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The forces mateerials by N. gonorrhoeae cells trigger accumulation of actin qntimicrobial other Macronutrient ratios, events that are critical for the antimiicrobial of the host Antimicrobail et al.
More materiaks, a potential mechanism antimicrobail action for E. coli mechanotransduction has been suggested, indicating that E. coli can sense anttimicrobial local mechanical environment through voltage-induced calcium Adxptive, then causing an electric pulse Bruni et al.
In fact, it has been proven that Adaptive antimicrobial materials amtimicrobial other mammalian cells such as human sensory neurons, share the electrical pathway as a common tool for sensing their environment.
Besides electrical cues, both mammalian and bacterial cells have been reported to sense and respond to different signals, namely magnetic field, electrical field, mechanical, and biochemical cues Figure 1. Bacteria are indeed extraordinary organisms, claimed to be one of the dominant forms of life on the planet.
It occupies a broad variety of ecological niches on Earth and were the first organisms reported in the fossil record Rasmussen, As a single and simple organism, it is remarkable the capacity of bacteria to adapt to different environments, tolerating a big range of temperatures, pressures and pHs, and having the ability to acquire resistance as a mechanism of survival.
Their long evolutionary history, being exposed to vastly different physicochemical environments, has made them a multifunctional organism able to detect and respond to a wide range of signals such as chemical, thermal, mechanical, electrical, and magnetic.
This adaptative behavior has been a valuable tool for developing novel strategies for obtaining effective infection control strategies or for potentiating the advantages of beneficial bacterial. By mimicking the bacterial cell microenvironment, the fate of bacterial cells may be tuned for either anti- or pro-microbial approaches.
The discovery of penicillin and its introduction in medical practice back in the middle of the last century saved countless lives and had a profound impact on the quality of human life, providing relief from pain and suffering Miller, It seems nowadays difficult to believe that nearly 70 years ago an infected wound could be close to a death sentence.
Figure 2. Schematic representation of A the timeline for the development of bacterial resistance and B its impact on the natural flora present in human intestine. Afterthe introduction of antibiotics allowed to cure previously deadly diseases and saved a large amount of lives, extending life span and allowing further medical procedures.
Nevertheless, the constant application of antibiotics soon resulted in antibiotic resistant strains. Harmless microbes from the flora in our guts are also killed giving space for the resistant strains to proliferate. Nevertheless, persistent use, overuse and misuse of antibiotics worldwide has led to an alarming increased incidence of resistant bacteria Tenover, ; Gould and Bal, ; Reardon, Bacterial infections caused by antibiotic-resistant strains and a lack of new drugs to replace old ones is a growing public health concern, considered by the World Health Organization WHO a priority health issue Collins, Dangerous bacterial species such as the methicillin-resistant Staphylococcus aureus MRSA and vancomycin-resistant enterococci VRE have emerged.
If no serious actions are taken in the immediate future, antimicrobial resistance AMR may cause many antibiotics to fail, which will become one of the biggest threats to human health Tenover, ; Rasko and Sperandio, ; Laxminarayan et al.
Currently, resistant bacteria infect millions of people worldwide and causes more thandeaths every year, also inflicting enormous costs to health systems. Byit is estimated that more than 10 million people may die per year due to resistant bacteria Collins, It was during Second World War in the s that antibiotics were introduced, saving innumerous wounded soldiers and fast became available for use in the general population.
It was then just a matter of time until the resistance to several antibiotics take place. In fact, nowadays, antibiotic resistance is already and should be a public health concern and novel strategies are indeed needed to fight AMR.
Human intestines are home for many different microbes, some of which create resistance to the antibiotics they are exposed to. These resistant strains then spread from person to person, in communities or in hospitals Figure 2Bultimately leading to the problem of bacteria resistance. Human body is constituted by trillion cells but only 1 in 10 is actually human.
The remaining cells are microorganisms such as bacteria Relman, These microorganisms are harmless and live in perfect balance with human body, playing an important role in supporting and maintaining vital functions such as our immune and digestive systems Relman, However, when this balance is broken and the delicate ecosystems that bacteria carefully construct in different parts of human body are disrupted, bacteria become pathogenic, causing infection diseases.
Pro-microbial approaches thus constitute one strategy that should call the attention of the scientific community. In a broader sense, the equilibrium between anti- and pro-microbial should be an important strategy.
Despite being often associated with virulence, infection and disease, bacteria are considered very important microorganisms to sustain human life. They are responsible for the correct functioning of our immune, respiratory, and digestive system Ichinohe et al.
That is why the right approach to obtain an effective strategy for infection control is to reinforce our beneficial microbial population, the microbiome, in a pro-microbial strategy, while providing an appropriate antimicrobial agent for full eradication of pathogenic bacterial anti-microbial strategywithout the possibility of developing resistance.
The equilibrium between these anti- and pro-microbial approaches is an important twin sustainable strategy for limiting AMR Jørgensen et al. Meanwhile, the benefits of microbiome are largely overlooked by the scientific community. The benefits derived from the diversity of beneficial microbes has only recently been proposed Jørgensen et al.
Among all antibiotics available on the market nowadays, the most recent class was discovered in the s, which demonstrates the difficulty on the process of finding new effective antimicrobials.
It is time to, together with focusing attention on the development of new antibiotics, give more relevance to the diversity of microbes present in the human body that assists on the eradication of harmful bacteria, which means the right balance between anti- and pro-microbial strategies.
In terms of pro-microbial strategies, a technique that is gaining more attention among the scientific community for the treatment and prevention of some infectious disease is the fecal microbiota transplantation FMT. The process consists in the infusion of beneficial bacteria from the stool of a healthy donor into a recipient with a disease related to an unhealthy gut microbiome Kim and Gluck, FMT has been successfully used to treat infections caused by Clostridium difficile C.
difficilebut also tested and recommended for other conditions such as inflammatory bowel disease IBDautoimmune disorders, certain allergic diseases, and metabolic disorders such as obesity Choi and Cho, The future of infectious disease treatment is thus the promotion of pro-microbial strategies such as FMT.
Future challenges regarding this technology are the safety in delivering FMT to patients, being imperative to standardize the methodologies and prepare highly specialized laboratories for stool preparation. Another challenge is to identify the effectiveness of microbiota-based medicines and identify the specific bacteria responsible for this effect.
Regarding anti-microbial strategies, synergistic approaches for inhibiting bacterial pathogenesis, i. Such strategies cause less evolutionary stress on bacteria population and thus prevent the emergence of resistance mechanisms.
The increasing understanding of bacterial pathogenesis and intercellular communication in a broader sense, both from the physical and biochemical points-of-view has been a valuable tool to develop new strategies that meets these challenges Hajipour et al.
Taking into consideration that bacteria can indeed feel the surrounding environment and modify their phenotype in response to it, the main cues responsible for the tailoring of bacteria behavior and thus assist the action of antibiotics are depicted in Figure 3.
Several studies have been performed on the effect of specific stimuli on bacteria, namely the mechanical, magnetic, electrical, and biochemical [quorum sensing QS mechanism] cues.
Their effects on bacterial cell, advantages and disadvantages are summarized in Table 1. Figure 3. Stimuli that bacteria sense and the mechanism of action of each bactericidal effect. Table 1. Summary of the effects of different physical and biochemical stimuli on bacteria.
The effect of mechanical vibrations on bacteria surface adhesion, proliferation and virulence has been mainly evaluated for the inhibition of biofilm formation, a protective mode of growth that confer pathogenic bacteria increased resistance to conventional antibiotics and host defenses mechanisms Mah and O'Toole, Surface acoustic waves generated from electrically activated piezo elements has been reported to be repulsive to bacteria and interfere with the docking and attachment of planktonic microorganisms to solid surfaces Hazan et al.
Also, vibration loads generated by magnetoelastic materials, which possess magnetostrictive properties, converting a magnetic stimulus into a mechanical deformation, were found to significantly reduce the adherent bacteria on samples exposed to different types of microorganisms Paces et al. The use of ultrasounds have also been used as a mean for preventing biofilm formation.
The application of ultrasound induce an acoustic cavitation phenomenon, which results in the formation of cavitation bubbles that implode and generate shock waves that cause mechanical damage to the bacteria and the formation of free radicals that creates oxidative stress to bacteria Figure 3 Gera and Doores, To obtain a relevant reduction on bacterial counts, ultrasound has been applied in combination with antibiotics as a synergistic approach.
Pseudomonas aeruginosa P. aeruginosa and E. coli biofilms were thus efficiently eradicated by gentamicin sulfate in combination with ultrasound Qian et al. Also, the killing effect of ZnO NPs against Staphylococcus aureus S.
: Adaptive antimicrobial materials| Surface Proteins and Systems: Porins | This review is focused on the development of highly efficient and specifically targeted biomaterials that release the antimicrobial agents or respond to bacteria on demands in body. The mechanisms of bacterial adhesion, biofilm formation, and antibiotic resistance are discussed, and the released substances accounting for implant infection are described. Strategies that have been used in past for the eradication of bacterial infections are also discussed. Different types of stimuli can be triggered only upon the existence of bacteria, leading to the release of antibacterial molecules that in turn kill the bacteria. In particular, the toxin-triggered, pH-responsive, and dual stimulus-responsive adaptive antibacterial biomaterials are introduced. Finally, the state of the art in fabrication of dual responsive antibacterial biomaterials and tissue integration in medical implants is discussed. The perturbation of these ion concentrations can lead to hyperpolarization, wherein the membrane potential becomes more negative or depolarization and the membrane potential becomes less negative toward zero Valič et al. The electropermeabilization effects from the application of low-strength electric fields has been shown to boost the effect of antibiotics against bacteria in biofilms, diminishing the concentration of antibiotic needed to kill bacteria Costerton et al. On the other hand, high strength electric pulses resulted in an efficient bactericidal effect on both gram-positive and gram-negative bacteria. These strategies however require the direct application of an electrical field on bacterial solution, which may not be recommendable for in vivo applications. Similarly to other stimuli previously mentioned the formation of reactive oxidative species ROS such as hydrogen peroxide H 2 O 2 and reactive nitrogen species RNS was also indicated as a possible mechanism of action for the bactericidal effect of low strength electric field Boda and Basu, Electrical stimulus has been mainly reported to impart bactericidal and bacteriostatic effect rather than a proliferation effect. Nevertheless, it has been also reported that low frequency mechanical stimuli leading to surface charge variations, induce similar effect to those occurring with eukaryotic cells, i. Besides chemotaxis, another important example of how bacteria sense its environment is the QS mechanism they use to express virulence factors, allowing bacteria to regulate community-wide behaviors including biofilm formation, virulence, conjugation, sporulation, and swarming motility Rutherford and Bassler, This mechanism of cell-to-cell communication is based on the production, secretion, and detection of small signaling molecules, called autoinducers AIs. In the QS-regulated communication, bacteria secrete signaling molecules, the AIs that are further recognized by specific receptors, allowing bacteria to act collectively as a multicellular microorganism. This knowledge is being increasingly used to develop new strategies for infection control Figure 4A. The inactivation of the QS signals in a process called quorum quenching QQ is an innovative strategy to control bacterial infections Hentzer and Givskov, ; Roche et al. Brominated furanones interfere with QS by acting as antagonists to receptors Rasmussen and Givskov, ; Kociolek, Similarly, enzymes such as acylase and lactonase have been shown to selectively degrade N-Acyl homoserine lactone AHL signals of Gram-negative bacteria Dong et al. In line with this knowledge, acylase has been successfully used to coat indwelling medical devices through functionalization techniques such as layer-by-layer technique. The enzyme multilayer coatings significantly reduced the bacterial load and biofilm formation on functionalized silicone-based urinary catheters, assessed with an in vitro catheterized bladder model Ivanova et al. Moreover, the QQ enzyme coatings were fully biocompatible since they were tested with mammalian cells such as fibroblasts over 7 days, the extended useful life of urinary catheters, and no toxicity was observed. Figure 4. A Simplified QS system of Gram-negative bacteria, general chemical formula of the signaling molecules and B strategies to QQ including the enzymatic degradation of AHL signals by AHL-lactonase and AHL-acylase. The main advantage of this approach is that it attenuates virulence, exerting less selective pressure on bacteria and reducing the risk of resistance development to drugs. Moreover, it affects bacterial behavior but does not kill or inhibits their growth, thus allowing the host defense system to eliminate attenuated bacteria or substantially increase the effect of co-administered antibiotics Ivanova et al. The action of such enzymes Figure 4B creates the conditions to eradicate the infection by the natural host immune system before virulence is established. The application of a magnetically and electrically active microenvironment is a strategy that has been widely explored in mammalian cells and that can be also used for tailoring specific bacterial responses. It is well-stablished that electroactive materials such as piezoelectric polymers and magnetoelectric composites develop voltage variations at the surface of the material when a mechanical stress Ribeiro et al. Knowing that bacteria are also able to sense these types of stimuli, these materials seem to constitute a suitable approach for both anti- and pro-microbial applications by developing active surfaces based on those materials. Examples of such materials are the ones possessing mechanoelectric, magnetostrictive, and magnetoelectric properties. Mechanoelectric materials are materials mainly constituted by, for example, piezoelectric polymers that respond to a mechanical stimulus, inducing an electrical charge variation in the material Figure 5A. Magnetic and magnetoelectric materials are composites comprising magnetic or magnetostrictive particles and a piezoelectric polymer. Due to their magnetic component, they sense a magnetic field that induce a mechanical stimulation on the material, due the incorporated magnetic or magnetostrictive properties, which further induce an electrical polarization variation due to the piezoelectric phase present in the composite Figures 5B,C. These materials thus respond to different stimuli, namely mechanical and magnetic field. Both stimuli are induced with the help of a bioreactor that provides specific cues on the materials and thus on the cells, for cell response investigation studies, or on active coatings through the surface functionalization of materials where those stimuli are present or can be induced, as for example in biomedical devices. Figure 5. Schematic representation of the A mechanoelectric properties of a material upon the application of mechanical stimuli and B,C magnetoelectric properties of scaffolds upon the application of magnetic stimuli. Piezoelectric synthetic polymers are the most widely used in the development of mechanoelectric materials for biomedical applications. Poly vinylidene fluoride PVDF and vinylidene fluoride VDF copolymers possess high electroactive properties, including piezoelectric, pyroelectric, and ferroelectric properties, which makes them particularly used Dubois, ; Serrado Nunes et al. Despite the fact that PVDF and its copolymers are not biodegradable, they are biostable and thus widely used. That is why poly L-lactid acid PLLA Preethi Soundarya et al. On the other hand, magnetic nanocomposites with magnetoelectric properties may be obtained by adding nanomaterials such as pure metals Co, Fe, Ni and metal oxides iron oxides Fe 2 O 3 or Fe 3 O 4 and ferrites such as BaFe 12 O 19 and CoFe 2 O 4 combined with the above-mentioned piezoelectric polymers Kudr et al. The possibility to remotely control tissue stimulation without the need of patient movement is certainly an innovative approach and is regarded as a breakthrough platform for tissue engineering applications Silva et al. In fact, the magnetic actuation ability of the magnetoelectric composite allows the mechanical and electrical stimuli of neighboring cells Martins and Lanceros-Méndez, In microbiology this approach could also be valuable, for example, for the prevention of infection of orthopedic indwelling devices by external stimulation. As previously mentioned, the development of these kind of materials has been explored in tissue engineering but poorly investigated in microbiology. The effect of a strong electrical field on the bacteria behavior, rather than acoustic mechanic waves, has been reported in a study where the effect of a piezoelectric material ceramics on bacteria was performed and the killing effect was due to the formation of ROS Tan et al. On the other hand, the proliferation effect of bacteria has been observed at the surface of electrically polarized hydroxyapatite Ueshima et al. Recent findings reported that bacterial cells behavior, which grow upon piezoelectric polymers, may be tailored according to the surface charge of the material and on the application of weak electrical field, promoted by a piezoelectric polymer under mechanical stimuli, demonstrating a different behavior between Gram-positive and Gram-negative cells Carvalho et al. The Gram-positive bacteria seems not adhere to positively charged surface, as opposed to the negatively charged surface. In the presence of an electrical stimuli, this strain shows a different behavior: the lower frequency promotes the antifouling and the higher stimulates the bacteria adhesion Figure 6 Carvalho et al. Figure 6. Such approaches are important to further define suitable anti- and pro-microbial strategies, intended for pathogenic and functional bacteria, respectively Carvalho et al. The ambition to create novel strategies to fight bacteria resistance using physical stimuli is an attractive and valid approach that relies on the fact that bacteria sense their environment and respond to it. This review thus calls the attention for the use of innovative electroactive smart materials as a novel tool for tailoring bacteria behavior and thus fight bacteria resistance using, not only anti-microbial strategies, but also pro-microbial ones. The most attractive feature of using such materials is the possibility for triggering the inhibition and proliferation of bacteria by changing the conditions applied, when bioreactors or smart and responsive surfaces and coatings are used. From one side, defining the conditions for antimicrobial strategies will allow these materials to be used synergistically with commonly applied antibiotics or other innovative elements that assist the antibacterial effect, reducing the quantity of antibiotic needed for killing bacteria. Such strategies cause less evolutionary selective pressure on bacteria population and thus prevent the emergence of resistance mechanisms. On the other side, defining the conditions for proper bacteria proliferation approaches will be the opportunity to pursue a pro-microbial activity, potentiating the function of human microbiome in assisting vital functions in our body. A healthy microbiome is essential for human and animal well-being since it has the ability to educate the immune system and keep the pathogenic bacteria out, apart from the obvious role it has on the digestive system. Therefore, novel materials or coatings that assist the antimicrobial effect and thus prevent the occurrence of nosocomial infections in clinical settings due to the contamination of medical devices such as stents and catheters or medical textiles are very appealing and needed to be used as first line defense against pathogenic bacteria, while potentiating the action of beneficial bacteria will allow to reinforce the microbiome, essential for disease prevention. More importantly, the application of electroactive materials may thus be the future for developing smart implantable devices, which due to their electric-sensitive properties may be used not only to promote anti- and pro-microbial strategies but also to take advantage of their characteristics for sensor applications. All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Amaro, L. Tailored biodegradable and electroactive poly Hydroxybutyrate-co-hydroxyvalerate based morphologies for tissue engineering applications. doi: PubMed Abstract CrossRef Full Text Google Scholar. Asadi, M. Bacterial inhibition by electrical stimulation. Wound Care 3, 91— Biais, N. Force-dependent polymorphism in type IV pili reveals hidden epitopes. Bidan, C. Magneto-active substrates for local mechanical stimulation of living cells. Binhi, V. Effect of static magnetic field on E. coli cells and individual rotations of ion—protein complexes. Bioelectromagnetics 22, 79— Boda, S. Engineered biomaterial and biophysical stimulation as combinatorial strategies to address prosthetic infection by pathogenic bacteria. Part B , — Bruni, G. Voltage-gated calcium flux mediates Escherichia coli mechanosensation. 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| Publication types | Tracking Nutrient-rich weight management dynamic ajtimicrobial between cellular Ginseng research studies and extracellular subproteomes in Pseudomonas aeruginosa Adatpive. Jochumsen N. Adaptive antimicrobial materials Materiials, Pagès J-M. Interaction of antibacterial compounds with RND efflux pumps in Pseudomonas aeruginosa. Masuda N. Rampant parasexuality evolves in a hospital pathogen during antibiotic selection. This suggests that there may be other mutations that occur prior to the pmrAB mutations which predispose PAO1 to colistin resistance. |
| Supplementary files | Gall, Metabolic health foods. aeruginosa found Adaptive antimicrobial materials the lungs of materals with CF. Mechanisms of Antimicrobiak resistance in bacteria. Note Acaptive these plots are qualitatively similar to the one shown in the main text Fig. AMEN: Conceived and designed the experiments, analyzed and interpreted the data, contributed reagents, materials, analysis tools, or data, wrote the paper. |
| How Antimicrobial Resistance Happens | The bioreactor was controlled using the Sartorius MFCSPID controller program. The controller was modified to use CO 2 output from the gas analyzer as a signal to regulate pumps. One pump maintained a constant flow of media to the culture. The second pump was designated to respond to CO 2 concentration and maintain mid-logarithmic growth. Turbidity measurements were made arbitrarily during the experiment using a sample of the effluent waste. As the effluent waste typically contains multi-drug resistant pathogens, the waste was collected and properly sterilized. The bioreactor was assembled as described above. The air inflow was set between 0. Antibiotic-free, sterile media was pumped through the media filters and into the vessel to a predetermined volume based upon the organism being studied. This volume can be varied from 0. depending on the organism. aeruginosa was adapted as a 0. Care was taken to prevent the system from having any unnecessary openings. All inlet and outlet lines were pinched off with metal clamps when not in use. The positive pressure inside the vessel established by the sterile air flow prevented contaminants from seeping into the system. The entire system was kept in a biosafety cabinet to further maintain sterility. The inoculum was prepared using a single colony from a freshly streaked plate of non-selective media. Post inoculation, the cells grew to mid-exponential phase before pumps were turned on. Once the desired cell density was reached, the pump feeding media at a steady rate was turned on to a speed which was sufficient for maintaining culture density. As the population density rises, the CO 2 produced by cellular metabolism also rises. If the CO 2 rose above the set point established for mid-log, a second pump was activated at a set speed, diluting the culture to maintain it at the set point. Once the appropriate dilution was achieved, the CO 2 concentration dropped, turning off the second pump. As the culture grew for longer periods of time in the vessel, biofilms started forming. The presence of the biofilms made it difficult to determine absolute cell density in the vessel. Since biofilms did not contribute to planktonic culture, the turbidity sample did not reflect the actual number of cells in the vessel. However, the biofilms contributed to the CO 2 generated in the vessel. Therefore, the CO 2 set point had to be re-set several times during the experiment based on the amount of accumulated biofilm. The culture was maintained in such a way that the planktonic population was always in mid-exponential phase. The sample was also streaked on the appropriate solid medium to observe colony morphology and check for contaminants. The supernatant was also frozen. The goal of the experiment is to identify mutations linked to antibiotic resistance and to minimize adaptation conditions that might be the result of growth within the bioreactor environment. To avoid adaptive mutations unrelated to antibiotic selection, we use non-limiting growth conditions such that the population is always experiencing excess nutrient and highly favorable growth conditions. Thus the bioreactors are not chemostats but instead allow for maximal exponential growth at all times during the experiment. After the culture established itself in the vessel, the first drug concentration was introduced in the vessel. This concentration was typically 0. Throughout the experiment the drug concentration is maintained well below the MIC of the population and it is this sub-MIC selection that favors the evolution of multiple simultaneous evolutionary trajectories, resulting in a complex polymorphic population within each vessel. At the end of the experiment, the sampling process was performed as described above. The remainder of the liquid contents of the vessel was collected through the sampling port. The vessel was then opened and the biofilm was collected from the vessel walls and the metal portions of the lid using a sterile spatula and collected in separate tubes. The biofilm was then suspended in a small volume of growth media and used for serial dilution. The rest was frozen as a glycerol stock. Serial dilutions of the planktonic and biofilm samples from the final day of the experiment were plated on appropriate non-selective, solid growth media. After incubation, single colonies end-point isolates were picked from the plates for phenotypic analysis. If obvious differences in morphology were observed, a few representative colonies from each morphology were picked. If not, colonies were picked randomly. Various phenotypic screens were performed to characterize the end-point isolates which included but were not limited to MIC testing, testing for cross-sensitivity or cross-resistance to other antibiotics, growth rate studies, biochemical tests and biofilm assays. Based on the diversity observed in these assays, end-point isolates were selected for whole genome sequencing. The ancestor strain for each experiment, samples collected from each day of the experiment, as well as end-point isolates were sequenced. End-point isolates were grown overnight and pelleted before DNA extraction. The frozen pellets from daily samples were thawed on ice and immediately extracted. The Illumina HiSeq platform was used for whole genome deep sequencing of the daily population samples to obtain at least × coverage. The end-point isolates and ancestors were sequenced to obtain at least × coverage. Comparison of the raw sequencing reads to the reference genome of the ancestor was done using Breseq. In cases where a closed reference genome was not available, the Pacific BioSciences sequencing platform was used to sequence the ancestor and assemble a closed reference. The pile-ups for called mutations were manually examined to verify their accuracy. The quantitative experimental evolution pipeline is composed of five key components: 1 Evolve resistant populations in a bioreactor under polymorphic selection conditions; 2 Identify the frequency and order of mutations correlated with antibiotic resistance as a function of time; 3 Identify the genotypes of end-point isolates to establish genetic linkages; 4 Validate the effect of mutations by physicochemical characterization; 5 Rank candidates for potential drug development Figure 2. Pipeline of quantitative experimental evolution to predict antibiotic resistance and identify targets for drug discovery. Each stage produces essential data and approaches that when taken together predict resistance, identify the most important targets, suggests potential biochemical mechanisms and leads to assay development. The stages are: 1 Evolve resistant populations in a bioreactor under polymorphic selection conditions; 2 Identify the frequency and order of mutations correlated with antibiotic resistance as a function of time; 3 Identify the genotypes of end-point isolates to establish genetic linkages; 4 Validate the effect of mutations by physicochemical characterization; 5 Rank candidates for potential drug development. aeruginosa , PAO1 was evolved to colistin resistance using our quantitative experimental evolution approach in our modified bioreactor. aeruginosa as determined by MIC Interpretive standards set by the Clinical and Laboratory Standards Institute CLSI. The mutation in MutS resulted in a strong hypermutator phenotype. Hypermutator phenotypes in P. aeruginosa have been observed at high frequency in clinical isolates of sputum from cystic fibrosis patients. In the presence of this rapid mutation rate, mutations specific to resistance were seen to accumulate in the two-component system, pmrAB , which has been clinically linked to colistin resistance. The deconvolution of all the mutations within this hypermutator will be discussed in later work. PmrAB is a two-component regulatory system that activates downstream lipopolysaccharide modification systems in response to cationic antimicrobial peptides. Quantitative analysis of the metagenomic deep sequencing data for each daily, heterogenous sample using Breseq 25 allowed us to identify the frequency at which mutations in pmrAB rose and spread throughout the population Figure 3. Based on the predicted domain structure of the membrane bound sensor kinase PmrB, 18 single nucleotide polymorphisms leading to amino acid substitutions in the transmembrane domains L17P, L18P, LP, LP , in the periplasmic domain D47G, VM , in the HAMP linker domain present in Histidine kinases, Adenyl cyclases, Methyl-accepting proteins and Phosphatases VA and in the C-terminal ATP binding domain AP, FL were observed in our evolved population. Mutations in these domains have also been observed in colistin-resistant clinical isolates 18 , 26 , 27 as well as lab-adapted colistin-resistant strains of P. Figure 3 traces the frequencies of these mutations and the point at which they arose during the experiment. This suggests that there may be other mutations that occur prior to the pmrAB mutations which predispose PAO1 to colistin resistance. One unintentional benefit of hypermutators is that they can provide a very extensive survey of the entire adaptive landscape and thus provide a comprehensive catalog of mutations that may facilitate resistance. Frequencies of mutant alleles associated with colistin resistance in P. aeruginosa PAO1. Mutations in the pmrAB operon were identified by analysis of whole genome sequencing data obtained from each daily population collected from the bioreactor. The corresponding day on which the mutation is observed is on the x axis. The gray dashed lines represent the distinct colistin concentrations the culture was exposed to during evolution. The mutations on the right are the amino acid changes in the protein caused by single nucleotide polymorphisms SNPs in the corresponding gene pmrA or pmrB. Note that some mutations such as pmrB AP have early success but then become extinct as other more successful pmrA alleles confer greater success to drug selection. Although there are several positions on the pmrB gene that developed single nucleotide polymorphisms during the course of adaptation, not all of them persisted till the end. The final end-point isolates we sequenced had only one L18P out of the nine mutations observed in the daily populations. Our results suggest that our approach provides a fairly comprehensive survey of all the mutations appearing throughout the course of adaptation and limits the role of population bottlenecks in limiting the accessible evolutionary trajectories. The modified bioreactor we use for adaptation provides several advantages over traditional serial transfer evolution experiments. Many clinically significant bacteria form thick biofilms and bioreactor culturing can select for this formation. This long-term establishment of biofilm more accurately mimics the natural ecology that many of these organisms create. The organism studied in this work, P. aeruginosa to colistin. Additionally, it is clear that the evolutionary trajectories obtained from these studies do not address the molecular mechanisms of pathogenesis. Pathogenesis requires an appropriate host or host cell line. In vitro experimental evolution is very informative, however, in determining the molecular basis for antibiotic resistance. Adaptive mutations conferring antibiotic resistance have very strong effects on the fitness of the organism that typically far out-weigh those of adapting to the bioreactor growth conditions since we do not limit critical resources such as carbon and nitrogen. Biofilm build up in the bioreactor vessel during evolution of P. The bioreactor design favors the development of strong biofilms. Since the evolution of the populations takes place over weeks in a single vessel, those adaptive alleles that favor biofilm formation have a selective advantage as they can adhere to surfaces and not be removed as new media is added to maintain a constant exponentially growing planktonic phase. Acinetobacter , enterococci and Pseudomonas have all exhibited strong biofilm formation in this experimental system. The bioreactor also maintains a continuous culture at its fastest growth rate while slowly increasing the antibiotic concentration in an empirically designed, stepwise manner. One of the major advantages of evolving resistance in a bioreactor is the evolution of a highly polymorphic population to study the subtle nuances of antibiotic resistance. This polymorphism arises from the large culture volume, continuous logarithmic growth, reduced bottleneck and growth in sub-inhibitory concentrations of antibiotic. Bioreactor experiments are carried out with culture volumes ranging from 0. Flask transfer experiments also enter stationary phase each day, reducing the number of doublings. While P. aeruginosa can only achieve 6—8 generations before reaching stationary phase in batch culture when growing in a rich medium, it experiences roughly 20 generations every day in the bioreactor. This increase in replication allows for a more thorough survey of possible mutations across the genome. Quantitative analysis of the deep sequencing data obtained from the daily populations provides us with a comprehensive list of all mutations occurring in the population during the process of evolution and their relative frequencies in the population. It also allows us to look at the rise and fall of genotypes that help in the early adaptation of the population but may not persist at higher antibiotic concentrations due to a more favorable mutation arising and taking over the population. This is clear in Figure 3 where early mutations like AP and LP within PmrB are seen at high frequencies during early adaptation but are replaced by other mutations at higher drug concentration. The appearance and persistence of a mutation relies on the fine balance between the resistance conferred by that mutation and the fitness cost associated with it. From our analysis, we can capture these unsuccessful mutations, which serve as progenitors for the more successful lineages. Having knowledge of these early mutations is useful in the clinic. With the decreasing cost of whole genome sequencing, clinicians are moving towards the sequencing approach to characterize pathogenic isolates from patients. Knowing which mutations predispose cells to becoming resistant to a particular drug is important information when deciding what antibiotics to administer as treatment. An essential component of our analysis is the establishment of the order of mutations as well as their frequency Figure 3. Targets for potential drug development are those identified in these critical first steps towards resistance. Work performed by C. Miller et al. faecalis shows that mutations specific to the liaFSR operon serve as an essential opening step to all the successful evolutionary trajectories leading to resistance. In another study by K. Beabout et al. showing the evolution of tigecycline TGC resistance in E. faecalis , metagenomic deep sequencing helped identify an increase in transconjugation that lead to the widespread presence of transposon Tn, containing the TGC resistance gene, tetM. baumannii to TGC resistance. baumannii in the clinic, as well as in our bioreactor evolved populations of P. The sheer number of mutations acquired in hypermutator populations poses a serious challenge for analysis. Metagenomic data from the daily populations as well as frequency data and statistical analysis were essential to identifying the key mutations associated with resistance in this complex genomic background. A holistic approach that uses experimental evolution, metagenomic deep sequencing and in vitro biochemistry is also very useful for deconstructing complex strategies of antibiotic resistance. The next step in the quantitative experimental evolution pipeline is the validation of targets identified from genome sequence analysis. However, many bacteria do not possess the genetic tools necessary for gene validation. Also, the epistatic relations between multiple adaptive alleles can prove to be nearly impossible as even five mutations will generate combinations of potential pair-wise interactions. The order of mutations from our time frequency metagenomics can help to establish the epistatic relationship of complex evolutionary trajectories. However, a combination of in vitro biochemistry, biophysics and modeling can be used to link physicochemical measurements to predictions of phenotypes such as drug resistance. Measured physicochemical data also provide vital information for the drug design process. From previous studies in our lab on daptomycin resistance in E. faecalis , 12 we showed that the LiaFSR three component system was crucial in conferring resistance. In a broader sense, the equilibrium between anti- and pro-microbial should be an important strategy. Despite being often associated with virulence, infection and disease, bacteria are considered very important microorganisms to sustain human life. They are responsible for the correct functioning of our immune, respiratory, and digestive system Ichinohe et al. That is why the right approach to obtain an effective strategy for infection control is to reinforce our beneficial microbial population, the microbiome, in a pro-microbial strategy, while providing an appropriate antimicrobial agent for full eradication of pathogenic bacterial anti-microbial strategy , without the possibility of developing resistance. The equilibrium between these anti- and pro-microbial approaches is an important twin sustainable strategy for limiting AMR Jørgensen et al. Meanwhile, the benefits of microbiome are largely overlooked by the scientific community. The benefits derived from the diversity of beneficial microbes has only recently been proposed Jørgensen et al. Among all antibiotics available on the market nowadays, the most recent class was discovered in the s, which demonstrates the difficulty on the process of finding new effective antimicrobials. It is time to, together with focusing attention on the development of new antibiotics, give more relevance to the diversity of microbes present in the human body that assists on the eradication of harmful bacteria, which means the right balance between anti- and pro-microbial strategies. In terms of pro-microbial strategies, a technique that is gaining more attention among the scientific community for the treatment and prevention of some infectious disease is the fecal microbiota transplantation FMT. The process consists in the infusion of beneficial bacteria from the stool of a healthy donor into a recipient with a disease related to an unhealthy gut microbiome Kim and Gluck, FMT has been successfully used to treat infections caused by Clostridium difficile C. difficile , but also tested and recommended for other conditions such as inflammatory bowel disease IBD , autoimmune disorders, certain allergic diseases, and metabolic disorders such as obesity Choi and Cho, The future of infectious disease treatment is thus the promotion of pro-microbial strategies such as FMT. Future challenges regarding this technology are the safety in delivering FMT to patients, being imperative to standardize the methodologies and prepare highly specialized laboratories for stool preparation. Another challenge is to identify the effectiveness of microbiota-based medicines and identify the specific bacteria responsible for this effect. Regarding anti-microbial strategies, synergistic approaches for inhibiting bacterial pathogenesis, i. Such strategies cause less evolutionary stress on bacteria population and thus prevent the emergence of resistance mechanisms. The increasing understanding of bacterial pathogenesis and intercellular communication in a broader sense, both from the physical and biochemical points-of-view has been a valuable tool to develop new strategies that meets these challenges Hajipour et al. Taking into consideration that bacteria can indeed feel the surrounding environment and modify their phenotype in response to it, the main cues responsible for the tailoring of bacteria behavior and thus assist the action of antibiotics are depicted in Figure 3. Several studies have been performed on the effect of specific stimuli on bacteria, namely the mechanical, magnetic, electrical, and biochemical [quorum sensing QS mechanism] cues. Their effects on bacterial cell, advantages and disadvantages are summarized in Table 1. Figure 3. Stimuli that bacteria sense and the mechanism of action of each bactericidal effect. Table 1. Summary of the effects of different physical and biochemical stimuli on bacteria. The effect of mechanical vibrations on bacteria surface adhesion, proliferation and virulence has been mainly evaluated for the inhibition of biofilm formation, a protective mode of growth that confer pathogenic bacteria increased resistance to conventional antibiotics and host defenses mechanisms Mah and O'Toole, Surface acoustic waves generated from electrically activated piezo elements has been reported to be repulsive to bacteria and interfere with the docking and attachment of planktonic microorganisms to solid surfaces Hazan et al. Also, vibration loads generated by magnetoelastic materials, which possess magnetostrictive properties, converting a magnetic stimulus into a mechanical deformation, were found to significantly reduce the adherent bacteria on samples exposed to different types of microorganisms Paces et al. The use of ultrasounds have also been used as a mean for preventing biofilm formation. The application of ultrasound induce an acoustic cavitation phenomenon, which results in the formation of cavitation bubbles that implode and generate shock waves that cause mechanical damage to the bacteria and the formation of free radicals that creates oxidative stress to bacteria Figure 3 Gera and Doores, To obtain a relevant reduction on bacterial counts, ultrasound has been applied in combination with antibiotics as a synergistic approach. Pseudomonas aeruginosa P. aeruginosa and E. coli biofilms were thus efficiently eradicated by gentamicin sulfate in combination with ultrasound Qian et al. Also, the killing effect of ZnO NPs against Staphylococcus aureus S. aureus and P. aeruginosa was boosted when ultrasound was applied to the medium Justin and Thomas, It is worth mentioning that the effect of the mechanical damage created by the acoustic cavitation of microbubbles in the blood, the possibility to induce rupture of blood vessel walls and interfere with blood flow may limit the application of this type of strategies in vivo Chen et al. The influence of magnetic field on biological systems, namely on biomolecules, cells and living organisms, is an important field of research due to the emergence of electromagnetic pollution in the form of low and high radio frequency magnetic fields from electronic devices Boda and Basu, It is believed that magnetic field interfere with the mechanism of ion transport via membrane channel proteins, leading to osmotic imbalance and membrane rupture. These phenomenon is attributed to the diamagnetic anisotropy exhibited by the large number of membrane proteins on bacteria cell, which result from the axial alignment of peptide bonds and specific amino acid residues containing aromatic groups Worcester, Therefore, the lipids and ion channel proteins that are present in bacterial membrane undergo conformational changes that lead to the dysfunction of these proteins, disrupting essential ion transport mechanisms on bacteria. One of the first reports of this effect on bacteria proved that magnetic field cause rotational motion of ion-protein complexes leading to the escape in E. coli Binhi et al. The generation of free radicals upon application of a magnetic field, leading to bacterial cell oxidative stress and genotoxicity Figure 3 has been another proposed mechanism of action involved in bacterial killing and possibly mammalian cell damage Ghodbane et al. Further studies need to be performed to assess the potential harm these approaches might induce to biological systems. It is known that direct application of strong electric fields may be bactericidal or a mean of preventing device-related infections, which are caused by biofilm formation, or even to disinfect contaminated liquids Poortinga et al. Moreover, the ability of electric fields to promote wound healing through angiogenesis while reducing microbial bioburden at the surface of material has already been proven Asadi and Torkaman, The electrical-based killing mechanism of action involves an increase in cell membrane permeability, known as electropermeabilization or irreversible electroporation. The perturbation of these ion concentrations can lead to hyperpolarization, wherein the membrane potential becomes more negative or depolarization and the membrane potential becomes less negative toward zero Valič et al. The electropermeabilization effects from the application of low-strength electric fields has been shown to boost the effect of antibiotics against bacteria in biofilms, diminishing the concentration of antibiotic needed to kill bacteria Costerton et al. On the other hand, high strength electric pulses resulted in an efficient bactericidal effect on both gram-positive and gram-negative bacteria. These strategies however require the direct application of an electrical field on bacterial solution, which may not be recommendable for in vivo applications. Similarly to other stimuli previously mentioned the formation of reactive oxidative species ROS such as hydrogen peroxide H 2 O 2 and reactive nitrogen species RNS was also indicated as a possible mechanism of action for the bactericidal effect of low strength electric field Boda and Basu, Electrical stimulus has been mainly reported to impart bactericidal and bacteriostatic effect rather than a proliferation effect. Nevertheless, it has been also reported that low frequency mechanical stimuli leading to surface charge variations, induce similar effect to those occurring with eukaryotic cells, i. Besides chemotaxis, another important example of how bacteria sense its environment is the QS mechanism they use to express virulence factors, allowing bacteria to regulate community-wide behaviors including biofilm formation, virulence, conjugation, sporulation, and swarming motility Rutherford and Bassler, This mechanism of cell-to-cell communication is based on the production, secretion, and detection of small signaling molecules, called autoinducers AIs. In the QS-regulated communication, bacteria secrete signaling molecules, the AIs that are further recognized by specific receptors, allowing bacteria to act collectively as a multicellular microorganism. This knowledge is being increasingly used to develop new strategies for infection control Figure 4A. The inactivation of the QS signals in a process called quorum quenching QQ is an innovative strategy to control bacterial infections Hentzer and Givskov, ; Roche et al. Brominated furanones interfere with QS by acting as antagonists to receptors Rasmussen and Givskov, ; Kociolek, Similarly, enzymes such as acylase and lactonase have been shown to selectively degrade N-Acyl homoserine lactone AHL signals of Gram-negative bacteria Dong et al. In line with this knowledge, acylase has been successfully used to coat indwelling medical devices through functionalization techniques such as layer-by-layer technique. The enzyme multilayer coatings significantly reduced the bacterial load and biofilm formation on functionalized silicone-based urinary catheters, assessed with an in vitro catheterized bladder model Ivanova et al. Moreover, the QQ enzyme coatings were fully biocompatible since they were tested with mammalian cells such as fibroblasts over 7 days, the extended useful life of urinary catheters, and no toxicity was observed. Figure 4. A Simplified QS system of Gram-negative bacteria, general chemical formula of the signaling molecules and B strategies to QQ including the enzymatic degradation of AHL signals by AHL-lactonase and AHL-acylase. The main advantage of this approach is that it attenuates virulence, exerting less selective pressure on bacteria and reducing the risk of resistance development to drugs. Moreover, it affects bacterial behavior but does not kill or inhibits their growth, thus allowing the host defense system to eliminate attenuated bacteria or substantially increase the effect of co-administered antibiotics Ivanova et al. The action of such enzymes Figure 4B creates the conditions to eradicate the infection by the natural host immune system before virulence is established. The application of a magnetically and electrically active microenvironment is a strategy that has been widely explored in mammalian cells and that can be also used for tailoring specific bacterial responses. It is well-stablished that electroactive materials such as piezoelectric polymers and magnetoelectric composites develop voltage variations at the surface of the material when a mechanical stress Ribeiro et al. Knowing that bacteria are also able to sense these types of stimuli, these materials seem to constitute a suitable approach for both anti- and pro-microbial applications by developing active surfaces based on those materials. Examples of such materials are the ones possessing mechanoelectric, magnetostrictive, and magnetoelectric properties. Mechanoelectric materials are materials mainly constituted by, for example, piezoelectric polymers that respond to a mechanical stimulus, inducing an electrical charge variation in the material Figure 5A. Magnetic and magnetoelectric materials are composites comprising magnetic or magnetostrictive particles and a piezoelectric polymer. Due to their magnetic component, they sense a magnetic field that induce a mechanical stimulation on the material, due the incorporated magnetic or magnetostrictive properties, which further induce an electrical polarization variation due to the piezoelectric phase present in the composite Figures 5B,C. These materials thus respond to different stimuli, namely mechanical and magnetic field. Both stimuli are induced with the help of a bioreactor that provides specific cues on the materials and thus on the cells, for cell response investigation studies, or on active coatings through the surface functionalization of materials where those stimuli are present or can be induced, as for example in biomedical devices. Figure 5. Schematic representation of the A mechanoelectric properties of a material upon the application of mechanical stimuli and B,C magnetoelectric properties of scaffolds upon the application of magnetic stimuli. Piezoelectric synthetic polymers are the most widely used in the development of mechanoelectric materials for biomedical applications. Poly vinylidene fluoride PVDF and vinylidene fluoride VDF copolymers possess high electroactive properties, including piezoelectric, pyroelectric, and ferroelectric properties, which makes them particularly used Dubois, ; Serrado Nunes et al. Despite the fact that PVDF and its copolymers are not biodegradable, they are biostable and thus widely used. That is why poly L-lactid acid PLLA Preethi Soundarya et al. On the other hand, magnetic nanocomposites with magnetoelectric properties may be obtained by adding nanomaterials such as pure metals Co, Fe, Ni and metal oxides iron oxides Fe 2 O 3 or Fe 3 O 4 and ferrites such as BaFe 12 O 19 and CoFe 2 O 4 combined with the above-mentioned piezoelectric polymers Kudr et al. The possibility to remotely control tissue stimulation without the need of patient movement is certainly an innovative approach and is regarded as a breakthrough platform for tissue engineering applications Silva et al. In fact, the magnetic actuation ability of the magnetoelectric composite allows the mechanical and electrical stimuli of neighboring cells Martins and Lanceros-Méndez, In microbiology this approach could also be valuable, for example, for the prevention of infection of orthopedic indwelling devices by external stimulation. As previously mentioned, the development of these kind of materials has been explored in tissue engineering but poorly investigated in microbiology. The effect of a strong electrical field on the bacteria behavior, rather than acoustic mechanic waves, has been reported in a study where the effect of a piezoelectric material ceramics on bacteria was performed and the killing effect was due to the formation of ROS Tan et al. On the other hand, the proliferation effect of bacteria has been observed at the surface of electrically polarized hydroxyapatite Ueshima et al. Recent findings reported that bacterial cells behavior, which grow upon piezoelectric polymers, may be tailored according to the surface charge of the material and on the application of weak electrical field, promoted by a piezoelectric polymer under mechanical stimuli, demonstrating a different behavior between Gram-positive and Gram-negative cells Carvalho et al. The Gram-positive bacteria seems not adhere to positively charged surface, as opposed to the negatively charged surface. In the presence of an electrical stimuli, this strain shows a different behavior: the lower frequency promotes the antifouling and the higher stimulates the bacteria adhesion Figure 6 Carvalho et al. Figure 6. Such approaches are important to further define suitable anti- and pro-microbial strategies, intended for pathogenic and functional bacteria, respectively Carvalho et al. The ambition to create novel strategies to fight bacteria resistance using physical stimuli is an attractive and valid approach that relies on the fact that bacteria sense their environment and respond to it. This review thus calls the attention for the use of innovative electroactive smart materials as a novel tool for tailoring bacteria behavior and thus fight bacteria resistance using, not only anti-microbial strategies, but also pro-microbial ones. The most attractive feature of using such materials is the possibility for triggering the inhibition and proliferation of bacteria by changing the conditions applied, when bioreactors or smart and responsive surfaces and coatings are used. From one side, defining the conditions for antimicrobial strategies will allow these materials to be used synergistically with commonly applied antibiotics or other innovative elements that assist the antibacterial effect, reducing the quantity of antibiotic needed for killing bacteria. Such strategies cause less evolutionary selective pressure on bacteria population and thus prevent the emergence of resistance mechanisms. On the other side, defining the conditions for proper bacteria proliferation approaches will be the opportunity to pursue a pro-microbial activity, potentiating the function of human microbiome in assisting vital functions in our body. A healthy microbiome is essential for human and animal well-being since it has the ability to educate the immune system and keep the pathogenic bacteria out, apart from the obvious role it has on the digestive system. Therefore, novel materials or coatings that assist the antimicrobial effect and thus prevent the occurrence of nosocomial infections in clinical settings due to the contamination of medical devices such as stents and catheters or medical textiles are very appealing and needed to be used as first line defense against pathogenic bacteria, while potentiating the action of beneficial bacteria will allow to reinforce the microbiome, essential for disease prevention. More importantly, the application of electroactive materials may thus be the future for developing smart implantable devices, which due to their electric-sensitive properties may be used not only to promote anti- and pro-microbial strategies but also to take advantage of their characteristics for sensor applications. All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Amaro, L. Tailored biodegradable and electroactive poly Hydroxybutyrate-co-hydroxyvalerate based morphologies for tissue engineering applications. doi: PubMed Abstract CrossRef Full Text Google Scholar. Asadi, M. Bacterial inhibition by electrical stimulation. Wound Care 3, 91— Biais, N. Force-dependent polymorphism in type IV pili reveals hidden epitopes. Bidan, C. Magneto-active substrates for local mechanical stimulation of living cells. Binhi, V. Effect of static magnetic field on E. coli cells and individual rotations of ion—protein complexes. Bioelectromagnetics 22, 79— Boda, S. Engineered biomaterial and biophysical stimulation as combinatorial strategies to address prosthetic infection by pathogenic bacteria. Part B , — Bruni, G. Voltage-gated calcium flux mediates Escherichia coli mechanosensation. Cars, O. Meeting the challenge of antibiotic resistance. BMJ a Cartmell, H. Kumar Weinheim: Wiley , — CrossRef Full Text Google Scholar. Carvalho, E. Tailoring bacteria response by piezoelectric stimulation. Different types of stimuli can be triggered only upon the existence of bacteria, leading to the release of antibacterial molecules that in turn kill the bacteria. In particular, the toxin-triggered, pH-responsive, and dual stimulus-responsive adaptive antibacterial biomaterials are introduced. Finally, the state of the art in fabrication of dual responsive antibacterial biomaterials and tissue integration in medical implants is discussed. Keywords: Anti-foulings; Antibacterial; Antibiotic resistance; Biofilm; Tissue engineering. |
| Adaptive antibacterial biomaterial surfaces and their applications | It was during Second World War in the s that antibiotics were introduced, saving innumerous wounded soldiers and fast became available for use in the general population. It was then just a matter of time until the resistance to several antibiotics take place. In fact, nowadays, antibiotic resistance is already and should be a public health concern and novel strategies are indeed needed to fight AMR. Human intestines are home for many different microbes, some of which create resistance to the antibiotics they are exposed to. These resistant strains then spread from person to person, in communities or in hospitals Figure 2B , ultimately leading to the problem of bacteria resistance. Human body is constituted by trillion cells but only 1 in 10 is actually human. The remaining cells are microorganisms such as bacteria Relman, These microorganisms are harmless and live in perfect balance with human body, playing an important role in supporting and maintaining vital functions such as our immune and digestive systems Relman, However, when this balance is broken and the delicate ecosystems that bacteria carefully construct in different parts of human body are disrupted, bacteria become pathogenic, causing infection diseases. Pro-microbial approaches thus constitute one strategy that should call the attention of the scientific community. In a broader sense, the equilibrium between anti- and pro-microbial should be an important strategy. Despite being often associated with virulence, infection and disease, bacteria are considered very important microorganisms to sustain human life. They are responsible for the correct functioning of our immune, respiratory, and digestive system Ichinohe et al. That is why the right approach to obtain an effective strategy for infection control is to reinforce our beneficial microbial population, the microbiome, in a pro-microbial strategy, while providing an appropriate antimicrobial agent for full eradication of pathogenic bacterial anti-microbial strategy , without the possibility of developing resistance. The equilibrium between these anti- and pro-microbial approaches is an important twin sustainable strategy for limiting AMR Jørgensen et al. Meanwhile, the benefits of microbiome are largely overlooked by the scientific community. The benefits derived from the diversity of beneficial microbes has only recently been proposed Jørgensen et al. Among all antibiotics available on the market nowadays, the most recent class was discovered in the s, which demonstrates the difficulty on the process of finding new effective antimicrobials. It is time to, together with focusing attention on the development of new antibiotics, give more relevance to the diversity of microbes present in the human body that assists on the eradication of harmful bacteria, which means the right balance between anti- and pro-microbial strategies. In terms of pro-microbial strategies, a technique that is gaining more attention among the scientific community for the treatment and prevention of some infectious disease is the fecal microbiota transplantation FMT. The process consists in the infusion of beneficial bacteria from the stool of a healthy donor into a recipient with a disease related to an unhealthy gut microbiome Kim and Gluck, FMT has been successfully used to treat infections caused by Clostridium difficile C. difficile , but also tested and recommended for other conditions such as inflammatory bowel disease IBD , autoimmune disorders, certain allergic diseases, and metabolic disorders such as obesity Choi and Cho, The future of infectious disease treatment is thus the promotion of pro-microbial strategies such as FMT. Future challenges regarding this technology are the safety in delivering FMT to patients, being imperative to standardize the methodologies and prepare highly specialized laboratories for stool preparation. Another challenge is to identify the effectiveness of microbiota-based medicines and identify the specific bacteria responsible for this effect. Regarding anti-microbial strategies, synergistic approaches for inhibiting bacterial pathogenesis, i. Such strategies cause less evolutionary stress on bacteria population and thus prevent the emergence of resistance mechanisms. The increasing understanding of bacterial pathogenesis and intercellular communication in a broader sense, both from the physical and biochemical points-of-view has been a valuable tool to develop new strategies that meets these challenges Hajipour et al. Taking into consideration that bacteria can indeed feel the surrounding environment and modify their phenotype in response to it, the main cues responsible for the tailoring of bacteria behavior and thus assist the action of antibiotics are depicted in Figure 3. Several studies have been performed on the effect of specific stimuli on bacteria, namely the mechanical, magnetic, electrical, and biochemical [quorum sensing QS mechanism] cues. Their effects on bacterial cell, advantages and disadvantages are summarized in Table 1. Figure 3. Stimuli that bacteria sense and the mechanism of action of each bactericidal effect. Table 1. Summary of the effects of different physical and biochemical stimuli on bacteria. The effect of mechanical vibrations on bacteria surface adhesion, proliferation and virulence has been mainly evaluated for the inhibition of biofilm formation, a protective mode of growth that confer pathogenic bacteria increased resistance to conventional antibiotics and host defenses mechanisms Mah and O'Toole, Surface acoustic waves generated from electrically activated piezo elements has been reported to be repulsive to bacteria and interfere with the docking and attachment of planktonic microorganisms to solid surfaces Hazan et al. Also, vibration loads generated by magnetoelastic materials, which possess magnetostrictive properties, converting a magnetic stimulus into a mechanical deformation, were found to significantly reduce the adherent bacteria on samples exposed to different types of microorganisms Paces et al. The use of ultrasounds have also been used as a mean for preventing biofilm formation. The application of ultrasound induce an acoustic cavitation phenomenon, which results in the formation of cavitation bubbles that implode and generate shock waves that cause mechanical damage to the bacteria and the formation of free radicals that creates oxidative stress to bacteria Figure 3 Gera and Doores, To obtain a relevant reduction on bacterial counts, ultrasound has been applied in combination with antibiotics as a synergistic approach. Pseudomonas aeruginosa P. aeruginosa and E. coli biofilms were thus efficiently eradicated by gentamicin sulfate in combination with ultrasound Qian et al. Also, the killing effect of ZnO NPs against Staphylococcus aureus S. aureus and P. aeruginosa was boosted when ultrasound was applied to the medium Justin and Thomas, It is worth mentioning that the effect of the mechanical damage created by the acoustic cavitation of microbubbles in the blood, the possibility to induce rupture of blood vessel walls and interfere with blood flow may limit the application of this type of strategies in vivo Chen et al. The influence of magnetic field on biological systems, namely on biomolecules, cells and living organisms, is an important field of research due to the emergence of electromagnetic pollution in the form of low and high radio frequency magnetic fields from electronic devices Boda and Basu, It is believed that magnetic field interfere with the mechanism of ion transport via membrane channel proteins, leading to osmotic imbalance and membrane rupture. These phenomenon is attributed to the diamagnetic anisotropy exhibited by the large number of membrane proteins on bacteria cell, which result from the axial alignment of peptide bonds and specific amino acid residues containing aromatic groups Worcester, Therefore, the lipids and ion channel proteins that are present in bacterial membrane undergo conformational changes that lead to the dysfunction of these proteins, disrupting essential ion transport mechanisms on bacteria. One of the first reports of this effect on bacteria proved that magnetic field cause rotational motion of ion-protein complexes leading to the escape in E. coli Binhi et al. The generation of free radicals upon application of a magnetic field, leading to bacterial cell oxidative stress and genotoxicity Figure 3 has been another proposed mechanism of action involved in bacterial killing and possibly mammalian cell damage Ghodbane et al. Further studies need to be performed to assess the potential harm these approaches might induce to biological systems. It is known that direct application of strong electric fields may be bactericidal or a mean of preventing device-related infections, which are caused by biofilm formation, or even to disinfect contaminated liquids Poortinga et al. Moreover, the ability of electric fields to promote wound healing through angiogenesis while reducing microbial bioburden at the surface of material has already been proven Asadi and Torkaman, The electrical-based killing mechanism of action involves an increase in cell membrane permeability, known as electropermeabilization or irreversible electroporation. The perturbation of these ion concentrations can lead to hyperpolarization, wherein the membrane potential becomes more negative or depolarization and the membrane potential becomes less negative toward zero Valič et al. The electropermeabilization effects from the application of low-strength electric fields has been shown to boost the effect of antibiotics against bacteria in biofilms, diminishing the concentration of antibiotic needed to kill bacteria Costerton et al. On the other hand, high strength electric pulses resulted in an efficient bactericidal effect on both gram-positive and gram-negative bacteria. These strategies however require the direct application of an electrical field on bacterial solution, which may not be recommendable for in vivo applications. Similarly to other stimuli previously mentioned the formation of reactive oxidative species ROS such as hydrogen peroxide H 2 O 2 and reactive nitrogen species RNS was also indicated as a possible mechanism of action for the bactericidal effect of low strength electric field Boda and Basu, Electrical stimulus has been mainly reported to impart bactericidal and bacteriostatic effect rather than a proliferation effect. Nevertheless, it has been also reported that low frequency mechanical stimuli leading to surface charge variations, induce similar effect to those occurring with eukaryotic cells, i. Besides chemotaxis, another important example of how bacteria sense its environment is the QS mechanism they use to express virulence factors, allowing bacteria to regulate community-wide behaviors including biofilm formation, virulence, conjugation, sporulation, and swarming motility Rutherford and Bassler, This mechanism of cell-to-cell communication is based on the production, secretion, and detection of small signaling molecules, called autoinducers AIs. In the QS-regulated communication, bacteria secrete signaling molecules, the AIs that are further recognized by specific receptors, allowing bacteria to act collectively as a multicellular microorganism. This knowledge is being increasingly used to develop new strategies for infection control Figure 4A. The inactivation of the QS signals in a process called quorum quenching QQ is an innovative strategy to control bacterial infections Hentzer and Givskov, ; Roche et al. Brominated furanones interfere with QS by acting as antagonists to receptors Rasmussen and Givskov, ; Kociolek, Similarly, enzymes such as acylase and lactonase have been shown to selectively degrade N-Acyl homoserine lactone AHL signals of Gram-negative bacteria Dong et al. In line with this knowledge, acylase has been successfully used to coat indwelling medical devices through functionalization techniques such as layer-by-layer technique. The enzyme multilayer coatings significantly reduced the bacterial load and biofilm formation on functionalized silicone-based urinary catheters, assessed with an in vitro catheterized bladder model Ivanova et al. Moreover, the QQ enzyme coatings were fully biocompatible since they were tested with mammalian cells such as fibroblasts over 7 days, the extended useful life of urinary catheters, and no toxicity was observed. Figure 4. A Simplified QS system of Gram-negative bacteria, general chemical formula of the signaling molecules and B strategies to QQ including the enzymatic degradation of AHL signals by AHL-lactonase and AHL-acylase. The main advantage of this approach is that it attenuates virulence, exerting less selective pressure on bacteria and reducing the risk of resistance development to drugs. Moreover, it affects bacterial behavior but does not kill or inhibits their growth, thus allowing the host defense system to eliminate attenuated bacteria or substantially increase the effect of co-administered antibiotics Ivanova et al. The action of such enzymes Figure 4B creates the conditions to eradicate the infection by the natural host immune system before virulence is established. The application of a magnetically and electrically active microenvironment is a strategy that has been widely explored in mammalian cells and that can be also used for tailoring specific bacterial responses. It is well-stablished that electroactive materials such as piezoelectric polymers and magnetoelectric composites develop voltage variations at the surface of the material when a mechanical stress Ribeiro et al. Knowing that bacteria are also able to sense these types of stimuli, these materials seem to constitute a suitable approach for both anti- and pro-microbial applications by developing active surfaces based on those materials. Examples of such materials are the ones possessing mechanoelectric, magnetostrictive, and magnetoelectric properties. Mechanoelectric materials are materials mainly constituted by, for example, piezoelectric polymers that respond to a mechanical stimulus, inducing an electrical charge variation in the material Figure 5A. Magnetic and magnetoelectric materials are composites comprising magnetic or magnetostrictive particles and a piezoelectric polymer. Due to their magnetic component, they sense a magnetic field that induce a mechanical stimulation on the material, due the incorporated magnetic or magnetostrictive properties, which further induce an electrical polarization variation due to the piezoelectric phase present in the composite Figures 5B,C. These materials thus respond to different stimuli, namely mechanical and magnetic field. Both stimuli are induced with the help of a bioreactor that provides specific cues on the materials and thus on the cells, for cell response investigation studies, or on active coatings through the surface functionalization of materials where those stimuli are present or can be induced, as for example in biomedical devices. Figure 5. Schematic representation of the A mechanoelectric properties of a material upon the application of mechanical stimuli and B,C magnetoelectric properties of scaffolds upon the application of magnetic stimuli. Piezoelectric synthetic polymers are the most widely used in the development of mechanoelectric materials for biomedical applications. Poly vinylidene fluoride PVDF and vinylidene fluoride VDF copolymers possess high electroactive properties, including piezoelectric, pyroelectric, and ferroelectric properties, which makes them particularly used Dubois, ; Serrado Nunes et al. Despite the fact that PVDF and its copolymers are not biodegradable, they are biostable and thus widely used. That is why poly L-lactid acid PLLA Preethi Soundarya et al. On the other hand, magnetic nanocomposites with magnetoelectric properties may be obtained by adding nanomaterials such as pure metals Co, Fe, Ni and metal oxides iron oxides Fe 2 O 3 or Fe 3 O 4 and ferrites such as BaFe 12 O 19 and CoFe 2 O 4 combined with the above-mentioned piezoelectric polymers Kudr et al. The possibility to remotely control tissue stimulation without the need of patient movement is certainly an innovative approach and is regarded as a breakthrough platform for tissue engineering applications Silva et al. In fact, the magnetic actuation ability of the magnetoelectric composite allows the mechanical and electrical stimuli of neighboring cells Martins and Lanceros-Méndez, In microbiology this approach could also be valuable, for example, for the prevention of infection of orthopedic indwelling devices by external stimulation. As previously mentioned, the development of these kind of materials has been explored in tissue engineering but poorly investigated in microbiology. The effect of a strong electrical field on the bacteria behavior, rather than acoustic mechanic waves, has been reported in a study where the effect of a piezoelectric material ceramics on bacteria was performed and the killing effect was due to the formation of ROS Tan et al. On the other hand, the proliferation effect of bacteria has been observed at the surface of electrically polarized hydroxyapatite Ueshima et al. Recent findings reported that bacterial cells behavior, which grow upon piezoelectric polymers, may be tailored according to the surface charge of the material and on the application of weak electrical field, promoted by a piezoelectric polymer under mechanical stimuli, demonstrating a different behavior between Gram-positive and Gram-negative cells Carvalho et al. The Gram-positive bacteria seems not adhere to positively charged surface, as opposed to the negatively charged surface. In the presence of an electrical stimuli, this strain shows a different behavior: the lower frequency promotes the antifouling and the higher stimulates the bacteria adhesion Figure 6 Carvalho et al. Figure 6. Such approaches are important to further define suitable anti- and pro-microbial strategies, intended for pathogenic and functional bacteria, respectively Carvalho et al. The ambition to create novel strategies to fight bacteria resistance using physical stimuli is an attractive and valid approach that relies on the fact that bacteria sense their environment and respond to it. This review thus calls the attention for the use of innovative electroactive smart materials as a novel tool for tailoring bacteria behavior and thus fight bacteria resistance using, not only anti-microbial strategies, but also pro-microbial ones. The most attractive feature of using such materials is the possibility for triggering the inhibition and proliferation of bacteria by changing the conditions applied, when bioreactors or smart and responsive surfaces and coatings are used. From one side, defining the conditions for antimicrobial strategies will allow these materials to be used synergistically with commonly applied antibiotics or other innovative elements that assist the antibacterial effect, reducing the quantity of antibiotic needed for killing bacteria. Such strategies cause less evolutionary selective pressure on bacteria population and thus prevent the emergence of resistance mechanisms. On the other side, defining the conditions for proper bacteria proliferation approaches will be the opportunity to pursue a pro-microbial activity, potentiating the function of human microbiome in assisting vital functions in our body. A healthy microbiome is essential for human and animal well-being since it has the ability to educate the immune system and keep the pathogenic bacteria out, apart from the obvious role it has on the digestive system. Therefore, novel materials or coatings that assist the antimicrobial effect and thus prevent the occurrence of nosocomial infections in clinical settings due to the contamination of medical devices such as stents and catheters or medical textiles are very appealing and needed to be used as first line defense against pathogenic bacteria, while potentiating the action of beneficial bacteria will allow to reinforce the microbiome, essential for disease prevention. More importantly, the application of electroactive materials may thus be the future for developing smart implantable devices, which due to their electric-sensitive properties may be used not only to promote anti- and pro-microbial strategies but also to take advantage of their characteristics for sensor applications. All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Amaro, L. Tailored biodegradable and electroactive poly Hydroxybutyrate-co-hydroxyvalerate based morphologies for tissue engineering applications. doi: PubMed Abstract CrossRef Full Text Google Scholar. Asadi, M. Bacterial inhibition by electrical stimulation. Wound Care 3, 91— Biais, N. Force-dependent polymorphism in type IV pili reveals hidden epitopes. Bidan, C. 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The effect of electric fields on bacterial attachment to conductive surfaces. Soft Matter 9, — Gera, N. Kinetics and mechanism of bacterial inactivation by ultrasound waves and sonoprotective effect of milk components. Food Sci. Ghasemi-Mobarakeh, L. Application of conductive polymers, scaffolds and electrical stimulation for nerve tissue engineering. Tissue Eng. Ghodbane, S. Bioeffects of static magnetic fields: oxidative stress, genotoxic effects, and cancer studies. Biomed Res. Gould, I. New antibiotic agents in the pipeline and how they can help overcome microbial resistance. Virulence 4, — Graczyk, E. The neural basis of perceived intensity in natural and artificial touch. Guduru, R. Magnetic field-controlled release of paclitaxel drug from functionalized magnetoelectric nanoparticles. Particle Particle Syst. Hajipour, M. Antibacterial properties of nanoparticles. Trends Biotechnol. Hazan, Z. Effective prevention of microbial biofilm formation on medical devices by low-energy surface acoustic waves. Specifically, it has been proposed that DNA methylation by the DAM methylase could be responsible for: i the presence and inheritance of different gene expression profiles [ 13 — 18 ] and ii the variability in expression observed for methylated genes. The activity of the DAM methylase gene has been found correlated with the emergence of adaptive resistance. In fact, high expression of the DAM methylase gene, increases the survival rate by a factor of five in cells treated with nalidixic acid [ 1 ]. Furthermore, this heightened resistance is consistent with a two fold increase in the expression of efflux pumps [ 1 ]. Therefore, DNA methylation affecting the activity of efflux pumps, could be a very likely explanation of the increased resistance observed experimentally, along with the fact that it will produce enough heterogeneity in the population for antibiotic selection to act on [ 1 , 13 , 15 , 16 ]. In the light of these experimental observations, we propose a theoretical model to quantitatively determine what specific elements are essential for the emergence of reversible resistance. One key hypothesis of the model is that the Efflux Pump Regulatory Network EPRN from now on is a target of epigenetic modifications. Specifically, these modifications will produce variability in the gene expression patterns of the EPRN transcription factors. After cell division, such gene expression patterns will be inherited from mother to daughter and will consequently impose correlations in the dynamics of the EPRN across generations. Our results indicate that this mechanism is central for the emergence of adaptive resistance. Our model also predicts that epigenetic variability and mother-daughter correlations are not sufficient to explain reversibility of resistance. Here, we demonstrate that reversibility is a consequence of a trade-off between the benefit of efflux pumps, keeping the antibiotic below lethal levels, and a cost associated with their activity, as they are known to pump out essential metabolites and therefore slow down cell growth [ 18 ]. Our theoretical framework aims at identifying and deciphering the role of each phenomenological observation in the emergence of adaptive resistance in order to provide a comprehensive and quantitative picture of this reversible phenomenon. There are several EPRNs present in bacteria, and most of them share a number of prototypic characteristics that make them qualitatively similar to each other. We have constructed a single-cell model based on the acrAB-tolC system present in Escherichia coli , since it has been historically the most widely studied and well characterized system [ 19 — 28 ]. This simplified version includes the main components of the acrAB-tolC system which are present not only in E. coli but also in other gram negative bacteria [ 9 ]. Note that the network depicted in Fig. For the reduced network we have chosen only the most dynamically important elements. This choice was not arbitrary but based on extensive numerical simulations that identified the essential components of the network, and eliminated the ones that did not provide additional information or present significant changes in the dynamics when removed see S1 Text and S1 Fig. For instance, we found that AcrR only reduces the expression of AcrA and AcrB linearly, and its role could be accounted for, by just changing the transcription rate of the AcrAB operon. This result is in agreement with previous research that indicates that AcrR only fine-tunes the expression of AcrAB to prevent unwanted expression [ 29 ], a scenario that becomes irrelevant when the antibiotic is introduced. All the results presented in this work were obtained with the simplified network. Nonetheless, the simplified network gives essentially the same results as the more complete version of this system see S1 Fig. Therefore, without loss of generality or biological realism, we use generic names such as Activator , Repressor , etc. It is worth mentioning that the acrAB-tolC system responds to several antibiotics and also to non-lethal chemicals such as salicylate , some of which were used in the adaptive resistance experiments mentioned earlier [ 20 — 22 ]. We will use the terms antibiotic and inducer indistinctly to refer to any chemical that promotes the activation of the EPRN. Arrows indicate positive regulation. Blunt arrows indicate repression. A Literature base reconstruction of the AcrAB-TolC efflux pump regulatory network of Escherichia coli as reported on [ 9 ]. B Simplified version of the AcrAB-TolC efflux pump regulatory network EPRN. The activator Act and repressor Rep are two Transcriptional Factors that belong to the same transcriptional unit EPRN operon, indicated by the dashed line. When the repressor occupies its DNA binding site, the expression of the operon is restrained. Nonetheless, when the antibiotic or inducer, Ind enters the cell, it inactivates the repressor by binding to it, allowing the operon to be actively transcribed, promoting the production of pumps and decreasing the synthesis of porins this last process is known to occur through an intermediary. Both food and inducer are expelled by the efflux pump system. In the population model, a reduction in food concentration implies an increase in the division time. The main idea behind the dynamics of the EPRN is the following. Two transcription factors are present: an activator and a repressor, both belonging to the same operon hereafter referred to as the EPRN-operon. They auto-regulate their own transcription by binding to the EPRN-operon promoter region. In the absence of antibiotic the EPRN-operon is expressed at low levels [ 22 , 23 ]. This is because the repressor molecule has a higher binding affinity at least four times than the activator [ 24 , 25 ] and therefore the operon transcription is mainly restrained [ 26 ]. When the antibiotic enters the cell, usually via membrane porins, it binds tightly to the aforementioned repressor molecule, inactivating it. This new repressor-inducer conformation is now unable to bind to the EPRN-operon promoter region, allowing the activator to be transcribed. Since the activator promotes its own expression, it increases its concentration rapidly, boosting the production of efflux pumps; at the same time it represses the expression of membrane porins which reduces the intake of antibiotics [ 24 , 27 , 28 ]. We use a system of stochastic differential equations see Supporting Information to model the single-cell dynamics of the EPRN shown in Fig. Validation and parameter calibration for this system were done by comparing our simulations with experimental data from E. coli wild-type cells and ΔtolC mutant strains [ 30 ] see S1 Text , S1 and S2 Tables and S2 and S3 Figs. The efflux of antibiotics depends on two main parameters: the transcription rate β 0 of the EPRN operon, which ultimately affects the amount of available pumps, and the pump efficiency ε I that controls how much antibiotic the pumps can expel at a certain time. We will see later that by introducing cell-to-cell variability and mother-daughter correlations in these two parameters, highly resistant populations can arise. Importantly, due to the large size of the parameter space, we do not perform an exhaustive parameter search to determine the complete regions over which our results hold. Rather, in S1 Table we present a set of parameters that qualitatively reproduce the experimental observations. Nonetheless, in the Supporting Information we provide a sampling of a wide region of the parameter space for which our results hold see S1 Text and S4 and S5 Figs. The population consists of a set of replicating cells, each one represented by a copy of the system of equations governing the dynamics of the EPRN formally presented in the SI. Each cell runs internally its own system of equations independently from other cells. These cells will grow or die depending on their internal concentration of nutrients and antibiotics, respectively. Cell-to-cell variability is implemented by slightly changing the two parameters that most affect the capability of the pumps to reduce the internal concentration of antibiotics. One such parameter is the efficiency ε I of the efflux pumps. A high efficiency will correspond to a decreased toxicity of the antibiotic, which is due to pumps operating more rapidly or with greater specificity. We assume that changes in ε I are caused by genetic mutations. However, since mutations alone cannot account for the rapid emergence of adaptive resistance [ 1 , 3 , 4 , 6 ], we also implement variability in the transcription rate β 0 of the EPRN operon. As stated in the introduction, we assume that cell-to-cell variations of this parameter are caused by epigenetic processes, most likely methylation [ 1 , 14 — 17 ]. It is known that different methylation patterns can produce different transcription rates by changing the DNA binding affinity of some transcription factors [ 14 — 17 ]. We found 12 DAM methylation sites GATC in the regulatory operon of the AcrAB-TolC efflux pump system in E. coli see SI. Each site can be in two states: either it is methylated or it is not. As there are several possible patterns, we will assume that β 0 the transcription rate changes as a continuous variable. However, this assumption is not crucial. We will see later that even under this scenario in which the values of β 0 are discrete and finite, the same qualitative results are obtained. Cells with different values of β 0 will produce pumps at different rates, which in turn affect their survival. Variability in the population is then introduced by selecting, for each cell, the parameters β 0 and ε I from Gaussian distributions G μ β , σ β and G μ ε , σ ε , respectively in each case μ is the mean and σ 2 is the variance. On the other hand, inheritance is implemented by correlating the mean values of these Gaussians across generations. To illustrate the inheritance mechanism in our model, let us consider the i th cell at generation t , which has a transcription rate β 0 i , t. In other words, at each generation and for each cell, β 0 is drawn from a Gaussian distribution G μ β , σ β whose average μ β is the value of β 0 previously owned by the corresponding mother. This mechanism, which clearly correlates the parameters β 0 along cell lineages, models the fact that methylation patterns that affect gene expression can be inherited with certain variability [ 14 — 17 ]. Inheritance in the pump efficiency ε I is implemented in an analogous way, but using the corresponding Gaussian distribution G μ ε , σ ε. However, since we are assuming that changes in β 0 are epigenetic whereas those in ε I are genetic, the time-scales at which significant modifications in these parameters occur, are very different. For it is known that phenotypic modifications due to epigenetic changes happen at rates at least one order of magnitude faster than those due to genetic changes [ 31 ]. Among the implications of using such small variances are that the changes in gene expression and pump efficiency between mother and daughter cells occur gradually. We also implemented for σ β a uniform distribution between 0 and 10, which allows abrupt changes in gene expression between the mother and daughter cells. However, when this type of abrupt changes are allowed in σ β , adaptive resistance is not observed see S1 Text and S6 Fig. We do not know, based on experimental measurements, which of the two mechanisms mentioned above i. uniform vs Gaussian randomness is more compatible with the effect caused by methylation. But, as we will see in the next section, our model predicts that when Gaussian distributions with small variances are used, adaptive resistance emerges, which is not the case for uniform distributions see S1 Text and S6 Fig. In order to quantify the effect that each type of inheritance has on the emergence of the resistance phenotype, we implemented four different scenarios: Control simulation: There is no inheritance, only variability. The distributions G μ β , σ β and G μ ε , σ ε remain the same for all the cells in the population and throughout generations. Genetic inheritance: Mother-daughter correlations are implemented only in the pump efficiency ε I but not in the transcription rate β 0. Epigenetic inheritance: Mother-daughter correlations are implemented only in the transcription rate β 0 but not in the pump efficiency ε I. Mixed inheritance: Mother-daughter correlations are implemented in both the transcription rate β 0 and the pump efficiency ε I. The synthesis and functioning of efflux pumps are associated with an energetic cost that must be taken into account. First, the pumps are very unspecific on its substrates [ 21 , 32 , 33 ]. Thus, in addition to antibiotics, they expel metabolites necessary for cell growth and division [ 34 ]. For instance, the acrAB-tolC efflux pump, is known to recognize a broad spectrum of chemicals. It also has a biased affinity towards phenolic rings, which are not only constituents of inducers of the system such as salicylic acid, but also of amino acids such as tyrosine [ 32 , 35 ]. Second, the synthesis of the pumps themselves large protein complexes and their functioning consume energy [ 9 , 32 , 35 ]. Therefore, it is reasonable to assume that the production and functioning of the pumps will slow down cell growth. This assumption is supported by experimental observations indicating that over-expression of efflux pumps is correlated with both high levels of resistance and decreased growth [ 18 , 36 , 37 ]. In our model we set this cost by making the internal concentration of nutrient in each cell depend inversely on the amount of pumps see Eq. The net result is a slowdown of the cell division rate, because a minimum internal nutrient concentration is required for division to happen. Thus, when the internal concentration of nutrients reaches a certain threshold θ F , the cell divides consuming the nutrient load, F. Clearly, the division time the time it takes to reach the threshold θ F depends on the amount of pumps, which in turn depends on the transcription rate β 0 and the concentration of inducer see S1 Text and S7 Fig. We have also included cell death in our population model. In order for the cell to survive, the efflux pumps need to keep the internal antibiotic concentration below the lethal level θ I. Whenever this threshold is reached, the cell dies and it is removed from the population. As in the experiments reported in Refs. Thus, after M shocks the external antibiotic concentration will be. After each antibiotic shock, indicated by downward arrows in Fig. As can be observed in Fig. By contrast when epigenetic inheritance is not present, every cell in the population dies after the first shock. The above results show that in our model variability alone is not enough for the emergence of adaptive resistance Fig. Analogously, genetic inheritance, which essentially consists of mother-daughter correlations occurring at long time scales, cannot give rise by itself to adaptive resistance either Fig. This figure shows tracking plots of populations growing in successively increasing concentrations of antibiotic. For each cell the concentration of Activator is plotted at constant time intervals dots. The four panels correspond to the four different inheritance scenarios mentioned in the main text. A Only epigenetic inheritance is implemented. B Mixed Inheritance. C Only genetic inheritance and D control no inheritance. The inset in A shows a zoomed in representation of the tracking plot, where one cell lineage is followed as it goes through several cell divisions and deaths. Since dead cells are removed immediately from the population, their expression is no longer visible and their curves terminate abruptly causing a step-like structure. Note that high levels of resistance can be achieved only when there is epigenetic inheritance A and B. Otherwise, the entire population dies after the first shock C and D. For such adaptive resistance to emerge, short-term mother-daughter correlations in the transcription rate β 0 of the EPRN operon need to be implemented in the model. Note that when both genetic and epigenetic inheritance are present Fig. It is important to mention that if the antibiotic shocks occur at high frequencies for instance less than 10 generations between two successive shocks , or if each antibiotic shock is much more intense e. twice or more than the previous one, we observe no surviving cells whatsoever in any of the four scenarios. It is also worth noting that if a discrete distribution for β 0 with 40 different values is used instead of a continuous Gaussian, the same qualitative results are obtained, as can be seen in S8 Fig. Therefore, adaptive resistance occurs even in the presence of moderate variability in gene expression, as long as there are mother-daughter correlations in such variability. So far, the difference between genetic and epigenetic inheritance consists on one hand, in the time scales at which these two processes produce phenotypic changes in the population, and on the other hand in the parameters they affect. Genetic inheritance affects the pump efficiency ε I whereas epigenetic inheritance affects the transcription rate β 0. Another important difference is that changes caused by genetic mutations are very unlikely to be reversible whereas epigenetic changes are much more likely to be reversible [ 31 , 38 ]. To test if our model can reproduce the experimentally observed reversibility, we replicate the simulations described in the previous section where levels of resistance are ramped higher for the mixed scenario. But now, after several rounds of selection, we remove the external antibiotic and allow the cells to grow and divide without stress. The population size decreases exponentially each time an antibiotic shock is applied. Note that when the antibiotic is removed the population growth returns to its wild-type behavior. These results are qualitatively similar to those observed experimentally [ 1 , 3 — 8 , 9 ]. However, this fact does not necessarily mean that the cells return to their wild type levels of susceptibility. For the cells that have reversed back to a sensitive phenotype could still have very high values of transcription rate, β 0 , and this high rate would imply that as soon as the antibiotic is applied again, the activator and the efflux pumps will be produced rapidly and at high concentrations. At this stage, most of the cells would be able to survive easily almost any antibiotic shock making the system non-reversible. A This tracking plot shows that the expression of the activator increases while the antibiotic shocks are applied as in Fig. Then, when the antibiotic is removed indicated by the tilted black arrow , the expression of the activator decreases abruptly and eventually reaches its basal level. B Size of the population as a function of time for the same simulation as in A. After each antibiotic shock small black arrows the population size decreases exponentially and the recovery time becomes longer with each shock. After the antibiotic is removed tilted black arrow the population comes back again to its wild-type WT growth rate. Note that the average increases while the shocks are applied and then gradually comes back to small values when the antibiotic is removed. Error bars indicate the standard deviation. It can be observed that the standard deviation increases with the antibiotic stress. The panels below show the full distribution G μ β , σ β at three different times: before any antibiotic is introduced circle ; after several antibiotic shocks star ; after a long period of time without antibiotic line. The only way for the population to truly reverse to its wild-type condition and become susceptible again is to return to their initial distribution G μ β , σ β , which is centered at low values of β 0. We expect this to happen because cells with a small β 0 duplicate faster than cells with large β 0 see S1 Text and S7 Fig. Since the values of β 0 are correlated across generations, cells with faster division rates low β 0 will eventually dominate the population, shifting the distribution G μ β , σ β towards the low β 0 region. Note that μ β increases as the antibiotic concentration is ramped higher. Then, when the external antibiotic is removed the average transcription rate across the population μ β decreases gradually, reaching the same value as in the original wild-type population. The lower panels in Fig. But then again, when the antibiotic is removed, the distribution eventually returns to its initial configuration. In our model the time-scale to produce a phenotypic change due to genetic mutations is one order of magnitude larger than that needed to produce a phenotypic change due to epigenetic modifications. Thus, to observe any significant increase in resistance produced by changes in the pump efficiency we need to run the simulation for a longer time. Interestingly, by doing this we obtain a nonreversible resistance, first driven by our mechanism of epigenetic inheritance which is reversible , and then fixed by genetic variation and inheritance of the pump efficiency. To observe this phenomenon, which can be considered analogous to genetic assimilation [ 39 , 40 , 41 ], we performed numerical experiments similar to the ones presented in the previous sections, where the population is first induced with M antibiotic shocks. The difference now is that we will let the population be in contact with the antibiotic for a very long time before removing it. A similar measure was used in [ 3 ]. In each case, the arrows indicate the time at which the antibiotic is removed. The results depicted in Fig. The blue curve deserves special attention. After this, the antibiotic concentration was kept constant until the time indicated by the blue arrow, at which the antibiotic was removed. Note that the RI keeps increasing even during the interval of steady antibiotic concentration. Note also that the final RI stationary value reached after the antibiotic is removed is five times larger for the blue curve than for all the other curves. It is worth noting that the black curve, corresponding to a control population growing in the absence of antibiotic, remains close to the initial low basal level throughout the entire simulation. Therefore, in our model antibiotic resistance occurs only as a response to the selective pressure imposed by the antibiotic and not by random genetic drift. A Resistance Index RI as a function of time for populations induced with M antibiotic shocks. The different curves correspond to different values of M , except by the black one which corresponds to a control population growing with no antibiotic. B Blow up showing the first generations. For each curve, the corresponding arrow indicates the time at which the antibiotic is removed. In the case of the blue curve, the asterisk indicates the time at which the last antibiotic shock is applied, after which the antibiotic concentration is kept constant. C Blow up of the last part of the simulation showing the point at which the antibiotic is removed from the population corresponding to the blue curve. It can be observed that in this case the final stationary value of the RI is about five times higher than that of the control population. D Evolution of the average transcription rate μ β and the average pump efficiency μ ε for the population corresponding to the blue curve. Notice that as soon as the antibiotic concentration is kept constant, μ β starts decreasing whereas μ ε keeps rising until the antibiotic is completely removed. This shows that the evolutionary process does not reach a stationary state or fixed point in the presence of antibiotic. It is important to mention that the increase in the basal level of the RI shown in Fig. Indeed, Fig. From Fig. However, as soon as the antibiotic concentration is kept constant, even at a high value, the average transcription rate μ β starts decreasing and reaches its initial low value at the end of the simulation. Contrary to this, the average pump efficiency μ ε keeps rising as long as there is antibiotic in the environment, reaching a steady value only when the antibiotic is removed. Thus, exposing the population to a high antibiotic concentration for a long time produces a non-reversible shift in the pump efficiency distribution P ε , permanently increasing the level of resistance of the population. It is also important to emphasize the difference between the survival rate SR and the resistance index RI. The former is defined as the fraction of cells that survive an induction, and this fraction ranges from 0 if no cell survives to 1 if all cells survive. On the other hand, the RI is the value of the antibiotic concentration at which the SR is 0. Therefore, the RI does not have to be between 0 and 1. Actually, its value depends on the units used to measure the antibiotic concentration in our case we use arbitrary units and the capability of the population to resist the antibiotic. This capability, in turn, depends on the way β 0 and ε I are distributed across the population. In each cell, these parameters determine the fixed points of the system only one fixed point exists for a given combination of β 0 and ε I in the range of concentrations explored in this work, see S1 Text and S9 Fig. The results presented in Fig. Adaptive resistance in bacteria is observed after subjecting a population to gradual increments of antibiotic concentration. Regardless of the level of resistance reached through this process, which can be very high , the resistance disappears after a few generations in the absence of antibiotic. Previous studies have independently identified epigenetic inheritance and phenotypic heterogeneity as important components involved in the emergence of adaptive resistance [ 1 , 3 , 4 , 6 , 7 , 8 , 11 ], but their role has never been evaluated quantitatively. Additionally, the molecular origin of reversibility observed in adaptive resistance has remained unclear. In this study we present a theoretical framework that identifies the essential mechanisms for the emergence, evolution and reversibility of adaptive resistance. We constructed a single-cell dynamic model of a prototypic efflux pump regulatory network EPRN that incorporates the most updated information available in the literature. We calibrated this model with experimental observations for wild type and mutant E. coli strains. We then grew a population of such single cells with growth dynamics obeying simple rules such as division, death, variability and inheritance of gene expression patterns. For each cell in the population we compute their EPRN temporal dynamics. Through this model we demonstrate that heterogeneity and mother-daughter correlations affecting transcription rates, specifically those of the EPRN main regulators, can explain the gradual amplification of the multidrug resistant phenotype. By contrast, mother-daughter correlations implemented in the pump efficiency, and developing at longer timescales, were not sufficient to make the population adapt and survive to successive antibiotic shocks but had a role in fixing resistance when the population had contact with antibiotics for a very long time. We also found that introducing a cost associated with the functioning of the EPRN was enough to explain the observed reversibility to the susceptible non-resistant phenotype. A previous report [ 11 ] proposed that adaptive resistance developed as a consequence of heterogeneity in gene expression because cells that randomly have a high production of efflux pumps survive, and those that did not, die. Through our model, we were able to show that although heterogeneity in gene expression is necessary, it is not sufficient to explain the emergence of resistance, nor its gradual response, as epigenetic inheritance of gene expression patterns is also necessary. Epigenetic modifications can change gene expression patterns at short time scales, providing a mechanism by which cells can adapt to changing environments quickly. At the same time, it allows for enough flexibility: if the environment returns to its earlier state, a population whose fitness is compromised by the new gene expression patterns can return to its previous state in a short time. Based on several experimental observations, another report [ 1 ] suggested that DNA methylation is a plausible mechanism driving this epigenetic inheritance. Methylation can indeed produce both the heterogeneity and epigenetic inheritance of gene expression patterns required for adaptive resistance to occur. Our results support this idea and specifically identify the regulatory regions of the main regulators of the EPRN as the most probable targets for the methylation process, as amplification of the antibiotic resistance do not occur without the mother-daughter correlations in gene transcription rates see Fig. Consequently, the process of DNA methylation in bacteria is potentially an important target for the development of therapeutic treatments in preventing the emergence of adaptive resistance. It is important to stress that variability in gene expression is known to be essential for adaptive resistance to occur [ 11 ]. So, our model incorporates this variability with the additional feature that it has to be inherited. Whether or not this variability is caused by methylation is not the central point. Nonetheless we propose DNA methylation of the marRAB operon as the possible cause of this variability because: i it can be inherited; ii mutant cells in which methylation is lacking are much more susceptible to antibiotics [ 1 ]; iii it provides the necessary variability in short periods of time required for adaptive resistance to emerge [ 1 ]. However, regardless of the precise mechanism behind this variability, the important point in our model is the existence of inheritable variability that can be quickly developed. For our results show that some heritable mechanism modifying the transcription rates of an efflux pump regulatory network must be present in order to observe adaptive resistance. Another interesting observation is the emergence of a stable form of resistance when the population is left in a medium with high concentrations of antibiotic for very long times. In our model this non-reversible resistance is produced by changes effectively improving the pump efficiency ε I , meaning that the pumps become better at distinguishing antibiotics from nutrients, so that they can pump out the former at a higher rate than the latter. Although these genetic modifications are rare and insufficient to save the population initially, they become important at longer times, transforming into an alternate source of resistance without adverse effects. Therefore, this heritable trait will, at longer time scales, permanently increase the basal levels of resistance of the population when it is under selective pressure see Fig. In our model genetic changes at each generation are small and increase the pump efficiency gradually Fig. In reality, genetic changes, although rare, may produce abrupt changes in the level of resistance of the population. The important point is that the fast epigenetic changes occurring in the transcription rate allow the population to survive long enough as to develop more stable and efficient forms of resistance. This behavior is consistent with experimental observations, showing that bacterial populations that have been continuously exposed to antibiotics are permanently much more resistant than populations that have been not [ 1 ]. We have based our simulations on the regulatory scheme of the widely known acrAB-tolC efflux pump system, for which many of the kinetic parameters are still unknown. In our study, we aimed to identify the essential mechanisms that could explain and reproduce adaptive resistance and our results hold in a significant region of the parameter space and not only for the particular values presented in S1 Table see S4 and S5 Figs. However, performing a deep search in the parameter space of the equations could reveal important constraints; such as the timescales at which epigenetic inheritance or genetic mutations must occur; or even the amount of pumps that the cell needs to produce which is to our current knowledge an unknown variable. We also explored alternative mechanisms that could yield resistance, such as uneven pump distribution in each cell division one daughter cells takes the majority of the pumps and increased mutation rates which increases the variability σ ε in the efficiency of the efflux pumps. The results, presented in the SI see S1 Text , S10 and S11 Figs. show that neither of these two mechanisms is able to produce adaptive resistance. Our model provides an explanation for the emergence of adaptive resistance based on the cost and benefit of the biological characteristics of an efflux pump system. It does not only predict the behavior of populations subjected to different antibiotic shocks and at different time, but also a number of different phenomena observed experimentally in bacterial populations, such as phenotypic reversibility, genetic assimilation, and even the survival rates of populations that have been pre-induced with non-lethal antibiotic concentrations see S1 Text and S12 Fig. Expression of the nodes corresponding to the activator MarA in the complete network and Activator in the simplified network and the pumps AcrAB-TolC in the complete network and Pumps in the simplified network. It can be observed that the curves for the complete and simplified networks are extremely similar in all cases. |
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So wird nicht gehen.
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