The impact of ColRS two-component system and TtgABC efflux pump on phenol tolerance of Pseudomonas putida becomes evident only in growing bacteria

<p>Abstract</p> <p>Background</p> <p>We have recently found that <it>Pseudomonas putida </it>deficient in ColRS two-component system is sensitive to phenol and displays a serious defect on solid glucose medium where subpopulation of bacteria lyses. The latter phenotype is significantly enhanced by the presence of phenol in growth medium. Here, we focused on identification of factors affecting phenol tolerance of the <it>colR</it>-deficient <it>P. putida</it>.</p> <p>Results</p> <p>By using transposon mutagenesis approach we identified a set of phenol-tolerant derivatives of <it>colR</it>-deficient strain. Surprisingly, half of independent phenol tolerant clones possessed miniTn5 insertion in the <it>ttgABC </it>operon. However, though inactivation of TtgABC efflux pump significantly enhanced phenol tolerance, it did not affect phenol-enhanced autolysis of the <it>colR </it>mutant on glucose medium indicating that phenol- and glucose-caused stresses experienced by the <it>colR</it>-deficient <it>P. putida </it>are not coupled. Inactivation of TtgABC pump significantly increased the phenol tolerance of the wild-type <it>P. putida </it>as well. Comparison of phenol tolerance of growing <it>versus </it>starving bacteria revealed that both ColRS and TtgABC systems affect phenol tolerance only under growth conditions and not under starvation. Flow cytometry analysis showed that phenol strongly inhibited cell division and to some extent also caused cell membrane permeabilization to propidium iodide. Single cell analysis of populations of the <it>ttgC- </it>and <it>colRttgC-</it>deficient strains revealed that their membrane permeabilization by phenol resembles that of the wild-type and the <it>colR </it>mutant, respectively. However, cell division of <it>P. putida </it>with inactivated TtgABC pump seemed to be less sensitive to phenol than that of the parental strain. At the same time, cell division appeared to be more inhibited in the <it>colR</it>-mutant strain than in the wild-type <it>P. putida</it>.</p> <p>Conclusions</p> <p>ColRS signal system and TtgABC efflux pump are involved in the phenol tolerance of <it>P. putida</it>. However, as they affect phenol tolerance of growing bacteria only, this indicates that they participate in the regulation of processes which are active during the growth and/or cell division. Single cell analysis data indicated that the cell division step of cell cycle is particularly sensitive to the toxic effect of phenol and its inhibition can be considered as an adaptive response under conditions of phenol stress.</p

Making Antimicrobial Susceptibility Testing More Physiologically Relevant with Bicarbonate?

Azithromycin is a clinically important drug for treating invasive salmonellosis despite poor activity in laboratory assays for MIC. Addition of the main buffer in blood, bicarbonate, has been proposed for more physiologically relevant and more predictive testing conditions. However, we show here that bicarbonate-triggered lowering of azithromycin MIC is entirely due to alkalization of insufficiently buffered media. In addition, bicarbonate is unlikely to be altering efflux pump activity

Monitoring of antimicrobial drug chloramphenicol release from electrospun nano-and microfiber mats using UV imaging and bacterial bioreporters

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

Pseudomonas putida vastused fenoolist tulenevatele metabolismi- ja stressisignaalidele

Käesolev doktoriväitekiri keskendub erinevatele signaalidele, mida tajub mullabakter Pseudomonas putida kokkupuutes aromaatse süsinikuühendiga fenool. P. putida on võimeline kasutama fenooli ainsa süsiniku- ja energiaallikana plasmiidse pheBA operoni ja kromosomaalse katehholi lagundamist võimaldava raja koosavaldumisel. Töö esimeseks eesmärgiks oli selgitada, milliste mehhanismide abil toimub pheBA promootorilt lähtuva transkriptsiooni pärssimine erinevates kasvukeskkondades. Keskonnas toimuvate muutuste tunnetamiseks kasutavad bakterid kahekonponendilisi signaaliülekande süsteeme. Minu teiseks töö eesmärgiks oli uurida signaaliülekande süsteemi ColRS rolli bakteris P. putida, täpsemalt selle seost rakkude fenoolitaluvusega. Doktoritöö osas, mis puudutab fenooli lagundamist võimaldavate geenide ekspressiooni, selgus, et aminohapete juuresolekul toimub pheBA operoni ekspressiooni pärssimine Crc valgu vahendusel. Uurimistöö teise osa andmed viitavad selle, et ColRS süsteem reguleerib bakterirakkude membraani läbitavust ja osaleb seetõttu fenoolitolerantsuse tagamises. Lisaks näitavad töö tulemused, et funktsionaalse ColRS süsteemi puudumine põhjustab rakkudele stressi ka glükoosi tardsöötmel. Kokkuvõtvalt näitasid käesoleva töö tulemused, et fenoolist tulenevad signaalid bakteris P. putida sõltuvad tugevalt nii fenooli kontsentratsioonist kui ka teistest keskkonnatingimustest.This study concentrates on different phenol-induced signals that are sensed by soil bacterium Pseudomonas putida. P. putida is able to degrade a wide variety of aromatic compounds. Coordinated expression of the plasmid-originated pheBA genes and the chromosomal catechol degradation pathway enables P. putida to degrade phenol and use it as a growth substrate. First, the study focuses on the mechanisms, which negatively affect transcription of the phenol degradation operon in P. putida. Many bacterial adaptive responses to environmental changes are controlled by two-component signal transduction systems. To unravel the function of ColRS two-component signal transduction pathway, the second part the thesis concentrates on the elucidation of the functions of the ColRS system and particularly on its role in phenol tolerance of P. putida. Study of regulation of phenol degradation genes showed that repressive effect on expression of pheBA genes caused by amino acids in the growth medium is mediated by Crc protein. In the second part of my thesis I show that ColRS system participates in phenol tolerance of P. putida and in addition the colR-deficient strain experiences serious stress on glucose medium. Altogether the study shows, that the nature of the phenol-induced signals, which are sensed by P. putida, strongly depend on the concentration of phenol in the medium as well as other growth conditions

Fluorescent reporters give new insights into antibiotics-induced nonsense and frameshift mistranslation

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

Uropathogenic Escherichia coli shows antibiotic tolerance and growth heterogeneity in an in-vitro model of intracellular infection

Uropathogenic Escherichia coli (UPEC), the major causative agent of urinary tract infections, can invade different types of host cells. To compare the pharmacodynamic properties of antibiotics against intra- and extracellular UPEC, an in vitro model of intracellular infection was established in J774 mouse macrophages infected by the UPEC strain CFT073. We tested antibiotics commonly prescribed against urinary tract infections (gentamicin, ampicillin, nitrofurantoin, trimethoprim, sulfamethoxazole, and ciprofloxacin) and the investigational fluoroquinolone finafloxacin. The metabolic activity of individual bacteria was assessed by expressing the fluorescent reporter protein TIMERbac within CFT073. Concentration-response experiments revealed that all tested antibiotics were much less effective against intracellular bacteria than extracellular ones. Most antibiotics, except fluoroquinolones, were unable to reach a bactericidal effect intracellularly at clinically achievable concentrations. Ciprofloxacin and finafloxacin killed 99.9% of extracellular bacteria at concentrations around the MIC, while for intracellular bacteria, concentrations more than 100× over the MIC were required to achieve a bactericidal effect. Time-kill curves showed that finafloxacin was more rapidly bactericidal in acidic medium than at neutral pH, while the reverse observation was made for ciprofloxacin. Intracellularly, kill curves showed biphasic kinetics for both fluoroquinolones, suggesting the presence of drug-tolerant subpopulations. Flow cytometry analysis of TIMERbac fluorescence revealed a marked heterogeneity in intracellular growth of individual bacteria, suggesting that the presence of subpopulations reaching a state of metabolic dormancy was the main reason for increased antibiotic tolerance of intracellular UPEC