Stationary Phase Cells Research Articles

Polymeric paint coated common-touch surfaces that can kill bacteria, fungi and influenza virus.

In the current situation of COVID-19 pandemic, the role of surfaces in transmitting pathogens is clearer than ever. Herein, we report an organo-soluble, quaternary antimicrobial paint (QAP) based on polyethyleneimine (PEI) which was coated on a wide range of surfaces such as polyvinylchloride (PVC), nylon, rubber, aluminum. The coating completely killed drug-resistant bacteria. It showed rapid bactericidal properties with complete killing in 45 min of exposure and lowered bacterial adherence, asserting self-sterilizing nature. The coating exhibited complete killing of stationary phase cells of bacteria. The coating killed drug-resistant C. albicans strains. Importantly, QAP coating showed complete killing of influenza virus (H1N1).Graphic abstract

Corynebacterium jeikeium Dormant Cell Formation and Photodynamic Inactivation.

Pathogenic non-spore forming bacteria enter a dormant state under stressful conditions, which likely allows them to acquire resistance to various antibiotics. This work revealed the efficient formation of dormant “non-culturable” (NC) Corynebacterium jeikeium cells in stationary phase upon gradual acidification of the growth medium. Such cells were unable to form colonies and existed in a prolonged stationary phase. At an early stage of dormancy (approximately 14 days post-inoculation), dormant cells are able for resuscitation in liquid medium. However, those stored for long time in dormant state needed addition of supernatant taking from active C. jeikeium cultures for successful resuscitation. NC cells possessed low RNA synthesis and significant tolerance to antibiotics (rifampicin and vancomycin). They also accumulated free porphyrins, and 5-aminolevulinic acid addition enhanced free porphyrin accumulation which makes them potentially sensitive to photodynamic inactivation (PDI). PDI of dormant bacteria was accomplished by exposing cells to a 565 nm wavelength of light using a SOLIS-4C light-emitting diode for 60 min. This revealed that increased porphyrin concentrations were correlated with elevated PDI sensitivity. Results shown here demonstrate the potential utility of employing PDI to minimize levels of dormant, persistent corynebacteria and the C. jeikeium dormancy model developed here may be useful for finding new drugs and techniques for combatting persistent corynebacteria.

Identification of a rate-limiting step in a metabolic pathway using the kinetic model and in vitro experiment

Identification of the rate-limiting step in a metabolic pathway is an important challenge in metabolic engineering for enhancing pathway flow. Although specific enzyme activities (Vmax) provide valuable clues for the identification, it is time-consuming and difficult to measure multiple enzymes in the pathway because different assay protocols are required for each enzyme. In the present study, we propose a method to simultaneously determine the Vmax values of multiple enzymes using a kinetic model with a time course of the intermediate concentrations through an invitro experiment. To demonstrate this method, nine glycolysis reactions for converting glucose-6-phosphate (G6P) to pyruvate in Escherichia coli were considered. In a reaction mixture containing G6P and cofactors, glycolysis was initiated by adding a crude cell extract obtained from stationary phase cells. The Vmax values were optimized to minimize the difference between the measured and simulated time-courses using a kinetic model. Metabolic control analysis using the kinetic model with the estimated Vmax values revealed that fructose bisphosphate aldolase (FBA) was the rate-limiting step in the upper part of glycolysis. The addition of FBA in the reaction mixture successfully increased the glycolytic flux invitro. Furthermore, invivo, the specific glucose consumption rate of an FBA overexpression strain was 1.4 times higher than that of the control strain during the stationary phase. These results confirmed that FBA was the rate-limiting step in glycolysis under the stationary phase. This approach provides Vmax values of multiple enzymes in a pathway for metabolic control analysis with a kinetic model.

Sublethal Paraquat Confers Multidrug Tolerance in Pseudomonas aeruginosa by Inducing Superoxide Dismutase Activity and Lowering Envelope Permeability.

Stressors and environmental cues shape the physiological state of bacteria, and thus how they subsequently respond to antibiotic toxicity. To understand how superoxide stress can modulate survival to bactericidal antibiotics, we examined the effect of intracellular superoxide generators, paraquat and menadione, on stationary-phase antibiotic tolerance of the opportunistic pathogen, Pseudomonas aeruginosa. We tested how pre-challenge with sublethal paraquat and menadione alters the tolerance to ofloxacin and meropenem in wild-type P. aeruginosa and mutants lacking superoxide dismutase (SOD) activity (sodAB), the paraquat responsive regulator soxR, (p)ppGpp signaling (relA spoT mutant), or the alternative sigma factor rpoS. We confirmed that loss of SOD activity impairs ofloxacin and meropenem tolerance in stationary phase cells, and found that sublethal superoxide generators induce drug tolerance by stimulating SOD activity. This response is rapid, requires de novo protein synthesis, and is RpoS-dependent but does not require (p)ppGpp signaling nor SoxR. We further showed that pre-challenge with sublethal paraquat induces a SOD-dependent reduction in cell-envelope permeability and ofloxacin penetration. Our results highlight a novel mechanism of hormetic protection by superoxide generators, which may have important implications for stress-induced antibiotic tolerance in P. aeruginosa cells.

Potentiating aminoglycoside antibiotics to reduce their toxic side effects.

The lack of new antibiotics necessitates the improvement of existing ones, many of which are limited by toxic side effects. Aminoglycosides, antibiotics with excellent activity and low bacterial resistance, are hampered by dose-dependent toxic effects in patients (nephrotoxicity, ototoxicity). High antibiotic concentrations are often required to treat dormant, non-dividing bacteria, though previous studies show that aminoglycosides can be activated against such bacteria by specific metabolites. Here, we employed this mechanism to greatly boost the activity of low concentrations of aminoglycosides against prevalent Gram-negative pathogens (Escherichia coli, Salmonella enterica, and Klebsiella pneumoniae), suggesting that less toxic drug concentrations might be used effectively in patients. We go on to show that this effect improved treatment of biofilms, did not increase aminoglycoside resistance, and was due to the generation of proton-motive force (PMF). By single-cell microscopy, we demonstrate that stationary-phase cells, while non-dividing, actively maintain a growth-arrested state that is not reversed by metabolite addition. Surprisingly, within starved populations, we observed rare cells (3%) that divided without added nutrients. Additionally, we discovered that mannitol could directly protect human kidney cells from aminoglycoside cytotoxicity, independent of the metabolite’s effect on bacteria. This work forwards a mechanism-based strategy to improve existing antibiotics by mitigating their toxic side effects.

Protocol for the transformation of Bacillus subtilis through inducing natural competence

Transformation is the key step in genetic engineering as it governs the facile introduction of plasmid DNA or genome editing tools into the microbial cells. More importantly, approach taken for transformation differs between different microbial chassis depending on the structure of the cell envelope and other barriers. In this aspect, Bacillus subtilis requires a different transformation technique compared to the heat shock approach commonly used for Escherichia coli. Given that B. subtilis enters competence state during stationary phase, DNA could theoretically be introduced into the Gram-positive bacterium during this differentiation program. This aspect of B. subtilis cell biology has been exploited in biotechnology through the use of xylose for inducing the comK master competence regulator. With xylose as inducer, comK gene is transcribed, which would set off a transcriptional program that guides B. subtilis stationary phase cells to enter the natural competence state, where the cell envelope of the bacterium is permeabilized to afford facile entry of plasmid DNA. This protocol details the experimental approach and procedure for inducing natural competence in B. subtilis cells for enabling plasmid DNA to enter the bacterial cell during transformation. One methodological innovation in the described protocol is the use of stationary phase B. subtilis cells as compared to exponential phase cells in earlier incarnations of the approach.

Escherichia coli Has a Unique Transcriptional Program in Long-Term Stationary Phase Allowing Identification of Genes Important for Survival.

Microbes live in complex and constantly changing environments, but it is difficult to replicate this in the laboratory. Escherichia coli has been used as a model organism in experimental evolution studies for years; specifically, we and others have used it to study evolution in complex environments by incubating the cells into long-term stationary phase (LTSP) in rich media. In LTSP, cells experience a variety of stresses and changing conditions. While we have hypothesized that this experimental system is more similar to natural environments than some other lab conditions, we do not yet know how cells respond to this environment biochemically or physiologically. In this study, we began to unravel the cells' responses to this environment by characterizing the transcriptome of cells during LTSP. We found that cells in LTSP have a unique transcriptional program and that several genes are uniquely upregulated or downregulated in this phase. Further, we identified two genes, cspB and cspI, which are most highly expressed in LTSP, even though these genes are primarily known to respond to cold shock. By competing cells lacking these genes with wild-type cells, we show that these genes are also important for survival during LTSP. These data can help identify gene products that may play a role in survival in this complex environment and lead to identification of novel functions of proteins.IMPORTANCE Experimental evolution studies have elucidated evolutionary processes, but usually in chemically well-defined and/or constant environments. Using complex environments is important to begin to understand how evolution may occur in natural environments, such as soils or within a host. However, characterizing the stresses that cells experience in these complex environments can be challenging. One way to approach this is by determining how cells biochemically acclimate to heterogenous environments. In this study, we began to characterize physiological changes by analyzing the transcriptome of cells in a dynamic complex environment. By characterizing the transcriptional profile of cells in long-term stationary phase, a heterogenous and stressful environment, we can begin to understand how cells physiologically and biochemically react to the laboratory environment, and how this compares to more-natural conditions.

Thiol-specific oxidant diamide downregulates whiA gene of Corynebacterium glutamicum, thereby suppressing cell division and metabolism

The whiA (NCgl1527) gene from Corynebacterium glutamicum plays a crucial role during cell growth, and WhiA is recognized as the transcription factor for genes involved in cell division. In this study, we assessed the regulatory role of the gene in cell physiology. Transcription of the gene was specifically downregulated by the thiol-specific oxidant, diamide, and by heat stress. Cells exposed to diamide showed decreased transcription of genes involved in cell division and these effects were more profound in ΔwhiA cells. In addition, the ΔwhiA cells showed sensitivity to thiol-specific oxidants, DNA-damaging agents, and high temperature. Further, downregulation of sigH (NCgl0733), the central regulator in stress responses, along with master regulatory genes in cell metabolism, was observed in the ΔwhiA strain. Moreover, the amount of cAMP in the ΔwhiA cells in the early stationary phase was only at 30% level of that for the wild-type strain. Collectively, our data indicate that the role of whiA is to downregulate genes associated with cell division in response to heat or thiol-specific oxidative stress, and may suggest a role for the gene in downshifting cell metabolism by downregulating global regulatory genes when growth condition is not optimal for cells.

In S. cerevisiae hydroxycitric acid antagonizes chronological aging and apoptosis regardless of citrate lyase

Caloric restriction mimetics (CRMs) are promising molecules to prevent age-related diseases as they activate pathways driven by a true caloric restriction. Hydroxycitric acid (HCA) is considered a bona fide CRM since it depletes acetyl-CoA pools by acting as a competitive inhibitor of ATP citrate lyase (ACLY), ultimately repressing protein acetylation and promoting autophagy. Importantly, it can reduce inflammation and tumour development. In order to identify phenotypically relevant new HCA targets we have investigated HCA effects in Saccharomyces cerevisiae, where ACLY is lacking. Strikingly, the drug revealed a powerful anti-aging effect, another property proposed to mark bona fide CRMs. Chronological life span (CLS) extension but also resistance to acetic acid of HCA treated cells were associated to repression of cell apoptosis and necrosis. HCA also largely prevented cell deaths caused by a severe oxidative stress. The molecule could act widely by negatively modulating cell metabolism, similarly to citrate. Indeed, it inhibited both growth reactivation and the oxygen consumption rate of yeast cells in stationary phase. Genetic analyses on yeast CLS mutants indicated that part of the HCA effects can be sensed by Sch9 and Ras2, two conserved key regulators of nutritional and stress signal pathways of primary importance. Our data together with published biochemical analyses indicate that HCA may act with multiple mechanisms together with ACLY repression and allowed us to propose an integrated mechanistic model as a basis for future investigations.

4-Ethoxybenzoic acid inhibits Staphylococcus aureus biofilm formation and potentiates biofilm sensitivity to vancomycin

The adverse health effects of Staphylococcus aureus biofilm infections coupled with an increased global prevalence of antibiotic resistance highlight the need for novel anti-pathogenic, anti-biofilm compounds. The authors recently determined that ethyl-4-ethoxybenzoic acid (EEB) had anti-pathogenic, anti-biofilm activity. Based on this finding, a structure-activity analysis was undertaken to identify more effective compounds. Microtitre crystal violet assays followed by plate counts were conducted to measure the dose-dependent anti-biofilm and antimicrobial activities of 13 phenolic compounds related to EEB. By displaying these characteristics on a two-component plot, 4-ethoxybenzoic acid (4EB) and methyl gallate were identified as two anti-pathogenic, anti-biofilm compounds of interest. To characterize their mechanisms of activity, their effects on cell hydrophobicity, hemolysis activity, membrane integrity, extracellular polymeric substance production and vancomycin sensitivity were examined. Both 4EB and methyl gallate inhibited up to 87% of biofilm formation with minimal impact on the viability of stationary-phase cells or bacterial growth. Combination treatments of 4EB and vancomycin decreased the viability of biofilm-dwelling cells by up to 85% compared with vancomycin alone, indicating a synergistic effect. Methyl gallate did not potentiate vancomycin. 4EB decreased the percentage of hydrophobic cells in culture from 78% to 49%, indicating that 4EB may prevent biofilm formation by altering cell membrane hydrophobicity. These findings suggest that 4EB has potential as an anti-pathogenic, anti-biofilm agent for the prevention of S. aureus biofilms, or as a treatment for established biofilms when combined with antibiotics.

One-Step Curable, Covalently Immobilized Coating for Clinically Relevant Surfaces That Can Kill Bacteria, Fungi, and Influenza Virus.

Microbial attachment and subsequent colonization onto surfaces lead to the spread of deadly community-acquired and hospital-acquired (nosocomial) infections. Cationic polymeric coatings have gained enormous attention to tackle this scenario. However, non-biodegradable cationic polymer coated surfaces suffer from accumulation of microbial debris leading to toxicity and consequent complexities. Synthetic reproducibility and sophisticated coating techniques further limit their application. In this present study, we have developed one-step curable, covalent coatings based on two organo- and water-soluble small molecules, quaternary benzophenone-based ester and quaternary benzophenone-based amide, which can cross-link on surfaces upon UV irradiation. Upon contact, the coating completely killed bacteria and fungi in vitro including drug-resistant pathogens methicillin-resistant Staphylococcus aureus (MRSA) and fluconazole-resistant Candida albicans spp. The coating also showed antiviral activity against notorious influenza virus with 100% killing. The coated surfaces also killed stationary-phase cells of MRSA, which cannot be eradicated by traditional antibiotics. Upon hydrolysis, the surfaces switched to an antifouling state displaying significant reduction in bacterial adherence. To the best of our knowledge, this is the first report of an antimicrobial coating which could kill all of bacteria, fungi, and influenza virus. Taken together, the antimicrobial coating reported herein holds great promise to be developed for further application in healthcare settings.

Analysis of biosorption and biotransformation mechanism of Pseudomonas chengduensis strain MBR under Cd(II) stress from genomic perspective.

Microbial treatment of heavy metal-polluted sites is considered an environmentally friendly bioremediation technology with high potential. This study shows that Pseudomonas chengduensis strain MBR, a bacterium that can potentially be applied in the treatment of heavy metal pollution, is most affected by Cd(II) stress at the beginning of its growth. Up to 100% of total Cd(II) adsorption occurs in the first 48h after treatment of stationary phase cells with Cd(II). A biofilm forms on the cell surface, Cd(II) adsorbs, and is reduced to Cd (0) in the form of nanoscale particles. The genome of strain MBR was sequenced, annotated and analyzed. We identified various genes potentially related to cadmium resistance, transport and metabolism. Analysis of the strain MBR genome is helpful to explore the mechanism of Cd(II) resistance, and can provide new ideas for cadmium pollution control.

Phylogeny, growth and toxicity of the noxious red-tide dinoflagellate Alexandrium leei in Japan

A red tide of the dinoflagellate Alexandrium leei occurred at the beginning of April 2017 in Nomi Bay, Japan, killing aquacultured fishes, such as red sea breams and great amberjacks. This was the first known reported case of an ichthyotoxic red tide of A. leei in Japan, so we collected basic information about this dinoflagellate in the field and laboratory. Wild and cultured A. leei cells from Japan had similar morphological characteristics to cells from foreign waters, and phylogenetic analysis revealed that the Japanese isolate belonged to a northeast Asian group. Meteorological data from 2014 to 2018 showed that precipitation was lowest and the duration of bright sunshine was longest during January–May 2017 than in any other year. Field studies further showed that the water temperature during the red tide was ∼18°C and the salinity and dissolved inorganic nitrogen concentrations during January–May were higher in 2017 than in any other year from 2014 to 2018. A strain of A. leei that was cultured at 10–25°C grew at photosynthetic photon flux densities >15–30μmol photons m−2 s−1, exhibiting a maximum growth rate at 150 μmol photons m−2 s−1 at 20°C. Toxicity bioassays showed that most red sea breams showed signs of death within 2 h of exposure to cell suspensions containing >3 × 103 cells ml−1 of A. leei in both the logarithmic (log) and stationary phases of growth, rotifers died in a cell suspension containing <3 × 103 cells ml−1, with log-phase cells exhibiting higher toxicity than stationary-phase cells, and haemolytic activity increased in a cell concentration-dependent manner during the log phase of growth to reach <90% at 5 × 103 cells ml−1. The present study indicates that A. leei may affect fishes and other organisms in Japanese coastal waters.

Identifying Metabolic Inhibitors to Reduce Bacterial Persistence.

Bacterial persisters are rare phenotypic variants that are temporarily tolerant to high concentrations of antibiotics. We have previously discovered that stationary-phase-cell subpopulations exhibiting high redox activities were less capable of producing proteins and resuming growth upon their dilution into fresh media. The redox activities of these cells were maintained by endogenous protein and RNA degradation, resulting in self-inflicted damage that transiently repressed the cellular functions targeted by antibiotics. Here, we showed that pretreatment of stationary-phase cells with an ATP synthase inhibitor, chlorpromazine hydrochloride (CPZ), significantly reduced stationary-phase-redox activities and protein degradation, and yielded cells that were more susceptible to cell death when exposed to antibiotics in fresh media. Leveraging this knowledge, we developed an assay integrating a degradable fluorescent protein system and a small library, containing FDA-approved drugs and antibiotics, to detect medically relevant drugs that potentially target persister metabolism. We identified a subset of chemical inhibitors, including polymyxin B, poly-L-lysine and phenothiazine anti-psychotic drugs, that were able to reduce the persistence phenotype in Escherichia coli. These chemical inhibitors also reduced Pseudomonas aeruginosa persistence, potentially verifying the existence of similar mechanisms in a medically relevant organism.

Vancomycin Derivative Inactivates Carbapenem-Resistant Acinetobacter baumannii and Induces Autophagy.

Vancomycin is a standard drug for the treatment of multidrug-resistant Gram-positive bacterial infections. Albeit, development of resistance (VRE, VRSA) and its inefficacy against persistent infections is a demerit. It is also intrinsically inactive against Gram-negative bacteria. Herein, we report a vancomycin derivative, VanQAmC10, that addresses these challenges. VanQAmC10 was rapidly bactericidal against carbapenem-resistant A. baumannii (6 log10 CFU/mL reduction in 6 h), disrupted A. baumannii biofilms, and eradicated their stationary phase cells. In MRSA infected macrophages, the compound reduced the bacterial burden by 1.3 log10 CFU/mL while vancomycin exhibited a static effect. Further investigation indicated that the compound, unlike vancomycin, promoted the intracellular degradative mechanism, autophagy, in mammalian cells, which may have contributed to its intracellular activity. The findings of the work provide new perspectives on the field of glycopeptide antibiotics.

Toxicity of UV filters on marine bacteria: Combined effects with damaging solar radiation

Organic UV filters are of emerging concern due to their occurrence and persistence in coastal ecosystems. Because marine bacteria are crucial in the major biogeochemical cycles, there is an urgent need to understand to what extent these microorganisms are affected by those chemicals. This study deciphers the impact of five common sunscreen UV filters on twenty-seven marine bacteria, combining both photobiology and toxicity analysis on environmentally relevant species. Seven bacteria were sensitive to different organic UV filters at 1000 μg L−1, including octinoxate and oxybenzone. This is the first report demonstrating inhibition of bacterial growth from 100 μg L−1. None of the UV filters showed any toxicity at 1000 μg L−1 on stationary phase cells, demonstrating that physiological state was found to be a key parameter in the bacterial response to UV-filters. Indeed, non-growing bacteria were resistant to UV filters whereas growing cells exhibited UV filter dependent sensitivity. Octinoxate was the most toxic chemical at 1000 μg L−1 on growing cells. Interestingly, photobiology experiments revealed that the toxicity of octinoxate and homosalate decreased after light exposure while the other compounds were not affected. In terms of environmental risk characterization, our results revealed that the increasing use of sun blockers could have detrimental impacts on bacterioplanktonic communities in coastal areas. Our findings contribute to a better understanding of the impact of the most common UV filters on bacterial species and corroborate the importance to consider environmental parameters such as solar radiation in ecotoxicology studies.

Protection from hydrogen peroxide stress relies mainly on AhpCF and KatA2 in Stenotrophomonas maltophilia

BackgroundAerobically-grown bacteria can be challenged by hydrogen peroxide stress from endogenous aerobic metabolism and exogenously generated reactive oxygen species. Catalase (Kat), alkyl hydroperoxidase (Ahp), and glutathione peroxidase (Gpx) systems are major adaptive responses to H2O2 stress in bacteria. Stenotrophomonas maltophilia is a ubiquitous Gram-negative bacterium equipped with four Kats (KatA1, KatA2, KatMn, and KatE), one Ahp (AhpCF), and three Gpxs (Gpx1, Gpx2, and Gpx3). Here, we systematically investigated how the eight H2O2 scavenging genes differentially contribute to the low-micromolar levels of H2O2 generated from aerobic metabolism and high-millimolar levels of H2O2 from exogenous sources.MethodsGene expression was assessed and quantified by reverse transcription-PCR (RT-PCR) and real time quantitative PCR (qRT-PCR), respectively. The contribution of these enzymes to H2O2 stress was assessed using mutant construction and functional investigation.ResultsOf the eight genes, katA2, ahpCF, and gpx3 were intrinsically expressed in response to low-micromolar levels of H2O2 from aerobic metabolism, and the expression of katA2 and ahpCF was regulated by OxyR. AhpCF and KatA2 were responsible for alleviating aerobic growth-mediated low concentration H2O2 stress and AhpCF played a critical role for stationary-phase cells. KatA2 was upregulated to compensate for AhpCF in the case of ahpCF inactivation. After exposure to millimolar levels of H2O2, katA2 and ahpCF were upregulated in an OxyR-dependent manner. KatA2 was the critical enzyme for dealing with high concentration H2O2. Loss-of-function of KatA2 increased bacterial susceptibility to high concentration H2O2.ConclusionsAhpCF and KatA2 are key enzymes protecting S. maltophilia from hydrogen peroxide stress.

Evaluation of Natural and Botanical Medicines for Activity Against Growing and Non-growing Forms of B. burgdorferi.

Lyme disease is the most common vector-borne disease in the US and Europe. Although the current recommended Lyme antibiotic treatment is effective for the majority of Lyme disease patients, about 10–20% of patients continue to suffer from persisting symptoms. There have been various anecdotal reports on the use of herbal extracts for treating patients with persisting symptoms with varying degree of improvements. However, it is unclear whether the effect of the herb products is due to their direct antimicrobial activity or their effect on host immune system. In the present study, we investigated the antimicrobial effects of 12 commonly used botanical medicines and three other natural antimicrobial agents for potential anti-Borrelia burgdorferi activity in vitro. Among them, 7 natural product extracts at 1% were found to have good activity against the stationary phase B. burgdorferi culture compared to the control antibiotics doxycycline and cefuroxime. These active botanicals include Cryptolepis sanguinolenta, Juglans nigra (Black walnut), Polygonum cuspidatum (Japanese knotweed), Artemisia annua (Sweet wormwood), Uncaria tomentosa (Cat's claw), Cistus incanus, and Scutellaria baicalensis (Chinese skullcap). In contrast, Stevia rebaudiana, Andrographis paniculata, Grapefruit seed extract, colloidal silver, monolaurin, and antimicrobial peptide LL37 had little or no activity against stationary phase B. burgdorferi. The minimum inhibitory concentration (MIC) values of Artemisia annua, Juglans nigra, and Uncaria tomentosa were quite high for growing B. burgdorferi, despite their strong activity against the non-growing stationary phase B. burgdorferi. On the other hand, the top two active herbs, Cryptolepis sanguinolenta and Polygonum cuspidatum, showed strong activity against both growing B. burgdorferi (MIC = 0.03–0.06% and 0.25–0.5%, respectively) and non-growing stationary phase B. burgdorferi. In subculture studies, only 1% Cryptolepis sanguinolenta extract caused complete eradication, while doxycycline and cefuroxime and other active herbs could not eradicate B. burgdorferi stationary phase cells as many spirochetes were visible after 21-day subculture. Further studies are needed to identify the active constituents of the effective botanicals and evaluate their combinations for more effective eradication of B. burgdorferi in vitro and in vivo. The implications of these findings for improving treatment of persistent Lyme disease are discussed.

Carotenoid Content and Composition in Exponential, Stationary and Biofilm States of Staphylococcus aureus and their Influence on Membrane Biophysical Properties

In Staphylococcus aureus, biofilms are implicated in the resilience of chronic infections, presenting a serious clinical problem world-wide. Here, S. aureus biofilms are grown under flow within clinical catheters at 37 °C. The lipid composition and biophysical properties of lipid extracts from these biofilms are compared with those from exponential growth and stationary phase cells. Biofilms show a reduction in iso and anteiso branching compensated by an increase in saturated fatty acids compared to stationary phase. A drastic reduction in carotenoid levels is also observed during biofilm formation. HPLC-MS shows the different species of carotenoids found in the stationary phase, where the different metabolic intermediates of the staphyloxanthin synthesis route are identified. Thermotropic measurements of Laurdan GP and DPH polarization, show a reduction of lipid packing at 37 °C for biofilms compared to stationary phase. We studied the effects of carotenoid content on DMPG and DPPG model membranes showing trends in thermotropic behavior consistent with those observed in bacterial isolates, indicating that carotenoids participate in modulating lipid packing. Additionally, bending elastic constant (kc) measurements using vesicle fluctuation analysis (VFA) show that the presence of carotenoids can increase membrane bending rigidity. The antimicrobial peptide Magainin H2 was less active on liposomes composed of stationary phase compared to biofilms or exponential growth isolates. This study contributes to an understanding of how Staphylococcus aureus modulates the composition of its membrane lipids, and how those changes affect the biophysical properties of membranes, which in turn may play a role in its virulence and its resistance to different membrane-active antimicrobial agents.

Growth Mode and Physiological State of Cells Prior to Biofilm Formation Affect Immune Evasion and Persistence of Staphylococcus aureus.

The present study investigated Staphylococcus aureus ATCC25923 surfaceomes (cell surface proteins) during prolonged growth by subjecting planktonic and biofilm cultures (initiated from exponential or stationary cells) to label-free quantitative surfaceomics and phenotypic confirmations. The abundance of adhesion, autolytic, hemolytic, and lipolytic proteins decreased over time in both growth modes, while an opposite trend was detected for many tricarboxylic acid (TCA) cycle, reactive oxygen species (ROS) scavenging, Fe-S repair, and peptidolytic moonlighters. In planktonic cells, these changes were accompanied by decreasing and increasing adherence to hydrophobic surface and fibronectin, respectively. Specific RNA/DNA binding (cold-shock protein CspD and ribosomal proteins) and the immune evasion (SpA, ClfA, and IsaB) proteins were notably more abundant on fully mature biofilms initiated with stationary-phase cells (SDBF) compared to biofilms derived from exponential cells (EDBF) or equivalent planktonic cells. The fully matured SDBF cells demonstrated higher viability in THP-1 monocyte/macrophage cells compared to the EDBF cells. Peptidoglycan strengthening, specific urea-cycle, and detoxification enzymes were more abundant on planktonic than biofilm cells, indicating the activation of growth-mode specific pathways during prolonged cultivation. Thus, we show that S. aureus shapes its surfaceome in a growth mode-dependent manner to reach high levofloxacin tolerance (>200-times the minimum biofilm inhibitory concentration). This study also demonstrates that the phenotypic state of the cells prior to biofilm formation affects the immune-evasion and persistence-related traits of S. aureus.