, following growth in GMM in the presence or absence of PYO (100 μM), to different aminoglycosides and fluoroquinolones . Data points represent replicates . Stationary-phase tolerance experiments in GMM are not shown for the aminoglycosides (GEN and TOB), as treatment with TOB in stationary phase under our conditions at this clinically relevant concentration [(C) Representative data showing CFUs counted for Δphz grown for 20 hours . These are the CFUs for the negative control (no antibiotic) for the experiment performed with CIP in (D) Experimental design for time-lapse microscopy experiments, in which cells were grown on agarose pads after exposure to CIP (10 μg/mL) in GMM. The strain/fluorescent protein examples shown  are the ones used in the images of (E) Quantification of microscopy data as done in (F) Experiment quantifying how PYO affects lag for CFUs appearing after treatment with CIP , and we observed an increased lag in the absence of PYO  (n = 4). (G) Effects of the efflux inhibitor PAβN on tolerance levels to CIP of Δphz cells grown in GMM in the presence or absence of PYO (100 μM). Cultures were treated with low (left) and high (right) CIP concentrations. Experiments for all the conditions were done in parallel (see n = 4). Statistics: B, C, E, G—Welch unpaired t tests. F—1-way ANOVA with Tukey HSD multiple comparison test, with asterisks showing the statistical significance of comparisons with the Δphz (no PYO) . Black horizontal lines mark the mean value for independent cultures . The data underlying this figure can be found in Table I in ntration  did not (TIF)Click here for additional data file.S5 Fig(A) Normalized cDNA levels measured by qRT-PCR. cDNA levels were normalized by the housekeeping gene oprI. (B) Fold change in expression upon arabinose induction. This dataset can be compared to the PYO-mediated induction of the same genes as shown in phz (white background); (2) Δphz Para:mexGHI-opmD (magenta background); (3) Δphz Para:ahpB (green background); and (4) Δphz Para:katB (blue background). +/− represent addition or not of 20 mM arabinose to the cultures for the artificial induction of expression. For additional experimental details and strain information, see n = 3). The data underlying this figure can be found in Table J in (TIF)Click here for additional data file.S6 Figphz strain and the 3 arabinose-inducible strains  grown in the presence or absence of 20 mM arabinose, without exposure to PYO. The tolerance experiments were performed for cultures in both log phase  and stationary phase  in GMM. In B, the experiment for mexGHI-opmD is the same as in t tests . Black horizontal lines mark the mean value for independent cultures. The data underlying this figure can be found in Table K in Survival relative to the no-antibiotic control is shown for the parent Δ(TIF)Click here for additional data file.S7 Fig(A) Apparent mutation rates of stationary-phase Δphz grown in liquid GMM and plated onto MH agar containing CIP (0.5 μg/mL), with or without pre- and/or co-exposure to 100 μM PYO relative to the antibiotic selection step (n = 8). (B-C) Apparent mutation rates of log-phase cells grown in GMM and plated onto MH agar containing CIP (0.5 μg/mL), with or without pre- and/or co-exposure to 20 mM arabinose relative to the antibiotic selection step. Data are shown for Δphz Para:mexGHI-opmD alone  and in comparison to Δphz  or Δphz Para:katB and Δphz Para:ahpB . In all panels, each data point represents 44 parallel cultures from a single biological replicate, and the vertical lines represent 84% confidence intervals, for which lack of overlap corresponds to statistical significance at the p < 0.05 level [05 level . The PYO(TIF)Click here for additional data file.S8 Fig(A) The percentage of CFUs recovered on CIP (0.5 μg/mL) either with or without 100 μM PYO in the agar, for log-phase cultures of representative resistant mutants of P.a. that were not enriched by exposure to PYO in the fluctuation tests. The mutants were pre-grown with or without 100 μM PYO in GMM before plating. On the x-axis, “pre” denotes the presence of PYO in the liquid cultures and “co” denotes the presence of PYO in the agar plates. Percentage recovery was calculated relative to total CFUs counted on nonselective plates (n = 4). (B) Tolerance to CIP (1 μg/mL) of partially resistant mutants grown in GMM to stationary phase with or without 100 μM PYO (n = 4). Experiments were performed as shown in (C) The percentage of CFUs recovered on CIP (0.5 μg/mL) for log-phase cultures of representative resistant mutants that were enriched by exposure to PYO in the fluctuation tests (n = 4). Experiments were performed in the same way as in panel A.  Growth curves performed for P. aeruginosa Δphz (P.a.) and B. multivorans 1 (B.m.) in GMM  is plotted in G, while PI fluorescence is plotted in H. Gray shaded regions represent the standard deviation of 4 biological replicates. In G, the dashed horizontal lines mark the cell density at which P. aeruginosa (lower line in left panel) or B. multivorans (upper line in left panel) would have been plated in our fluctuation tests. Note that these OD500 values differ from those reported in the Methods section for fluctuation tests due to the use of a microtiter plate reader in this experiment, whereas a different spectrophotometer was used in the fluctuation tests. In H, the vertical dashed lines mark the time at which the cultures would have been plated in the fluctuation tests . The increase in fluorescence seen for B. multivorans prior to stationary phase likely reflects the production of a fluorescent metabolite rather than early cell death, as fluorescence was initially higher for the cultures not treated with PYO and the exponential phase growth rates were identical regardless of PYO treatment. Statistics: A–C—Welch unpaired t tests . Unless indicated otherwise with brackets, statistical significance is shown for the comparison with the untreated (no PYO) condition. In A–C, data points represent independent biological cultures, with horizontal black lines marking the mean value for each condition. The data underlying this figure can be found in Table M in P.a., P. aeruginosa; PI, propidium iodide; PYO, pyocyanin; SCFM, synthetic cystic fibrosis sputum medium.(TIF)Click here for additional data file.S9 Figm (the expected number of mutations per culture) and w (the relative fitness of mutant cells vs. WT), as implemented by Hamon and Ycart [m and d (the number of generations that occur post-plating), allowing for the possibility of post-plating mutations, as implemented by Lang and colleagues [m, as implemented by Lang and colleagues [p-value  for the Hamon and Ycart model. The data underlying this figure can be found in Table N in Data are plotted for different combinations of PYO in liquid (pretreatment) and PYO in agar (co-exposure to antibiotic selection). The empirical CDFs of the data (black) are plotted against (1) a variation of the LD model fit with 2 parameters, nd Ycart  (pink); lleagues  (blue); lleagues  (gray). (TIF)Click here for additional data file.S1 Table2 transformed), where high ratio values = increased fitness and low ratio values = decreased fitness. CIP Tn-seq values are from Cameron and colleagues [2-transformed ratio” column). CIP, ciprofloxacin; PYO, pyocyanin; Tn-seq, transposon sequencing.Analysis was done using ARTIST software see . Ratios lleagues  (see Met(XLSX)Click here for additional data file.S2 Tablep-values were adjusted with the Benjamini–Hochberg correction to control the false discovery rate. LD, Luria–Delbrück; LRT, likelihood ratio test.Mutation rates reported in this table were calculated using the rSalvador function newton.LD.plating and were compared using the LRT.LD.plating function to determine statistical significance. Mutant frequencies were compared using the Mann–Whitney U test. Reported (XLSX)Click here for additional data file.S3 TableModel parameters: μ, apparent mutation rate per generation; m, expected number of mutational events per cultures; w, fitness ratio of mutants/WT; d, number of post-plating generations. See (XLSX)Click here for additional data file.S4 TableP. aeruginosa UCBPP-PA14 genome obtained from BioProject accession number PRJNA38507. Pseudogenes and synonymous substitution mutations were omitted from the table. CIP, ciprofloxacin.Mutations were detected using breseq, with the reference set as the (XLSX)Click here for additional data file.S5 TableThis table contains a list of the strains, plasmids, and primers used in this study.(XLSX)Click here for additional data file.S6 TableB. multivorans AU42096 as the reference. Only mutations unique to each isolate are included. CIP, ciprofloxacin.Mutations were detected using breseq, with a draft assembly of the genome for (XLSX)Click here for additional data file.S7 Table5 CFUs/mL. CFU, colony-forming unit; CIP, ciprofloxacin; MIC, minimum inhibitory concentration.MICs were determined in a microbroth dilution assay according to standard clinical methods see . Where a(XLSX)Click here for additional data file.S1 Moviephz cells  are shown in green. Recovery in the absence of PYO after exposure to ciprofloxacin was imaged every 15 minutes for 15 hours. Scale bar: 20 μm. CIP, ciprofloxacin; PYO, pyocyanin; WT, wild-type.Four time points of this movie are shown in (MP4)Click here for additional data file.S1 DataThis zipped folder contains Excel tables organized by figure, within which the raw data for individual panels are displayed in separate tabs, as well as a text file containing the R code for all functions that were written especially for this manuscript.(ZIP)Click here for additional data file.