Multi-targeting oligopyridiniums: Rational design for biofilm dispersion and bacterial persister eradication.

The development of effective antibacterial drugs to combat bacterial infections, particularly the biofilm-related infections, remains a challenge. There are two important features of bacterial biofilms, which are well-known critical factors causing biofilms hard-to-treat in clinical, including the dense and impermeable extracellular polymeric substances (EPS) and the metabolically repressed dormant and persistent bacterial population embedded. These characteristics largely increase the difficulty for regular antibiotic treatment due to insufficient penetration into EPS. In addition, the dormant bacteria are insensitive to the growth-inhibiting mechanism of traditional antibiotics. Herein, we explore the potential of a series of new oligopyridinium-based oligomers bearing a multi-biomacromolecule targeting function as the potent bacterial biofilm eradication agent. These oligomers were rationally designed to be "charge-on-backbone" that can offer a special alternating amphiphilicity. This novel and unique feature endows high affinity to bacterial membrane lipids, DNAs as well as proteins. Such a broad multi-targeting nature of molecules not only enables its penetration into EPS, but also plays vital roles in the bactericidal mechanism of action that is highly effective against dormant and persistent bacteria. Our in vitro, ex vivo, and in vivo studies demonstrated that OPc3, one of the most effective derivatives, was able to offer excellent antibacterial potency against a variety of bacteria and effectively eliminate biofilms in zebrafish models and mouse wound biofilm infection models.

Influence of Nε-Lysine Acetylation on the Formation of Protein Aggregates and Antibiotic Persistence in E. coli.

Numerous studies indicate that reversible Nε-lysine acetylation in bacteria may play a key role in the regulation of metabolic processes, transcription and translation, biofilm formation, virulence, and drug resistance. Using appropriate mutant strains deficient in non-enzymatic acetylation and enzymatic acetylation or deacetylation pathways, we investigated the influence of protein acetylation on cell viability, protein aggregation, and persister formation in Escherichia coli. Lysine acetylation was found to increase protein aggregation and cell viability under the late stationary phase. Moreover, increased lysine acetylation stimulated the formation of persisters. These results suggest that acetylation-dependent aggregation may improve the survival of bacteria under adverse conditions (such as the late stationary phase) and during antibiotic treatment. Further experiments revealed that acetylation-favorable conditions may increase persister formation in Klebsiella pneumoniae clinical isolate. However, the exact mechanisms underlying the relationship between acetylation and persistence in this pathogen remain to be elucidated.

Can mesenchymal stem/stromal cells and their secretomes combat bacterial persisters?

The increasing number of life-threatening infections caused by persister bacteria is associated with various issues, including antimicrobial resistance and biofilm formation. Infections due to persister cells are often difficult to suppress without the use of last-resort antibiotics. Throughout the world, bacterial persistence and resistance create an unmet clinical demand for the exploration of newly introduced therapeutic approaches. Mesenchymal stem / stromal cells (MSCs) have an antimicrobial activity to protect against bacterial infections, including those caused by bacterial persisters. MSCs have substantial potential to secrete antimicrobial peptides (AMPs), including cathelicidin, beta-defensins, lipocalin-2, hepcidin, indoleamine 2,3-dioxygenase (IDO), cysteine proteases, and inducible nitric oxide synthases (iNOS). MSCs possess the potential to contribute to innate immunity by regulating the immune response. Recently, MSCs and their secreted components have been reported to improve antimicrobial activity. Bactericidal activity by MSCs and their secretomes has been shown to be mediated in part by the secretion of AMPs. Even though they were discovered more than 80 years ago, therapeutic options for persisters are restricted, and there is an urgent need for alternative treatment regimens. Hence, this review intends to critically assess the current literature on the effects of MSCs and their secretomes on persister bacteria. MSCs and their secretome-based therapies could be preferred as an up-and-coming approach to reinforce the antimicrobial efficiency in persister infections.

The physiological and ecological properties of bacterial persisters discovered from municipal sewage sludge and the potential risk.

Bacterial persisters are a special microbial population and are considered to be the bacterial reservoir of antibiotic-resistant bacteria. They can survive antibiotic treatment even in high concentrations of antibiotics and revive in the appropriate conditions. However, the characteristics of bacterial persisters in the municipal sewage sludge and their potential environmental risks have not yet been paid much attention to. In this study, bacterial persisters were discovered from the sludge of wastewater treatment plants in four different regions (Jilin, Lhasa, Shenzhen, and Yili), and the metagenomic analysis confirmed that bacterial persisters were ubiquitous in all four municipal sewage sludge and positively related to the protobacterium populations. At the taxonomic genus level, a total of 57 genera of bacterial persisters were shared by the four sewage sludge, and the genera with abundance exceeding 2% were Acinetobacter, Lysinibacillus, Aeromonas, Brevundimonas, Pseudomonas, and Alcaligenes, among which Acinetobacter accounted for 57.24%. Genus Lysinibacillus and Aeromonas were significant in Jilin and Lhasa, respectively. The persistence mechanism of bacterial persisters derived from sludge was also clarified, among which, Aeromonas, Brevundimonas, and Alcaligenes rely on the hipBA toxin-antitoxin system, while Acinetobacter enters the persistence state mainly through the stringent response system based on (p)ppGpp. Moreover, it was found that a typical bacterial persister originated from Acinetobacter, named T9-9, could tolerate a variety of antibiotics, such as 1000 µg/mL of kanamycin, 160 µg/mL of tetracycline, and 30 µg/mL of ciprofloxacin. Even if the ultraviolet intensity was 6-36 times the usual dosage of ultraviolet disinfection in wastewater treatment plants, it could not completely kill T9-9, but the killing efficiency by chlorine disinfection technology could reach 100%. This study pointed out an environmental risk of bacterial persisters that existed in sewage sludge that had been neglected and strongly recommended to improve the disinfection process in the wastewater treatment plant.

Persisting cancer cells are different from bacterial persisters.

The persistence of drug-sensitive tumors poses a significant challenge in cancer treatment. The concept of bacterial persisters, which are a subpopulation of bacteria that survive lethal antibiotic doses, is frequently used to compare to residual disease in cancer. Here, we explore drug tolerance of cancer cells and bacteria. We highlight the fact that bacteria, in contrast to cancer cells, have been selected for survival at the population level and may therefore possess contingency mechanisms that cancer cells lack. The precise mechanisms of drug-tolerant cancer cells and bacterial persisters are still being investigated. Undoubtedly, by understanding common features as well as differences, we, in the cancer field, can learn from microbiology to find strategies to eradicate persisting cancer cells.

Tea Tree Essential Oil Kills Escherichia coli and Staphylococcus epidermidis Persisters.

Persister cells are a small subpopulation of non-growing bacteria within a population that can survive long exposures to antibiotic treatment. Following antibiotic removal, persister cells can regrow and populate, playing a key role in the chronic reoccurrence of bacterial infections. The development of new molecules and methods to kill bacterial persisters is critical. Essential oils and other natural products have long been studied for their antimicrobial effects. Here, we studied the effectiveness of tea tree essential oil (TTO), a common component in many commercial care products, against Escherichia coli and Staphylococcus epidermidis persister cells. Using biphasic kill curve assays, we found that concentrations of 0.5% and 1.0% TTO for E. coli and S. epidermidis, respectively, completely eradicated persister cells over a period of 24 h, with the component terpinen-4-ol responsible for most of the killing. Using a colorimetric assay, it was determined that the TTO exhibited its anti-persister effects through a membrane disruption mechanism.

The top 100 cited studies on bacterial persisters: A bibliometric analysis.

Background: Bacterial persisters are thought to be responsible for the recalcitrance and relapse of persistent infections, and they also lead to antibiotic treatment failure in clinics. In recent years, researches on bacterial persisters have attracted worldwide attention and the number of related publications is increasing. The purpose of this study was to better understand research trends on bacterial persisters by identifying and bibliometrics analyzing the top 100 cited publications in this field. Methods: The Web of Science Core Collection was utilized to retrieve the highly cited publications on bacterial persisters, and these publications were cross-matched with Google Scholar and Scopus. The top 100 cited publications were identified after reviewing the full texts. The main information of each publication was extracted and analyzed using Excel, SPSS, and VOSviewer. Results: The top 100 cited papers on bacterial persisters were published between 1997 and 2019. The citation frequency of each publication ranged from 147 to 1815 for the Web of Science Core Collection, 153 to 1883 for Scopus, and 207 to 2,986 for Google Scholar. Among the top 100 cited list, there were 64 original articles, 35 review articles, and 1 editorial material. These papers were published in 51 journals, and the Journal of Bacteriology was the most productive journal with 8 papers. A total of 14 countries made contributions to the top 100 cited publications, and 64 publications were from the United States. 15 institutions have published two or more papers and nearly 87% of them were from the United States. Kim Lewis from Northeastern University was the most influential author with 18 publications. Furthermore, keywords co-occurrence suggested that the main topics on bacterial persisters were mechanisms of persister formation or re-growth. Finally, "Microbiology" was the most frequent category in this field. Conclusion: This study identified and analyzed the top 100 cited publications related to bacterial persisters. The results provided a general overview of bacterial persisters and might help researchers to better understand the classic studies, historical developments, and new findings in this field, thus providing ideas for further research.

Homology Model of a Catalytically Competent Bifunctional Rel Protein.

Bacteria have developed different bet hedging strategies to survive hostile environments and stressful conditions with persistency being maybe the most elegant yet still poorly understood one. Persisters' temporary tolerance to antibiotic treatment hints at their role not only in chronic and recurrent infections but also in the insurgence of resistant strains. Therefore, hampering persisters formation might represent an innovative strategy in the quest for new effective antimicrobial compounds. Among the molecular mechanisms postulated for the persister phenotypic switch, we decided to focus our attention on the stringent response and, in particular, on the upstream triggering step that is the accumulation of guanosine tetra- and pentaphosphate, collectivity called (p)ppGpp. Intracellular levels of (p)ppGpp are regulated by a superfamily of enzymes called RSH (RelA/SpoT homologue) that are able to promote its synthesis via pyrophosphate transfer from an ATP molecule to the 3' position of either GDP or GTP. These enzymes are classified based on the structural domain(s) present (only synthetase, only hydrolase, or both). Here we present our work on Rel Seq (from S. equisimilis), still the only bifunctional Rel protein for which a GDP-bound "synthetase-ON" structure is available. Analysis of the synthetase site, occupied only by GDP, revealed a partially active state, where the supposed ATP binding region is not conformationally apt to accommodate it. In order to achieve a protein model that gets closer to a fully active state, we generated a chimera structure of Rel Seq by homology modeling, starting from the crystal structure of the catalytically competent state of RelP, a smaller, single-domain, Rel protein from S. aureus. Molecular dynamics simulations allowed verifying the stability of the generated chimera model. Virtual screening and ligand design studies are underway.

Cellular Self-Digestion and Persistence in Bacteria.

Cellular self-digestion is an evolutionarily conserved process occurring in prokaryotic cells that enables survival under stressful conditions by recycling essential energy molecules. Self-digestion, which is triggered by extracellular stress conditions, such as nutrient depletion and overpopulation, induces degradation of intracellular components. This self-inflicted damage renders the bacterium less fit to produce building blocks and resume growth upon exposure to fresh nutrients. However, self-digestion may also provide temporary protection from antibiotics until the self-digestion-mediated damage is repaired. In fact, many persistence mechanisms identified to date may be directly or indirectly related to self-digestion, as these processes are also mediated by many degradative enzymes, including proteases and ribonucleases (RNases). In this review article, we will discuss the potential roles of self-digestion in bacterial persistence.

Eradicating Bacterial Persisters with Combinations of Strongly and Weakly Metabolism-Dependent Antibiotics.

The vast majority of bactericidal antibiotics display poor efficacy against bacterial persisters, cells that are in a metabolically repressed state. Molecules that retain their bactericidal functions against such bacteria often display toxicity to human cells, which limits treatment options for infections caused by persisters. Here, we leverage insight into metabolism-dependent bactericidal antibiotic efficacy to design antibiotic combinations that sterilize both metabolically active and persister cells, while minimizing the antibiotic concentrations required. These rationally designed antibiotic combinations have the potential to improve treatments for chronic and recurrent infections.

Cysteine Potentiates Bactericidal Antibiotics Activity Against Gram-Negative Bacterial Persisters.

PURPOSE: Bacterial metabolism regulators offer a novel productive strategy in the eradication of antibiotic refractory bacteria, particularly bacterial persisters. However, the potential of amino acids in the fight against Gram-negative bacterial persisters has not been fully explored. The aim of this study is to investigate the potentiation of amino acids to antibiotics in combating Gram-negative bacterial persisters and to reveal the underlying mechanisms of action. METHODS: Bactericidal activity of antibiotics in the absence or presence of amino acids was evaluated through detecting the reduction of bacterial CFUs. The ratio of NAD+/NADH in E. coli B2 persisters was determined using assay kit with WST-8. Bacterial respiration and ROS production were measured by the reduction of iodonitrotetrazolium chloride and fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate, respectively. RESULTS: In this study, we found that cysteine possesses excellent synergistic bactericidal activity with ciprofloxacin against multiple Gram-negative bacterial persisters. Furthermore, the potentiation of cysteine was evaluated in exponential and stationary-phase E. coli ATCC 25922 and E. coli B2. Interestingly, cysteine significantly improves three bactericidal antibiotics killing against stationary-phase bacteria, but not exponential-phase bacteria, implying that the effect of cysteine correlates with the metabolic state of bacteria. Mechanistic studies revealed that cysteine accelerates the bacterial TCA cycle and promotes bacterial respiration and ROS production. These metabolic regulation effects of cysteine re-sensitive bacterial persisters to antibiotic killing. CONCLUSION: Collectively, our study highlights the synergistic bactericidal activity of bacterial metabolism regulators such as cysteine with commonly used antibiotics against Gram-negative bacterial persisters.

Anti-MRSA agent discovery using Caenorhabditis elegans-based high-throughput screening.

Staphylococcus aureus is a leading cause of hospital- and community-acquired infections. Despite current advances in antimicrobial chemotherapy, the infections caused by S. aureus remain challenging due to their ability to readily develop resistance. Indeed, antibiotic resistance, exemplified by methicillin-resistant S. aureus (MRSA) is a top threat to global health security. Furthermore, the current rate of antibiotic discovery is much slower than the rate of antibiotic-resistance development. It seems evident that the conventional in vitro bacterial growth-based screening strategies can no longer effectively supply new antibiotics at the rate needed to combat bacterial antibiotic-resistance. To overcome this antibiotic resistance crisis, screening assays based on host-pathogen interactions have been developed. In particular, the free-living nematode Caenorhabditis elegans has been used for drug screening against MRSA. In this review, we will discuss the general principles of the C. elegans-based screening platform and will highlight its unique strengths by comparing it with conventional antibiotic screening platforms. We will outline major hits from high-throughput screens of more than 100,000 small molecules using the C. elegans-MRSA infection assay and will review the mode-of-action of the identified hit compounds. Lastly, we will discuss the potential of a C. elegans-based screening strategy as a paradigm shift screening platform.

Viable bacteria persist on antibiotic spacers following two-stage revision for periprosthetic joint infection.

Treatment in periprosthetic joint infection (PJI) remains challenging. The failure rate of two-stage revision and irrigation and debridement with component retention in PJI suggests that biofilm cells have a high tolerance to antibiotic chemotherapy. Previous work has demonstrated that biofilm cells have high antibiotic tolerance in vitro, but there is little clinical evidence to support these observations. The aim of this study was to determine if retrieved antibiotic spacers from two-stage revision total knee arthroplasty for PJI have evidence of remaining viable bacteria. Antibiotic poly (methyl methacrylate) (PMMA) spacers from two-stage revision total knee arthroplasty for PJI were prospectively collected and analyzed for bacterial 16s rRNA using polymerase chain reaction (PCR), reverse transcription (RT)-PCR, quantitative RT-PCR (qRT-PCR), and single genome analysis (SGA). PCR and RT-PCR identified bacterial species on 53.8% (7/13) of these samples. When initial culture negative cases are excluded, 68% (6/9) samples were identified with bacterial species. A more rigorous qRT-PCR analysis showed a strong positive signal for bacterial contamination in 30.7% (4/13) of cases. These patients did not show any clinical evidence of PJI recurrence after 15 months of follow-up. Because the half-life of bacterial rRNA is approximately a few days, the identification of bacteria rRNA on antibiotic PMMA spacers suggests that viable bacteria were present after conclusion of antibiotic therapy. This study provides evidence for the high tolerance of biofilm cells to antibiotics in vivo and the important role of bacterial persisters in PJI. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:452-458, 2018.

Formation, physiology, ecology, evolution and clinical importance of bacterial persisters.

Persisters are transiently tolerant variants that allow populations to avoid eradication by antibiotic treatment. Their antibiotic tolerance is non-genetic, not inheritable and results from a phenotypic switch from the normal, sensitive cell type to the tolerant, persister state. Here we give a comprehensive overview on bacterial persistence. We first define persistence, summarize the various aspects of persister physiology and show their heterogeneous nature. We then focus on the role of key cellular processes and mechanisms controlling the formation of a subpopulation of tolerant cells. Being a prime example of a risk-spreading strategy, we next discuss the eco-evolutionary aspects of persistence, e.g. how persistence evolves in the face of treatment with antibiotics. Finally, we illustrate the clinical importance of persisters, as persistence is worsening the worldwide antibiotic crisis by prolonging antibiotic treatment, causing therapy failure or catalyzing the development of genetically encoded antibiotic resistance. A better understanding of this phenotype is critical in our fight against pathogenic bacteria and to obtain a better outlook on future therapies.