Persistent Bacteria Research Articles

Toxin-Antitoxin Systems and Bacterial Persistence (Review)

Persistence is a phenomenon in which bacteria avoid the lethal effects of antibiotics due to their transition to a physiological state, which enables tolerance to antibacterial drugs. Among the factors that contribute to persistence, a special role belongs to toxin–antitoxin (TA) systems, which are involved in the regulation of a wide range of physiological processes and have a significant effect on the results of antibiotic therapy. This review examines the mechanisms of the participation of TA systems in the formation of persistent bacteria, as well as the clinical importance of the phenomenon of persistence and methods for the control of phenotypically tolerant cells via regulation of the activity level of TA modules.

Slow growth determines nonheritable antibiotic resistance in Salmonella enterica.

Bacteria can withstand killing by bactericidal antibiotics through phenotypic changes mediated by their preexisting genetic repertoire. These changes can be exhibited transiently by a large fraction of the bacterial population, giving rise to tolerance, or displayed by a small subpopulation, giving rise to persistence. Apart from undermining the use of antibiotics, tolerant and persistent bacteria foster the emergence of antibiotic-resistant mutants. Persister formation has been attributed to alterations in the abundance of particular proteins, metabolites, and signaling molecules, including toxin-antitoxin modules, adenosine triphosphate, and guanosine (penta) tetraphosphate, respectively. Here, we report that persistent bacteria form as a result of slow growth alone, despite opposite changes in the abundance of such proteins, metabolites, and signaling molecules. Our findings argue that transitory disturbances to core activities, which are often linked to cell growth, promote a persister state regardless of the underlying physiological process responsible for the change in growth.

Revegetation on abandoned salt ponds relieves the seasonal fluctuation of soil microbiomes

BackgroundSalt pond restoration aims to recover the environmental damages that accumulated over the long history of salt production. Of the restoration strategies, phytoremediation that utilizes salt-tolerant plants and soil microorganisms to reduce the salt concentrations is believed to be environmentally-friendly. However, little is known about the change of bacterial community during salt pond restoration in the context of phytoremediation. In the present study, we used 16S metagenomics to compare seasonal changes of bacterial communities between the revegetated and barren salterns at Sicao, Taiwan.ResultsIn both saltern types, Proteobacteria, Planctomycetes, Chloroflexi, and Bacteroidetes were predominant at the phylum level. In the revegetated salterns, the soil microbiomes displayed high species diversities and underwent a stepwise transition across seasons. In the barren salterns, the soil microbiomes fluctuated greatly, indicating that mangroves tended to stabilize the soil microorganism communities over the succession. Bacteria in the order Halanaerobiaceae and archaea in the family Halobacteriaceae that were adapted to high salinity exclusively occurred in the barren salterns. Among the 441 persistent operational taxonomic units detected in the revegetated salterns, 387 (87.5%) were present as transient species in the barren salterns. Only 32 persistent bacteria were exclusively detected in the revegetated salterns. Possibly, salt-tolerant plants provided shelters for those new colonizers.ConclusionsThe collective data indicate that revegetation tended to stabilize the microbiome across seasons and enriched the microbial diversity in the salterns, especially species of Planctomycetes and Acidobacteria.

Plausible role of bacterial toxin–antitoxin system in persister cell formation and elimination

Although, a large proportion of pathogenic bacteria gets eliminated from hosts after antibiotic treatment, a fraction of population confronts against such effects and undergoes growth arrest to form persisters. Persistence in bacteria is a dormant physiological state where cells escape the effects of antimicrobials as well as other host immune defences without any genetic mutations. The state of dormancy is achieved through various complex phenomena and it is known that a gene pair named as toxin-antitoxin (TA) acts as a key player of persister cell formation where the toxin is activated either stochastically or after an environmental insult, thereby silencing the physiological processes. However, the controversial role of TA modules in persister cell formation has also been documented with reasonable clarity. Persisters may revert back from state of quiescence and regrow when conditions become favourable for their propagation. Therefore, the elimination of dormant bacteria is crucial, and currently, research interest is highly focussed on developing several antipersister strategies that may kill persister bacteria by targeting different molecules. It is worth examining these targets to develop appropriate therapeutic interventions against bacterial infections and it is believed that earmarking TA system can be a novel approach for resuscitation of persisters. In this review, we discussed the role of TA modules in mediating persistence with highlighting on the debatable issues regarding contribution of these modules in dormant bacteria formation. Furthermore, we discussed if these modules in bacteria can be targeted for successful elimination of dormant persister cells.

Enhanced disinfection of bacterial populations by nutrient and antibiotic challenge timing

Several difficult to treat illnesses like tuberculosis, chronic pneumonia, and inner ear infections are caused by tolerant bacteria enmeshed in a biofilm. Bacterial tolerance can be genotypic (resistance-i.e. MRSA), phenotypic (non-heritable) or environmental (e.g. nutrient gradients). Persister formation is a phenotypic expression and this phenotype is highly tolerant of disinfection. Constant dosing is typically ineffective and to generate an effective treatment protocol, we need to understand the dynamics of persister cells. In this study, we investigate how manipulating the application of antibiotics and addition of nutrients enhances the disinfection of a bacterial population in batch culture. Eliminating persister bacteria is considered as a challenge for the food industry or wastewater treatment, since the failure may result in food contamination and disease transmission. Previous studies focused on the antimicrobial agent as a control variable to eliminate the bacterial population. In addition to antibiotic, we consider the significance of the nutrient in eradicating the susceptible and persister cells since the disinfection of susceptible population depends on the nutrient intake. We present a mathematical model that captures the dynamics between susceptible and persister bacteria with antibiotic and nutrient control variables. We investigate the optimal dose-withdrawal of antibiotic timing in several cases including constant nutrient in time, dynamic nutrient in time and piecewise constant nutrient in time.

A recombinant fungal defensin-like peptide-P2 combats multidrug-resistant Staphylococcus aureus and biofilms.

There is an urgent need to discover new active drugs to combat methicillin-resistant Staphylococcus aureus, which is a serious threat to humans and animals and incompletely eliminated by antibiotics due to its intracellular accumulation in host cells, production of biofilms, and persisters. Fungal defensin-like peptides (DLPs) are emerging as a potential source of new antibacterial drugs due to their potent antibacterial activity. In this study, nine novel fungal DLPs were firstly identified by querying against UniProt databases and expressed in Pichia pastoris, and their antibacterial and anti-biofilm ability were tested against multidrug-resistant (MDR)S. aureus. Results showed that among them, P2, the highest activity and expression level, showed low toxicity, no resistance, and high stability. Minimal inhibitory concentrations (MICs) of P2 against Gram-positive bacteria were < 2μg/mL. P2 exhibited the potent activity against intracellular MDR S. aureus (bacterial reduction in 80-97%) in RAW264.7 macrophages. P2 bound to/disrupted bacterial DNA, wrinkled outer membranes and permeabilized cytoplasmic membranes, but maintained the integrity of bacterial cells. P2 inhibited/eradicated the biofilm and killed 99% persister bacteria, which were resistant to 100× MIC vancomycin. P2 upregulated the anti-inflammatory cytokine (IL-10) and downregulated pro-inflammatory cytokines (TNF-α/IL-1β) and chemokine (MCP-1) levels in RAW 264.7 macrophages and in mice, respectively. Five milligram per kilogram P2 enhanced the survival of S. aureus-infected mice (100%), superior to vancomycin (30mg/kg), inhibited the bacterial translocation, and alleviated multiple-organ injuries (liver, spleen, kidney, and lung). These data suggest that P2 may be a candidate for novel antimicrobial agents against MDR staphylococcal infections.

Hydroalcoholic extract from Origanum vulgare induces a combined anti-mycobacterial and anti-inflammatory response in innate immune cells.

In nature, many plants or their extracted compounds have been found to possess anti-inflammatory features and therapeutic properties against infectious as well as non-infectious diseases, including cancer. In this study, we analysed the immunomodulatory effects on innate immune cells of hydroalcoholic extract from Origanum vulgare L. ssp. hirtum (HyE-Ov), a plant traditionally known for its anti-oxidative properties. The effects of HyE-Ov were tested on human monocyte derived dendritic cells (DC), type-1 (M1) and type-2 macrophages (M2) infected with M. bovis Bacille Calmette-Guérin (BCG), used as a model of persistent intracellular bacterium. DC, M1 and M2 treated with HyE-Ov significantly enhanced their mycobactericidal activity, which was associated with phagosomal acidification in M1 and M2 and increase of phagosomal, but not mitochondrial ROS production in M1, M2, and DC. Treatment of BCG-infected DC with HyE-Ov significantly reduced TNF-α and IL-12 production and increased TGF-β synthesis. Finally, experiments were repeated using eight different HPLC fractions of HyE-Ov. Results showed that the capability to activate anti-microbial and anti-inflammatory response is shared by different fractions, suggesting that diverse bioactive molecules are present within the hydroalcoholic extract. Altogether, these results show that HyE-Ov promotes anti-mycobacterial innate immunity and limits inflammatory response in vitro and suggest that this plant extract may be exploitable as phytocomplex or nutraceutical for novel host-directed therapeutic approaches.

Isothermal Microcalorimetry Detects the Presence of Persister Cells in a Staphylococcus aureus Biofilm After Vancomycin Treatment.

Staphylococcus aureus biofilm plays a major role in implant-associated infections. Here, the susceptibility of biofilm S. aureus to daptomycin, fosfomycin, vancomycin, trimethoprim/sulfamethoxazole, linezolid, and rifampicin was investigated by isothermal microcalorimetry (IMC). Moreover, the persister status of cells isolated from S. aureus biofilm after treatment with vancomycin was also analyzed. S. aureus biofilm was tolerant to all the antibiotics tested [minimum biofilm bactericidal concentration (MBBC) > 256 μg/ml], except to daptomycin [MBBC and minimum biofilm eradicating concentration (MBEC) = 32 μg/ml] and rifampin (MBBC and MBEC = 128 μg/ml). After the treatment of MRSA biofilm with 1024 μg/ml vancomycin, ∼5% cells survived, although metabolically inactive (persisters). Interestingly, IMC revealed that persister bacteria reverted to a normal-growing phenotype when inoculated into fresh medium without antibiotics. A staggered treatment of MRSA biofilm with vancomycin to kill all the metabolically active cells and daptomycin to kill persister cells eradicated the whole bacterial population. These results support the use in the clinical practice of a therapeutic regimen based on the use of two antibiotics to kill persister cells and eradicate MRSA biofilms. IMC represents a suitable technique to characterize in real-time the reversion from persister to metabolically-active cells.

Bedaquiline kills persistent Mycobacterium tuberculosis with no disease relapse: an in vivo model of a potential cure.

Non-replicating persistent Mycobacterium tuberculosis is difficult to kill since the organisms become undetectable using our conventional diagnostic methods and tolerant to anti-TB drugs. Resuscitation-promoting factors (RPFs) have been used to 'wake up' non-replicating persisters, making them easy to detect. Bedaquiline is a novel bactericidal and sterilizing anti-TB drug with the potential to eradicate RPF-dependent persistent M. tuberculosis. We present the first head-to-head comparison between the standard anti-TB regimen and a bedaquiline-modified regimen in eradicating RPF-dependent persistent M. tuberculosis, using the well-defined Cornell Model. M. tuberculosis-infected mice were treated for 14 weeks with either the standard regimen (rifampicin, isoniazid, pyrazinamide and ethambutol) or the same regimen where ethambutol was replaced by bedaquiline. The efficacy of both drug regimens was measured by cfu count elimination and eradication of persistent bacteria, which was evaluated using culture filtrate (CF) containing RPFs. At the end of treatment, the remaining cfu count-negative mice were administered hydrocortisone for 8 weeks. The induced disease relapse rates were determined by the percentage of mice that became positive for M. tuberculosis in the lung, spleen or both. The bedaquiline-containing regimen achieved total organ cfu count clearance at 8 weeks after treatment initiation, faster than the standard regimen (14 weeks). Importantly, the bedaquiline-containing regimen removed CF-dependent persistent bacilli at 8 weeks, leading to no disease relapse. A bedaquiline regimen eradicated persistent TB infections and completely prevented disease relapse in mice. These findings offer the potential for a faster cure for TB, with reduced relapse rate.

Lack of evidence for the necessity of root canal obturation.

Root canal obturation still is a relevant research topic and patients spend substantial amounts of financial resources for this step of endodontic treatment. Three experiments were conducted challenging the necessity of root canal obturation. Applying micro computed tomography, the volume of dentin tubules that cannot be instrumented during root canal therapy was determined. Using a simple biofilm model of human tooth segments, the effect of root canal obturation on the persistency of bacteria was evaluated and freshly extracted root canal treated teeth were examined for bacteria remaining in dentin. The volume of dentinal canals was found to be at least three times greater than the volume of the root canal itself. Bacterial growth was observed both in specimens with and without root canal obturation implying that the treatment rendered was ineffective in removing bacterial biofilm and the obturation material was incapable of hindering bacterial regrowth. Despite showing adequate root canal obturation radiographically, persistent bacteria could be identified in all teeth extracted. While perfect disinfection of root canals is mandatory, root canal obturation seems questionable as current materials have no antibacterial activity, do not stabilize the tooth, and cannot seal the canal system if a coronal restoration is missing.

A novel probiotic, Lactobacillus johnsonii 456, resists acid and can persist in the human gut beyond the initial ingestion period

ABSTRACT Probiotics are considered to have multiple beneficial effects on the human gastrointestinal tract, including immunomodulation, pathogen inhibition, and improved host nutrient metabolism. However, extensive characterization of these properties is needed to define suitable clinical applications for probiotic candidates. Lactobacillus johnsonii 456 (LBJ 456) was previously demonstrated to have anti-inflammatory and anti-genotoxic effects in a mouse model. Here, we characterize its resistance to gastric and bile acids as well as its ability to inhibit gut pathogens and adhere to host mucosa. While bile resistance and in vitro host attachment properties of LBJ 456 were comparable to other tested probiotics, LBJ 456 maintained higher viability at lower pH conditions compared to other tested strains. LBJ 456 also altered pathogen adhesion to LS 174T monolayers and demonstrated contact-dependent and independent inhibition of pathogen growth. Genome analyses further revealed possible genetic elements involved in host attachment and pathogen inhibition. Importantly, we show that ingestion of Lactobacillus johnsonii 456 over a one week yogurt course leads to persistent viable bacteria detectable even beyond the period of initial ingestion, unlike many other previously described probiotic species of lactic acid bacteria.

Spiky nanohybrids of titanium dioxide/gold nanoparticles for enhanced photocatalytic degradation and anti-bacterial property

We present a facile two-step procedure for synthesizing spiky nanohybrids of titanium dioxide/gold (TiO2/Au) nanoparticles. In this process, spiky TiO2 is obtained using a hydrothermal method, followed by the introduction of plasmonic Au nanoparticles (AuNPs) via a photoreduction approach in which titanium fluoride and chloroauric acid tetrahydrate are used as raw materials. The photodegradation property of the resulting sample was evaluated according to the removal of Rhodamine B (RhB) and ciprofloxacin (CIP) via excitation with visible light. Additionally, the antimicrobial property of the spiky TiO2/Au nanoparticles was examined with respect to the suppression of the growth of Escherichia coli (E. coli). Compared with commercial TiO2, the spiky TiO2/Au nanoparticles exhibited a significantly enhanced photocatalytic efficiency in persistent organic pollutant degradation and bacteria inactivation under simulated environmental conditions. The photocatalysis mechanism primarily entails the combination of AuNPs with spiky TiO2 nanoparticles, which increases the optical path owing to the unique spiky structures of the latter. This results in an improved light-harvesting efficiency based on the localized surface plasmon resonance (LSPR) of AuNPs and the promotion of the charge-separation efficiency through electron-trap processes. These nanoparticles realize the objective of effectively addressing the inherent weaknesses of bare TiO2 and potentially facilitate new fitting approaches for applications in sewage treatment and marine antifouling paint.

Disrupting folate metabolism reduces the capacity of bacteria in exponential growth to develop persisters to antibiotics.

Bacteria can survive high doses of antibiotics through stochastic phenotypic diversification. We present initial evidence that folate metabolism could be involved with the formation of persisters. The aberrant expression of the folate enzyme gene fau seems to reduce the incidence of persisters to antibiotics. Folate-impaired bacteria had a lower generation rate for persisters to the antibiotics ampicillin and ofloxacin. Persister bacteria were detectable from the outset of the exponential growth phase in the complex media. Gene expression analyses tentatively showed distinctive profiles in exponential growth at times when bacteria persisters were observed. Levels of persisters were assessed in bacteria with altered, genetically and pharmacologically, folate metabolism. This work shows that by disrupting folate biosynthesis and usage, bacterial tolerance to antibiotics seems to be diminished. Based on these findings there is a possibility that bacteriostatic antibiotics such as anti-folates could have a role to play in clinical settings where the incidence of antibiotic persisters seems to drive recalcitrant infections.

Discovery and Optimization of nTZDpa as an Antibiotic Effective Against Bacterial Persisters.

Conventional antibiotics are not effective in treating infections caused by drug-resistant or persistent nongrowing bacteria, creating a dire need for the development of new antibiotics. We report that the small molecule nTZDpa, previously characterized as a nonthiazolidinedione peroxisome proliferator-activated receptor gamma partial agonist, kills both growing and persistent Staphylococcus aureus cells by lipid bilayer disruption. S.aureus exhibited no detectable development of resistance to nTZDpa, and the compound acted synergistically with aminoglycosides. We improved both the potency and selectivity of nTZDpa against MRSA membranes compared to mammalian membranes by leveraging synthetic chemistry guided by molecular dynamics simulations. These studies provide key insights into the design of selective and potent membrane-active antibiotics effective against bacterial persisters.

Persistent Intracellular Staphylococcus aureus in Keratinocytes Lead to Activation of the Complement System with Subsequent Reduction in the Intracellular Bacterial Load.

The complement system is an ancient part of the innate immune system important for both tissue homeostasis and host defense. However, bacteria like Staphylococcus aureus (SA) possess elaborative mechanisms for evading both the complement system and other parts of the immune system. One of these evasive mechanisms—important in causing chronic and therapy resistant infections—is the intracellular persistence in non-immune cells. The objective of our study was to investigate whether persistent intracellular SA infection of epidermal keratinocytes resulted in complement activation. Using fluorescence microscopy, we found that persistent SA, surviving intracellularly in keratinocytes, caused activation of the complement system with formation of the terminal complement complex (TCC) at the cell surface. Skin samples from atopic dermatitis patients analyzed by bacterial culture and microscopy, demonstrated that SA colonization was associated with the presence of intracellular bacteria and deposition of the TCC in epidermis in vivo. Complement activation on keratinocytes with persistent intracellular bacteria was found with sera deficient/depleted of the complement components C1q, Mannan-binding lectin, or complement factor B, demonstrating involvement of more than one complement activation pathway. Viable bacterial counts showed that complement activation at the cell surface initiated cellular responses that significantly reduced the intracellular bacterial burden. The use of an inhibitor of the extracellular signal-regulated kinase (ERK) abrogated the complement-induced reduction in intracellular bacterial load. These data bridge the roles of the complement system in tissue homeostasis and innate immunity and illustrate a novel mechanism by which the complement system combats persistent intracellular bacteria in epithelial cells.

Infection Is Not Required for Mucoinflammatory Lung Disease in CFTR-Knockout Ferrets.

Classical interpretation of cystic fibrosis (CF) lung disease pathogenesis suggests that infection initiates disease progression, leading to an exuberant inflammatory response, excessive mucus, and ultimately bronchiectasis. Although symptomatic antibiotic treatment controls lung infections early in disease, lifelong bacterial residence typically ensues. Processes that control the establishment of persistent bacteria in the CF lung, and the contribution of noninfectious components to disease pathogenesis, are poorly understood. To evaluate whether continuous antibiotic therapy protects the CF lung from disease using a ferret model that rapidly acquires lethal bacterial lung infections in the absence of antibiotics. CFTR (cystic fibrosis transmembrane conductance regulator)-knockout ferrets were treated with three antibiotics from birth to several years of age and lung disease was followed by quantitative computed tomography, BAL, and histopathology. Lung disease was compared with CFTR-knockout ferrets treated symptomatically with antibiotics. Bronchiectasis was quantified from computed tomography images. BAL was evaluated for cellular differential and features of inflammatory cellular activation, bacteria, fungi, and quantitative proteomics. Semiquantitative histopathology was compared across experimental groups. We demonstrate that lifelong antibiotics can protect the CF ferret lung from infections for several years. Surprisingly, CF animals still developed hallmarks of structural bronchiectasis, neutrophil-mediated inflammation, and mucus accumulation, despite the lack of infection. Quantitative proteomics of BAL from CF and non-CF pairs demonstrated a mucoinflammatory signature in the CF lung dominated by Muc5B and neutrophil chemoattractants and products. These findings implicate mucoinflammatory processes in the CF lung as pathogenic in the absence of clinically apparent bacterial and fungal infections.

Optimal doses of rifampicin in the standard drug regimen to shorten tuberculosis treatment duration and reduce relapse by eradicating persistent bacteria.

Although high-dose rifampicin holds promise for improving tuberculosis disease control by eradication of persistent bacteria, the optimal dose of rifampicin that kills persistent bacteria and shortens the treatment duration is unknown. The Cornell mouse model was used to test the efficacy of rifampicin at elevated doses combined with isoniazid and pyrazinamide to kill actively growing and persistent bacilli and to measure relapse rate. Persistent bacteria were evaluated using Mycobacterium tuberculosis culture supernatant containing resuscitation-promoting factors. Pharmacokinetic parameters and dose-dependent activity for cultivable and persistent bacilli were determined. Increasing doses of rifampicin in combination with isoniazid and pyrazinamide resulted in dose-dependent faster bacterial clearance. Evaluated both on solid media and in culture filtrate containing resuscitation-promoting factors, a regimen containing a standard dose of rifampicin at 10 mg/kg over 14 weeks failed to achieve organ sterility. In contrast, higher doses of rifampicin achieved organ sterility in a much shorter time of 8-11 weeks. Disease relapse, which occurred in 86% of mice treated with the standard regimen for 14 weeks, was completely prevented by rifampicin doses of ≥ 30 mg/kg. In the treatment of murine tuberculosis, a rifampicin dose of 30 mg/kg was sufficient to eradicate persistent M. tuberculosis, allowing shorter treatment duration without disease relapse.

Raman-Deuterium Isotope Probing for in-situ identification of antimicrobial resistant bacteria in Thames River

The emergence and widespread distribution of antimicrobial resistant (AMR) bacteria has led to an increasing concern with respect to potential environmental and public health risks. Culture-independent and rapid identification of AMR bacteria in-situ in complex environments is important in understanding the role of viable but non-culturable and antibiotic persistent bacteria and in revealing potential pathogens without waiting for colony formation. In this study, a culture-independent and non-destructive phenotyping approach, so called Raman Deuterium Stable Isotope Probing (Raman-DIP), was developed to identify AMR bacteria in the River Thames. It is demonstrated that Raman-DIP was able to accurately identify resistant and susceptible bacteria within 24 hours. The work shows that, in the River Thames, the majority of the bacteria (76 ± 2%) were metabolically active, whilst AMR bacteria to carbenicillin, kanamycin and both two antibiotics were 35 ± 5%, 28 ± 3%, 25 ± 1% of the total bacterial population respectively. Raman activated cell ejection (RACE) was applied to isolate single AMR bacteria for the first time, linking AMR phenotype (reistance to antibiotics) and genotype (DNA sequence). The sequences of the RACE sorted cells indicate that they were potential human pathogens Aeromonas sp., Stenotrophomonas sp. and an unculturable bacterium. This work demonstrates Raman-DIP and RACE are effective culture-independent approach for rapid identification of AMR bacteria at the single cell level in their natural conditions.

Removal of bacteria Legionella pneumophila, Escherichia coli, and Bacillus subtilis by (super)cavitation

In sufficient concentrations, the pathogenic bacteria L. pneumophila can cause a respiratory illness that is known as the “Legionnaires” disease. Moreover, toxic Shiga strains of bacteria E. coli can cause life-threatening hemolytic-uremic syndrome. Because of the recent restrictions imposed on the usage of chlorine, outbreaks of these two bacterial species have become more common. In this study we have developed a novel rotation generator and its effectiveness against bacteria Legionella pneumophila and Escherichia coli was tested for various types of hydrodynamic cavitation (attached steady cavitation, developed unsteady cavitation and supercavitation). The results show that the supercavitation was the only effective form of cavitation. It enabled more than 3 logs reductions for both bacterial species and was also effective against a more persistent Gram positive bacteria, B. subtilis. The deactivation mechanism is at present unknown. It is proposed that when bacterial cells enter a supercavitation cavity, an immediate pressure drop occurs and this results in bursting of the cellular membrane. The new rotation generator that induced supercavitation proved to be economically and microbiologically far more effective than the classical Venturi section (super)cavitation.

Lethal Effect of Photodynamic Treatment on Persister Bacteria.

Persister bacteria tolerate bactericidal antibiotics due to transient and reversible phenotypic changes. As these bacteria can limit the effectiveness of antibiotics to eradicate certain infections, their elimination is a relevant issue. Photodynamic therapy seems suitable for this purpose, but phenotypic tolerance to it has also been reported for Pseudomonas aeruginosa. To test whether any phenotypic feature could confer tolerance against both antibiotics and photoinactivation, survivors from exposures to light in the presence of methylene blue were treated with ofloxacin, an antibiotic effective on nongrowing bacteria. Susceptibility to ofloxacin was normal in these bacteria in spite of their increased ability to survive photodynamic inactivation, suggesting the absence of cross-tolerance. It thus seemed possible to use one of these treatments to eliminate bacteria which had phenotypic tolerance to the other. To test this strategy, persister bacteria emerging from ofloxacin treatments were submitted to the action of light and methylene blue while the antibiotic remained in the bacterial suspension. Persisters lost their clonogenic ability under these conditions and the effects of the treatments seemed to be synergistic. These observations suggest that photodynamic antimicrobial therapy could be used as a complement to antibiotic treatments to eliminate persister bacteria from localized infections.