Stationary Phase Cultures Research Articles

The Mla pathway promotes Vibrio cholerae re-expansion from stationary phase.

Bacteria regularly encounter conditions with nutrient scarcity, where cell growth and division are minimal. Knowledge of the pathways that enable re-growth following nutrient restriction is limited. Here, using the cholera pathogen, we uncovered a role for the Mla pathway, a system that enables phospholipid re-cycling, in promoting Vibrio cholerae re-expansion from stationary phase cultures. Cells labeled with DNA barcodes or fluorophores were useful to demonstrate that though the abundances of wild-type and Mla mutant cells were similar in stationary phase cultures, they had marked differences in their capacities to regrow on plates. Of note, Mla mutant cells lose cell envelope components including high-energy phospholipids due to OMV shedding. Our findings suggest that the defects in cellular energy homeostasis that emerge in the absence of the Mla pathway underlie its importance in maintaining V. cholerae culturability.

Quorum sensing regulation of Psl polysaccharide production by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a common opportunistic pathogen and a model organism for studying bacterial sociality. A social behavior of P. aeruginosa that is critical for its success as a pathogen is its ability to form protective biofilms. Many of P. aeruginosa's social phenotypes are regulated by quorum sensing-a type of cell-cell communication that allows bacteria to respond to population density. Although biofilm formation is known to be affected by quorum sensing, evidence for direct regulation of biofilm production by quorum regulators has remained elusive. In this work, we show that production of the major biofilm matrix polysaccharide Psl in P. aeruginosa PAO1 is regulated by the quorum regulators LasR and RhlR in stationary-phase cultures. Secretion of Psl into the culture medium requires LasR, RhlR, and the quorum signal molecules N-3-oxo-dodecanoyl-homoserine lactone and N-butanoyl homoserine lactone. Psl production in strains unable to synthesize the homoserine lactone signals can be restored by exogenous introduction of the signal molecules. We found that LasR and RhlR perform different roles in the regulation of Psl production: LasR acts at the promoter of the psl operon and activates transcription of the Psl biosynthetic genes, while RhlR activates translation of the psl transcripts. This work contributes to our understanding of the overlapping but distinct functions of the Las and Rhl quorum-sensing systems and implicates both in the direct regulation of biofilm matrix production.IMPORTANCEPseudomonas aeruginosa biofilms are responsible for many treatment-resistant infections in humans. Many cooperative behaviors in P. aeruginosa are controlled by quorum sensing, but evidence for a direct role of quorum sensing in the regulation of biofilm matrix production has been scant. In this work, we show that the Las and Rhl quorum-sensing systems have distinct roles in regulating production of the matrix polysaccharide Psl and that this regulation happens at the level of transcription (Las) and translation (Rhl) of the psl operon. These findings deepen our understanding of overlapping functions of Las and Rhl quorum sensing and the complex regulation of biofilm development in P. aeruginosa.

The Mla pathway promotes Vibrio cholerae re-expansion from stationary phase.

Bacteria have evolved diverse strategies to ensure survival under nutrient-limited conditions, where rapid energy generation is not achievable. Here, we performed a transposon insertion site sequencing loss-of-function screen to identify Vibrio cholerae genes that promote the pathogen's fitness in stationary phase. We discovered that the Mla (maintenance of lipid asymmetry) pathway, which is crucial for transferring phospholipids from the outer to the inner membrane, is critical for stationary phase fitness. Competition experiments with barcoded and fluorophore labeled wild-type and mlaE mutant V. cholerae revealed that the Mla pathway promotes re-expansion from 48h stationary phase cultures. The mutant's defect in transitioning out of stationary phase into active growth (culturability) was also observed in monocultures at 48h. However, by 96h the culturability of the mutant and wild-type strains were equivalent. By monitoring the abundances of genomically barcoded libraries of wild-type and ∆mlaE strains, we observed that a few barcodes dominated the mutant culture at 96h, suggesting that the similarity of the population sizes at this time was caused by expansion of a subpopulation containing a mutation that suppressed the mlaE mutant's defect. Whole genome sequencing revealed that mlaE suppressors inactivated flagellar biosynthesis. Additional mechanistic studies support the idea that the Mla pathway is critical for the maintenance of V. cholerae's culturability as it promotes energy homeostasis, likely due to its role in regulating outer membrane vesicle shedding. Together our findings provide insights into the cellular processes that control re-expansion from stationary phase and demonstrate a previously undiscovered role for the Mla pathway.

Transcriptional response of Methanosarcina acetivorans to repression of the energy-conserving methanophenazine: CoM-CoB heterodisulfide reductase enzyme HdrED.

Methane-producing archaea are key organisms in the anaerobic carbon cycle. These organisms, also called methanogens, grow by converting substrate to methane gas in a process called methanogenesis. Previous research showed that the reduction of the terminal electron acceptor is the rate-limiting step in methanogenesis by Methanosarcina acetivorans. In order to gain insight into how the cells sense and respond to the availability of the terminal electron acceptor, we designed an experiment to deplete cells of the essential terminal oxidase enzyme, HdrED. We found that the depletion of HdrED in vivo results in a higher abundance of transcripts for methyltransferases (mtaC2, mtaB3, mtaC3), coenzyme B biosynthesis, C1 metabolism, and pyrimidine compounds. In most cases, these changes were distinct from transcript abundance changes observed during the transition from exponential growth to stationary phase cultures. These data implicate the methylotrophic methanogenesis regulator MsrC (MA4383) in CoM-S-S-CoB heterodisulfide sensing and indicate cells have a specific mechanism to sense intracellular ratio of CoM-S-S-CoB, coenzyme M, and coenzyme B thiols and further suggest transcripts encoding translation and methanogenesis functions are controlled by feed-forward regulation depending on substrate availability.IMPORTANCEMethanosarcina is an emerging model archaeon and synthetic biology platform for the production of renewable energy and sustainable chemicals to reduce dependence on petroleum. Research into metabolic networks and gene regulation in this organism and other methanogens will inform genome-scale metabolic modeling and microbial function prediction in uncultured or non-model anaerobes and archaea. This study suggests methanogens use unknown mechanisms to efficiently couple methanogenesis to gene regulation via CoM-S-S-CoB and ATP availability.

Ubiquitin-like and ubiquitinylated proteins associated with the maternal cell walls of Scenedesmus obliquus 633 as identified by immunochemistry and LC-MS/MS proteomics.

The cell walls of green algae Scenedesmus obliquus are complex, polymeric structures including an inner cellulose layer surrounded by an algaenan-containing trilaminar sheath. The process of autosporulation leads to the formation of sporangial (maternal) cell walls, which are released into the medium after sporangial autolysis. In this study, a fraction of maternal cell wall material (CWM) was isolated from the stationary phase cultures of Scenedesmus obliquus 633 and subjected to immunofluorescence microscopy using polyclonal anti-ubiquitin antibodies. The water-extracted polypeptide fraction from the maternal cell walls was then analyzed using immunoblotting and LC-MS/MS. An immunoanalysis showed the presence of several peptides reactive with polyclonal anti-ubiquitin serum, with apparent molecular masses of c. 12, 70, 120, 200, and > 250kDa. Cell wall-associated peptides were identified on the basis of LC-MS/MS spectra across NCBI databases, including the Scenedesmaceae family (58 records), the Chlorophyceae class (37 records), and Chlamydomonas reinhardtii (18 records) corresponding to the signatures of 95 identified proteins. In particular, three signatures identified ubiquitin and ubiquitin-related proteins. In the maternal cell walls, immunoblotting analysis, immunofluorescence microscopy, and LC-MS/MS proteomics collectively demonstrated the presence of ubiquitin-like epitopes, ubiquitin-specific peptide signatures, and several putative ubiquitin conjugates of a higher molecular mass. These results support the presence of ubiquitin-like proteins in the extramembranous compartment of Scenedesmus obliquus 633 and suggest that protein ubiquitination plays a significant role in the formation and functional integrity of the maternal cell walls in green algae.

Bacterial cell size modulation along the growth curve across nutrient conditions.

Under stable growth conditions, bacteria maintain cell size homeostasis through coordinated elongation and division. However, fluctuations in nutrient availability result in dynamic regulation of the target cell size. Using microscopy imaging and mathematical modelling, we examine how bacterial cell volume changes over the growth curve in response to nutrient conditions. We find that two rod-shaped bacteria, Escherichia coli and Salmonella enterica, exhibit similar cell volume distributions in stationary phase cultures irrespective of growth media. Cell resuspension in rich media results in a transient peak with a five-fold increase in cell volume ≈ 2h after resuspension. This maximum cell volume, which depends on nutrient composition, subsequently decreases to the stationary phase cell size. Continuous nutrient supply sustains the maximum volume. In poor nutrient conditions, cell volume shows minimal changes over the growth curve, but a markedly decreased cell width compared to other conditions. The observed cell volume dynamics translate into non-monotonic dynamics in the ratio between biomass (optical density) and cell number (colony-forming units), highlighting their non-linear relationship. Our findings support a heuristic model comparing modulation of cell division relative to growth across nutrient conditions and providing novel insight into the mechanisms of cell size control under dynamic environmental conditions.

Downregulation of Klebsiella pneumoniae RND efflux pump genes following indole signal produced by Escherichia coli

BackgroundMore than a century has passed since it was discovered that many bacteria produce indole, but research into the actual biological roles of this molecule is just now beginning. The influence of indole on bacterial virulence was extensively investigated in indole-producing bacteria like Escherichia coli. To gain a deeper comprehension of its functional role, this study investigated how indole at concentrations of 0.5-1.0 mM found in the supernatant of Escherichia coli stationary phase culture was able to alter the virulence of non-indole-producing bacteria, such as Pseudomonas aeruginosa, Proteus mirabilis, and Klebsiella pneumoniae, which are naturally exposed to indole in mixed infections with Escherichia coli.ResultsBiofilm formation, antimicrobial susceptibility, and efflux pump activity were the three phenotypic tests that were assessed. Indole was found to influence antibiotic susceptibly of Pseudomonas aeruginosa, Proteus mirabilis and Klebsiella pneumoniae to ciprofloxacin, imipenem, ceftriaxone, ceftazidime, and amikacin through significant reduction in MIC with fold change ranged from 4 to 16. Biofilm production was partially abrogated in both 32/45 Pseudomonas aeruginosa and all eight Proteus mirabilis, while induced biofilm production was observed in 30/40 Klebsiella pneumoniae. Moreover, acrAB and oqxAB, which encode four genes responsible for resistance-nodulation-division multidrug efflux pumps in five isolates of Klebsiella pneumoniae were investigated genotypically using quantitative real-time (qRT)-PCR. This revealed that all four genes exhibited reduced expression indicated by 2^−ΔΔCT < 1 in indole-treated isolates compared to control group.ConclusionThe outcomes of qRT-PCR investigation of efflux pump expression have established a novel clear correlation of the molecular mechanism that lies beneath the influence of indole on bacterial antibiotic tolerance. This research provides novel perspectives on the various mechanisms and diverse biological functions of indole signaling and how it impacts the pathogenicity of non-indole-producing bacteria.

Green synthesis and antibacterial-antibiofilm properties of biogenic silver nanoparticles

The biosynthesis of metallic nanoparticles is gaining prominence as an alternative to traditional physicochemical methods, offering several advantages such as simplicity, non-toxicity, lower energy requirements and short reaction times leading to environmentally sustainable processes. The aims of this work were: to study the extracellular biosynthesis of silver nanoparticles (AgNPs) by Pseudomonas extremaustralis 2E-UNGS, to characterise the shape, monodispersity and size of AgNPs, to explore their antimicrobial and antibiofilm activities, and to evaluate the role of nitrate reductase activity in the biosynthesis process. The novelty of this work relies on the development of a green and sustainable method for the synthesis of stable AgNPs with optimal properties for potential applications in antimicrobial materials, especially when incorporated into polymeric matrices or used as agrochemical substitutes. Optimal conditions for the biosynthesis of spherical AgNPs were determined to be pH 7, 38 °C, 4 h of darkness and 120 rpm using stationary phase culture supernatants of P. extremaustralis 2E-UNGS. The involvement of extracellular nitrate reductase in AgNP biosynthesis was confirmed by enzymatic assays and supported by bioinformatics analysis, which identified the presence of the napA2 gene linked to the nirBD cluster. Antimicrobial assays demonstrated the inhibitory effect of AgNPs against both Gram-positive and Gram-negative bacteria, including Pseudomonas aeruginosa PA01 in both planktonic and biofilm states. In addition, the potential application of AgNPs in innovative antibacterial polymers was explored by incorporating them into polyurethane matrices either alone (PU-AgNP) or in combination with crystal violet as a photosensitizer (PU-AgNP-CV). Subsequent inoculation with a clinical isolate of Pseudomonas aeruginosa resulted in significant reductions in viable bacterial counts on both PU-AgNP-CV and PU-AgNP. Biogenic AgNPs showed antibacterial and antibiofilm properties for new antimicrobial material development.

The mycobacterial glycoside hydrolase LamH enables capsular arabinomannan release and stimulates growth

Mycobacterial glycolipids are important cell envelope structures that drive host-pathogen interactions. Arguably, the most important are lipoarabinomannan (LAM) and its precursor, lipomannan (LM), which are trafficked from the bacterium to the host via unknown mechanisms. Arabinomannan is thought to be a capsular derivative of these molecules, lacking a lipid anchor. However, the mechanism by which this material is generated has yet to be elucidated. Here, we describe the identification of a glycoside hydrolase family 76 enzyme that we term LamH (Rv0365c in Mycobacterium tuberculosis) which specifically cleaves α−1,6-mannoside linkages within LM and LAM, driving its export to the capsule releasing its phosphatidyl-myo-inositol mannoside lipid anchor. Unexpectedly, we found that the catalytic activity of this enzyme is important for efficient exit from stationary phase cultures, potentially implicating arabinomannan as a signal for growth phase transition. Finally, we demonstrate that LamH is important for M. tuberculosis survival in macrophages.

Low-dose ionizing radiation generates a hormetic response to modify lipid metabolism in Chlorella sorokiniana

Algal biomass is a viable source of chemicals and metabolites for various energy, nutritional, medicinal and agricultural uses. While stresses have commonly been used to induce metabolite accumulation in microalgae in attempts to enhance high-value product yields, this is often very detrimental to growth. Therefore, understanding how to modify metabolism without deleterious consequences is highly beneficial. We demonstrate that low-doses (1–5 Gy) of ionizing radiation in the X-ray range induces a non-toxic, hormetic response in microalgae to promote metabolic activation. We identify specific radiation exposure parameters that give reproducible metabolic responses in Chlorella sorokiniana caused by transcriptional changes. This includes up-regulation of >30 lipid metabolism genes, such as genes encoding an acetyl-CoA carboxylase subunit, phosphatidic acid phosphatase, lysophosphatidic acid acyltransferase, and diacylglycerol acyltransferase. The outcome is an increased lipid yield in stationary phase cultures by 25% in just 24 hours, without any negative effects on cell viability or biomass.

A high-throughput assay identifies molecules with antimicrobial activity against persister cells.

Introduction. Persister cells are transiently non-growing antibiotic-tolerant bacteria that cause infection relapse, and there is no effective antibiotic therapy to tackle these infections.Gap statement. High-throughput assays in drug discovery are biased towards detecting drugs that inhibit bacterial growth rather than killing non-growing bacteria. A new and simple assay to discover such drugs is needed.Aim. This study aims to develop a simple and high-throughput assay to identify compounds with antimicrobial activity against persister cells and use it to identify molecular motifs with such activity.Methodology. We quantified Staphylococcus aureus persister cells by enumeration of colony forming units after 24 h ciprofloxacin treatment. We first quantified how the cell concentration, antibiotic concentration, growth phase and presence/absence of nutrients during antibiotic exposure affected the fraction of persister cells in a population. After optimizing these parameters, we screened the antimicrobial activity of compound fragments to identify molecular structures that have activity against persister cells.Results. Exponential- and stationary-phase cultures transferred to nutrient-rich media displayed a bi-phasic time-kill curve and contained 0.001-0.07% persister cells. A short rifampicin treatment resulted in 100% persister cells for 7 h, after which cells resumed activity and became susceptible. Stationary-phase cultures displayed a low but constant death rate but ultimately resulted in similarly low survival rates as the exponential-phase cultures after 24 h ciprofloxacin treatment. The persister phenotype was only maintained in most of the population for 24 h if cells were transferred to a carbon-free minimal medium before exposure to ciprofloxacin. Keeping cells starved enabled the generation of high concentrations of S. aureus cells that tolerate 50× MIC ciprofloxacin, and we used this protocol for rapid screening for biocidal antibiotics. We identified seven compounds from four structural clusters with activity against antibiotic-tolerant S. aureus. Two compounds were moderately cytotoxic, and the rest were highly cytotoxic.Conclusion. Transferring a stationary-phase culture to a carbon-free minimal medium for antimicrobial testing is a simple strategy for high-throughput screening for new antibiotics that kill persister cells. We identified molecule fragments with such activity, but further screening is needed to identify motifs with lower general cytotoxicity.

Anti-Persisters Activity of Lacticaseibacillus rhamnosus Culture Filtrates against Pseudomonas aeruginosa in Artificial Sputum Medium.

Persisters are antibiotic-tolerant bacteria, playing a role in the recalcitrance and relapse of many bacterial infections, including P. aeruginosa pulmonary infections in Cystic Fibrosis (CF) patients. Among novel antimicrobial strategies, the use of probiotics and their products is emerging as a particularly promising approach. The aim of this study was to evaluate the anti-persisters activity of culture filtrate supernatants of Lacticaseibacillus rhamnosus (LRM-CFS) against P. aeruginosa in artificial sputum medium (ASM), which resembles the CF lung environment. Planktonic persisters of two clinical strains of P. aeruginosa (PaCF1 and PaCF4) were obtained following two different procedures: (i) exposing stationary-phase cultures to cyanide m-chlorophenylhydrazone (CCCP) in LB medium; (ii) incubating stationary-phase cultures with high doses of tobramycin (128-fold MIC) in ASM. In addition, persisters from biofilm were obtained by exposing 48 h old biofilm of P. aeruginosa to 128 x MIC of ciprofloxacin. LRM-CFS at dilutions of 1:6 and 1:4 resulted in being bactericidal in ASM against both PaCF1 and PaCF4 persisters obtained after CCCP or tobramycin treatment. Moreover, LRM-CFS at dilution 1:4 caused a reduction of antibiotic-tolerant bacteria in the biofilm of both P. aeruginosa strains. Overall, LRM-CFS represents a promising adjuvant therapeutic strategy against P. aeruginosa recalcitrant infections in CF patients.

Fate of in vitro cultured Mycobacterium abscessus populations when exposed to moxifloxacin.

Given the current need for predictive persisting model for Mycobacterium abscessus, we adopted a classical assay to study drug-tolerant bacterial persisters, focusing on the behavior of a small antibiotic-insensitive subpopulation during prolonged exposure to moxifloxacin. Our study showed a wide-ranging response of M. abscessus, depending on antibiotic concentration, growth stage of mycobacterial cultures, and the availability of potassium ions in the medium. Mid-logarithmic cultures, initially grown in either balanced or K+-free medium, contained small sup-populations capable of prolonged and stable survival in the presence of moxifloxacin. The response of these mid-log cultures to antibiotic exposure involved initial killing, followed by regrowth at 1-2 MBCs of moxifloxacin or a substantial reduction of the antibiotic-insensitive subpopulation to fewer than 102 CFU/mL at 16 MBCs. In stationary-phase cultures grown in a complete medium, a consistent number of viable cells was observed when exposed to a high dose of moxifloxacin. In contrast, antibiotic-insensitive subpopulations in stationary-phase M. abscessus cultures under potassium-deficient conditions experienced gradual killing across a wide range of moxifloxacin concentrations (1-16 MBCs). Studies on electron microscopy demonstrated that singular cells were rapidly destroyed after relatively short-term exposure to moxifloxacin, while cells in aggregates or clumps persisted longer, explaining the delayed biocidal effect. The small subpopulation that survived under intense moxifloxacin pressure was notably heterogeneous in cell morphology and fine structure, consisting of ovoid forms and cell-wall-deficient cells with reduced size. These findings suggest that the same antibiotic dose may have varying effects on M. abscessus cells, depending on their physiological state and abundance within infected cells or tissues. Taken together, our study may contribute to the development of strategies to combat recalcitrant survivor subpopulations.

Identification of a Shewanella halifaxensis Strain with Algicidal Effects on Red Tide Dinoflagellate Prorocentrum triestinum in Culture.

The dinoflagellate Prorocentrum triestinum forms high biomass blooms that discolor the water (red tides), which may pose a serious threat to marine fauna and aquaculture exploitations. In this study, the algicidal effect of a bacterial strain (0YLH) belonging to the genus Shewanella was identified and evaluated against P. triestinum. The algicidal effects on the dinoflagellate were observed when P. triestinum was exposed to cell-free supernatant (CFS) from stationary-phase cultures of the 0YLH strain. After 24 h exposure, a remarkable reduction in the photosynthetic efficiency of P. triestinum was achieved (55.9%), suggesting the presence of extracellular bioactive compounds produced by the bacteria with algicidal activity. Furthermore, the CFS exhibited stability and maintained its activity across a wide range of temperatures (20-120 °C) and pH values (3-11). These findings highlight the algicidal potential of the bacterium Shewanella halifaxensis 0YLH as a promising tool for the environmentally friendly biological control of P. triestinum blooms.

Distinct types of multicellular aggregates in Pseudomonas aeruginosa liquid cultures

Pseudomonas aeruginosa forms suspended multicellular aggregates when cultured in liquid media. These aggregates may be important in disease, and/or as a pathway to biofilm formation. The polysaccharide Psl and extracellular DNA (eDNA) have both been implicated in aggregation, but previous results depend strongly on the experimental conditions. Here we develop a quantitative microscopy-based method for assessing changes in the size distribution of suspended aggregates over time in growing cultures. For exponentially growing cultures of P. aeruginosa PAO1, we find that aggregation is mediated by cell-associated Psl, rather than by either eDNA or secreted Psl. These aggregates arise de novo within the culture via a growth process that involves both collisions and clonal growth, and Psl non-producing cells do not aggregate with producers. In contrast, we find that stationary phase (overnight) cultures contain a different type of multicellular aggregate, in which both eDNA and Psl mediate cohesion. Our findings suggest that the physical and biological properties of multicellular aggregates may be very different in early-stage vs late-stage bacterial cultures.

Studying the metabolic factors that may impact the growth of co-cultured Listeria monocytogenes strains at low temperature

The simultaneous presence of more than one strains of Listeria monocytogenes in the same food product may affect the growth capacity of each strain. The present study evaluated the metabolites composition that may potentially influence the growth of individual L. monocytogenes strains in a dual strain composite. Based on previous studies, L. monocytogenes strains, C5 (4b) and 6179 (1/2a) were selected due to the remarkable interaction, which was observed during their co-culture. The selected strains were inoculated (2.0 - 3.0 log CFU/mL) in Tryptic Soy Broth with 0.6% Yeast Extract (TSB-YE) in single and two-strain cultures (1:1 strain ratio). Bacterial growth was assessed during storage at 7 °C, under aerobic conditions (AC). Their resistance to different antibiotics enabled the selective enumeration of each strain in the co-culture. After reaching stationary phase, single and dual cultures were centrifuged and filtered. The cell-free spent medium (CFSM) was either characterized by Fourier transform infrared (FTIR-ATR) spectrometry or re-inoculated, after the addition of concentrated TSB-YE (for nutrient replenishment), with single and two-strain cultures for the evaluation of growth under the influence of metabolites produced from the same singly and co-cultured strains in the different combinations of strains and CFSM origin (7 °C/AC) (n = 2x3). By the end of storage, singly-cultured C5 and 6179 had reached 9.1 log CFU/mL, while in dual culture, 6179 was affected by the presence of C5 attaining only 6.4 ± 0.8 log CFU/mL. FTIR-ATR spectra of CFSM produced by singly-cultured 6179 and the co-culture were almost identical. Characteristic peaks in FTIR-ATR spectrum of CFSM of singly-cultured C5 at 1741, 1645 and 1223 cm−1 represent functional groups which were not present in the CFSM of the co-culture. These molecules may be located intracellularly or mounted on bacterial cell surface and removed from the supernatant during cell filtration of the co-culture. Both singly- and co-cultured 6179 managed to grow similarly regardless of CFSM origin. Contrarily, both singly- and co-cultured C5 managed to outgrow 6179 in CFSM which contained high concentration of C5 metabolites, while in CFSM produced by singly-cultured 6179, C5 did not grow, suggesting that the produced metabolites of strain 6179 appears to be harmful to strain C5. However, during co-culture, C5 may produce molecules that counteract the inhibitory effect of 6179. The findings shed more light on the mechanism behind the inter-strain interactions of L. monocytogenes indicating that both contact of cells and extracellular metabolites may influence the behavior of the different co-existing strains.

Staphylococcus aureus persisters are associated with reduced clearance in a catheter-associated biofilm infection.

Staphylococcus aureus causes a wide variety of infections, many of which are chronic or relapsing in nature. Antibiotic therapy is often ineffective against S. aureus biofilm-mediated infections. Biofilms are difficult to treat partly due to their tolerance to antibiotics, however the underlying mechanism responsible for this remains unknown. One possible explanation is the presence of persister cells-dormant-like cells that exhibit tolerance to antibiotics. Recent studies have shown a connection between a fumC (fumarase C, a gene in the tricarboxylic acid cycle) knockout strain and increased survival to antibiotics, antimicrobial peptides, and in a Drosophila melanogaster model. It remained unclear whether a S. aureus high persister strain would have a survival advantage in the presence of innate and adaptive immunity. To further investigate this, a fumC knockout and wild type strains were examined in a murine catheter-associated biofilm model. Interestingly, mice struggled to clear both S. aureus wild type and the fumC knockout strains. We reasoned both biofilm-mediated infections predominantly consisted of persister cells. To determine the persister cell population within biofilms, expression of a persister cell marker (Pcap5A::dsRED) in a biofilm was examined. Cell sorting of biofilms challenged with antibiotics revealed cells with intermediate and high expression of cap5A had 5.9-and 4.5-fold higher percent survival compared to cells with low cap5A expression. Based on previous findings that persisters are associated with reduced membrane potential, flow cytometry analysis was used to examine the metabolic state of cells within a biofilm. We confirmed cells within biofilms had reduced membrane potential compared to both stationary phase cultures (2.5-fold) and exponential phase cultures (22.4-fold). Supporting these findings, cells within a biofilm still exhibited tolerance to antibiotic challenge following dispersal of the matrix through proteinase K. Collectively, these data show that biofilms are largely comprised of persister cells, and this may explain why biofilm infections are often chronic and/or relapsing in clinical settings.

Inference of transcriptome signatures of Escherichia coli in long-term stationary phase

“Non-growing” is a dominant life form of microorganisms in nature, where available nutrients and resources are limited. In laboratory culture systems, Escherichia coli can survive for years under starvation, denoted as long-term stationary phase, where a small fraction of cells manages to survive by recycling resources released from nonviable cells. Although the physiology by which viable cells in long-term stationary phase adapt to prolonged starvation is of great interest, their genome-wide response has not been fully understood. In this study, we analyzed transcriptional profiles of cells exposed to the supernatant of 30-day long-term stationary phase culture and found that their transcriptome profiles displayed several similar responses to those of cells in the 16-h short-term stationary phase. Nevertheless, our results revealed that cells in long-term stationary phase supernatant exhibit higher expressions of stress-response genes such as phage shock proteins (psp), and lower expressions of growth-related genes such as ribosomal proteins than those in the short-term stationary phase. We confirmed that the mutant lacking the psp operon showed lower survival and growth rate in the long-term stationary phase culture. This study identified transcriptional responses for stress-resistant physiology in the long-term stationary phase environment.

Stimulating Transcription in Antibiotic-Tolerant Escherichia coli Sensitizes It to Fluoroquinolone and Nonfluoroquinolone Topoisomerase Inhibitors.

Antibiotic tolerant bacteria and persistent cells that remain alive after a course of antibiotic treatment can foster the chronicity of infections and the development of antibiotic resistance. Elucidating how bacteria overcome antibiotic action and devising strategies to bolster a new drug's activity can allow us to preserve our antibiotic arsenal. Here, we investigate strategies to potentiate the activities of topoisomerase inhibitors against nongrowing Escherichia coli that are often recalcitrant to existing antibiotics. We focus on sensitizing bacteria to the fluoroquinolone (FQ) levofloxacin (Levo) and to the spiropyrimidinetrione zoliflodacin (Zoli)-the first antibiotic in its class of compounds in clinical development. We found that metabolic stimulation either alone or in combination with inhibiting the AcrAB-TolC efflux pump sensitized stationary-phase E. coli to Levo and Zoli. We demonstrate that the added metabolites increased proton motive force generation and ATP production in stationary-phase cultures without restarting growth. Instead, the stimulated bacteria increased transcription and translation, which rendered the populations more susceptible to topoisomerase inhibitors. Our findings illuminate potential vulnerabilities of antibiotic-tolerant bacteria that can be leveraged to sensitize them to new and existing classes of topoisomerase inhibitors. These approaches enable us to stay one step ahead of adaptive bacteria and safeguard the efficacy of our existing antibiotics.

Pseudomonas Phage ZCPS1 Endolysin as a Potential Therapeutic Agent.

The challenge of antibiotic resistance has gained much attention in recent years due to the rapid emergence of resistant bacteria infecting humans and risking industries. Thus, alternatives to antibiotics are being actively searched for. In this regard, bacteriophages and their enzymes, such as endolysins, are a very attractive alternative. Endolysins are the lytic enzymes, which are produced during the late phase of the lytic bacteriophage replication cycle to target the bacterial cell walls for progeny release. Here, we cloned, expressed, and purified LysZC1 endolysin from Pseudomonas phage ZCPS1. The structural alignment, molecular dynamic simulation, and CD studies suggested LysZC1 to be majorly helical, which is highly similar to various phage-encoded lysozymes with glycoside hydrolase activity. Our endpoint turbidity reduction assay displayed the lytic activity against various Gram-positive and Gram-negative pathogens. Although in synergism with EDTA, LysZC1 demonstrated significant activity against Gram-negative pathogens, it demonstrated the highest activity against Bacillus cereus. Moreover, LysZC1 was able to reduce the numbers of logarithmic-phase B. cereus by more than 2 log10 CFU/mL in 1 h and also acted on the stationary-phase culture. Remarkably, LysZC1 presented exceptional thermal stability, pH tolerance, and storage conditions, as it maintained the antibacterial activity against its host after nearly one year of storage at 4 °C and after being heated at temperatures as high as 100 °C for 10 min. Our data suggest that LysZC1 is a potential candidate as a therapeutic agent against bacterial infection and an antibacterial bio-control tool in food preservation technology.