Bacterial Persistence Research Articles

Bacterial adhesion inhibition by microalgal EPSs from Cylindrotheca closterium and Tetraselmis suecica biofilms.

In the food industry, successful bacterial pathogen colonization and persistence begin with their adhesion to a surface, followed by the spatial development of mature biofilm of public health concerns. Compromising bacterial settlement with natural inhibitors is a promising alternative to conventional anti-fouling treatments typically based on chemical biocides that contribute to the growing burden of antimicrobial resistance. In this study, three extracellular polymeric substance (EPS) fractions extracted from microalgae biofilms of Cylindrotheca closterium (fraction C) and Tetraselmis suecica (fraction Ta rich in insoluble scale structure and fraction Tb rich in soluble EPS) were screened for their anti-adhesive properties, against eight human food-borne pathogens belonging to Escherichia coli, Staphylococcus aureus, Salmonella enterica subsp. enterica, and Listeria monocytogenes species. The results showed that the fraction Ta was the most effective inducing statistically significant reduction for three strains of E. coli, S. aureus, and L. monocytogenes. Overall, EPSs coating on polystyrene surfaces of the different fractions increased the hydrophilic character of the support. Differences in bacterial adhesion on the different coated surfaces could be explained by several dissimilarities in the structural and physicochemical EPS compositions, according to HPLC and ATR-FTIR analysis. Interestingly, while fractions Ta and Tb were extracted from the same microalgal culture, distinct adhesion patterns were observed, highlighting the importance of the extraction process. Overall, the findings showed that EPS extracted from microalgal photosynthetic biofilms can exhibit anti-adhesive effects against food-borne pathogens and could help develop sustainable and non-toxic anti-adhesive surfaces for the food industry. KEY POINTS: •EPSs from a biofilm-based culture of C. closterium/T. suecica were characterized. •Microalgal EPS extracted from T. suecica biofilms showed bacterial anti-adhesive effects. •The anti-adhesive effect is strain-specific and affects both Gram - and Gram + bacteria.

Vancomycin-Polyguanidino Dendrimer Conjugates Inhibit Growth of Antibiotic-Resistant Gram-Positive and Gram-Negative Bacteria and Eradicate Biofilm-Associated S.aureus.

The global challenge of antibiotic resistance necessitates the introduction of more effective antibiotics. Here we report a potentially general design strategy, exemplified with vancomycin, that improves and expands antibiotic performance. Vancomycin is one of the most important antibiotics in use today for the treatment of Gram-positive infections. However, it fails to eradicate difficult-to-treat biofilm populations. Vancomycin is also ineffective in killing Gram-negative bacteria due to its inability to breach the outer membrane. Inspired by our seminal studies on cell penetrating guanidinium-rich transporters (e.g., octaarginine), we recently introduced vancomycin conjugates that effectively eradicate Gram-positive biofilm bacteria, persister cells and vancomycin-resistant enterococci (with V-r8, vancomycin-octaarginine), and Gram-negative pathogens (with V-R, vancomycin-arginine). Having shown previously that the spatial array (linear versus dendrimeric) of multiple guanidinium groups affects cell permeation, we report here for the first time vancomycin conjugates with dendrimerically displayed guanidinium groups that exhibit superior efficacy and breadth, presenting the best activity of V-r8 and V-R in single broad-spectrum compounds active against ESKAPE pathogens. Mode-of-action studies reveal cell-surface activity and enhanced vancomycin-like killing. The vancomycin-polyguanidino dendrimer conjugates exhibit no acute mammalian cell toxicity or hemolytic activity. Our study introduces a new class of broad-spectrum vancomycin derivatives and a general strategy to improve or expand antibiotic performance through combined mode-of-action and function-oriented design studies.

Immune evasion by proteolytic shedding of natural killer group 2, member D ligands in Helicobacter pylori infection.

Helicobacter pylori (H. pylori) uses various strategies that attenuate mucosal immunity to ensure its persistence in the stomach. We recently found evidence that H. pylori might modulate the natural killer group 2, member 2 (NKG2D) system. The NKG2D receptor and its ligands are a major activation system of natural killer and cytotoxic T cells, which are important for mucosal immunity and tumor immunosurveillance. The NKG2D system allows recognition and elimination of infected and transformed cells, however viruses and cancers often subvert its activation. Here we aimed to identify a potential evasion of the NKG2D system in H. pylori infection. We analyzed expression of NKG2D system genes in gastric tissues of H. pylori gastritis and gastric cancer patients, and performed cell-culture based infection experiments using H. pylori isogenic mutants and epithelial and NK cell lines. In biopsies of H. pylori gastritis patients, NKG2D receptor expression was reduced while NKG2D ligands accumulated in the lamina propria, suggesting NKG2D evasion. In vitro, H. pylori induced the transcription and proteolytic shedding of NKG2D ligands in stomach epithelial cells, and these effects were associated with specific H. pylori virulence factors. The H. pylori-driven release of soluble NKG2D ligands reduced the immunogenic visibility of infected cells and attenuated the cytotoxic activity of effector immune cells, specifically the anti-tumor activity of NK cells. H. pylori manipulates the NKG2D system. This so far unrecognized strategy of immune evasion by H. pylori could potentially facilitate chronic bacterial persistence and might also promote stomach cancer development by allowing transformed cells to escape immune recognition and grow unimpeded to overt malignancy.

Antimicrobial activity of nature-inspired molecules against multidrug-resistant bacteria.

Multidrug-resistant bacterial infections present a serious challenge to global health. In addition to the spread of antibiotic resistance, some bacteria can form persister cells which are tolerant to most antibiotics and can lead to treatment failure or relapse. In the present work, we report the discovery of a new class of small molecules with potent antimicrobial activity against Gram-positive bacteria and moderate activity against Gram-negative drug-resistant bacterial pathogens. The lead compound SIMR 2404 had a minimal inhibitory concentration (MIC) of 2 μg/mL against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-intermediate Staphylococcus aureus (VISA). The MIC values against Gram-negative bacteria such as Escherichia coli and Actinobacteria baumannii were between 8-32 μg/mL. Time-kill experiments show that compound SIMR 2404 can rapidly kill tested bacteria. Compound SIMR 2404 was also found to rapidly kill MRSA persisters which display high levels of tolerance to conventional antibiotics. In antibiotic evolution experiments, MRSA quickly developed resistance to ciprofloxacin but failed to develop resistance to compound SIMR 2404 even after 24 serial passages. Compound SIMR 2404 was not toxic to normal human fibroblast at a concentration of 4 μg/mL which is twice the MIC concentration against MRSA. However, at a concentration of 8 μg/mL or higher, it showed cytotoxic activity indicating that it is not ideal as a candidate against Gram-negative bacteria. The acceptable toxicity profile and rapid antibacterial activity against MRSA highlight the potential of these molecules for further studies as anti-MRSA agents.

Trained immunity in recurrent Staphylococcus aureus infection promotes bacterial persistence.

Bacterial persister cells, a sub-population of dormant phenotypic variants highly tolerant to antibiotics, present a significant challenge for infection control. Investigating the mechanisms of antibiotic persistence is crucial for developing effective treatment strategies. Here, we found a significant association between tolerance frequency and previous infection history in bovine mastitis. Previous S. aureus infection led to S. aureus tolerance to killing by rifampicin in subsequent infection in vivo and in vitro. Actually, the activation of trained immunity contributed to rifampicin persistence of S. aureus in secondary infection, where it reduced the effectiveness of antibiotic treatment and increased disease severity. Mechanically, we found that S. aureus persistence was mediated by the accumulation of fumarate provoked by trained immunity. Combination therapy with metformin and rifampicin promoted eradication of persisters and improved the severity of recurrent S. aureus infection. These findings provide mechanistic insight into the relationship between trained immunity and S. aureus persistence, while providing proof of concept that trained immunity is a therapeutic target in recurrent bacterial infections involving persistent pathogens.

Goethite adaptation prompts alterations in antibiotic susceptibility and suppresses development of antibiotic resistance in bacteria

Understanding the impact of environmental factors on antibiotic sensitivity and the emergence of antibiotic resistance in microorganism is crucial for antibiotics management and environmental risk assessment. Natural materials, like mineral particles, are prevalent in aquatic and terrestrial ecosystems. However, it remains unclear how microorganism adapt to the physical stress of mineral particles and whether this adaptation influences antibiotic sensitivity and the evolution of antibiotic resistance. In this study, the model bacterium Escherichia coli (E. coli) was exposed to the mineral particle goethite for 30 generations. Adaptive morphogenesis, including an increase in the fraction of spherical bacteria, variations in bacterial mobility, a slightly increased cell membrane thickness, and genome-wide changes in the transcriptomic profile, were observed in adapted E. coli samples to counteract the stress. Moreover, the goethite adapted E. coli showed increased susceptibility to antibiotics including amoxicillin and tetracycline, and decreased susceptibility to kanamycin compared to its ancestral counterparts. These alterations in antibiotic susceptibility in the adapted E. coli were not heritable, as evidenced by the gradual recovery of antibiotic tolerance in cells with the cessation of goethite exposure. Transcriptomic data and a series of experiments suggested that these changes may be associated with variations in cell membrane property and iron metabolism. In addition, the evolution of antibiotic resistance in adapted cells occurred at a slower rate compared to their ancestral counterparts. For instance, E. coli adapted to goethite at a concentration of 1 mg/mL did not acquire antibiotic resistance even after 13 generations, probably due to its poor biofilm-formation capacity. Our findings underscore the occurrence of microbial adaptation to goethite, which influenced antibiotic sensitivity and decelerated the development of resistance in microorganisms. This insight contributes to our comprehension of the natural dynamics surrounding the evolution of antibiotic resistance and opens new perspectives for addressing this issue through nanotechnology-based approaches.

Development of a Standardised International Protocol for Evaluation of the Disinfection Efficacy of Healthcare Laundry Wash Processes

This research aims to develop a standardised protocol for monitoring the disinfection efficacy of healthcare laundry processes in view of numerous differential methodologies currently being employed within the healthcare laundry sector, including agitation and surface sampling for post-laundering decontamination assessment and swatch and bioindicator testing for in-wash-process efficacy. Enterococcus faecium as an indicator species within industrial wash systems is preferable due to its high thermal and disinfectant tolerance. Methods for measuring laundry disinfection were compared; commercially available E. faecium bioindicators and contaminated cotton swatches (loose, in cloth bags or within nylon membranes) were laundered industrially at ambient temperature and microbial recovery determined. E. faecium was lost from cotton during laundering but retained by the bioindicator membrane, which allows disinfection efficacy to be measured without loss of microorganisms from the test swatch. Commercially available bioindicators were only permeable to disinfectants and detergents at ≥60 °C. Subsequently, polyethersulphone membranes for enclosing contaminated swatches were developed for low-temperature laundering, with permeability to industrial laundry chemistries at below ≤60 °C. This study demonstrates that bioindicators are the recommended methodology for laundry disinfection validation. The use of a universal healthcare laundry disinfection methodology will lead to standardised microbiological testing across the industry and improvements in infection control.

Bacterial Persistence in Urinary Tract Infection Among Postmenopausal Population.

Urinary tract infections (UTIs) are common in older-aged women. Our study examined bacterial persistence with commonly prescribed antibiotics. Bacterial growth was demonstrated despite antibiotic treatment. The aims of this study were to quantify the bacterial persister phenotype in urine collected from postmenopausal women with acute and recurrent UTI and to determine the capabilities of first-line antibiotics to effectively treat persister cells. This was an institutional review board-approved cross-sectional analysis within a large academic referral center. Uropathogens were cultured from postmenopausal women with acute or recurrent UTI and screened for persister cells using persistence assays. Demographic and clinical variables were collected and analyzed. The entire experimental process was repeated in triplicate. Data were analyzed for significance (P < 0.05) between the persister culture and antibiotic treatments using a 1-way analysis of variance with multiple comparisons in Prism 9.3.0. Forty participants were included: 62.5% White, 22.5% Black, 3% Asian, and 2% Hispanic with a mean age of 72.3 ± 11.62 years. The persister phenotype was demonstrated in all of Escherichia coli isolates. Treatment with fosfomycin demonstrated reduced colony-forming units per milliliter compared with control (P < 0.01). Among recurrent isolates, there was a statistically significant decrease in colony-forming units per milliliter after antibiotic treatment with all 4 antibiotics (P < 0.05). This study demonstrated in vitro bacterial persistence in uropathogens from urogynecology patients despite treatment with commonly prescribed antibiotics. Fosfomycin generated the least amount of persister cells. Results suggest that persistence may be one bacterial defense mechanism involved in UTIs. Further research is needed to understand the clinical implications.

Brain glucose induces tolerance of Cryptococcus neoformans to amphotericin B during meningitis.

Antibiotic tolerance is the ability of a susceptible population to survive high doses of cidal drugs and has been shown to compromise therapeutic outcomes in bacterial infections. In comparison, whether fungicide tolerance can be induced by host-derived factors during fungal diseases remains largely unknown. Here, through a systematic evaluation of metabolite-drug-fungal interactions in the leading fungal meningitis pathogen, Cryptococcus neoformans, we found that brain glucose induces fungal tolerance to amphotericin B (AmB) in mouse brain tissue and patient cerebrospinal fluid via the fungal glucose repression activator Mig1. Mig1-mediated tolerance limits treatment efficacy for cryptococcal meningitis in mice via inhibiting the synthesis of ergosterol, the target of AmB, and promoting the production of inositolphosphorylceramide, which competes with AmB for ergosterol. Furthermore, AmB combined with an inhibitor of fungal-specific inositolphosphorylceramide synthase, aureobasidin A, shows better efficacy against cryptococcal meningitis in mice than do clinically recommended therapies.

Exploiting phage-antibiotic synergies to disrupt Pseudomonas aeruginosa PAO1 biofilms in the context of orthopedic infections.

Biofilm-related infections are among the most difficult-to-treat infections in all fields of medicine due to their antibiotic tolerance and persistent character. In the field of orthopedics, these biofilms often lead to therapeutic failure of medical implantable devices and urgently need novel treatment strategies. This forthcoming article aims to explore the dynamic interplay between newly isolated bacteriophages and routinely used antibiotics and clearly indicates synergetic patterns when used as a dual treatment modality. Biofilms were drastically more reduced when both active agents were combined, thereby providing additional evidence that phage-antibiotic combinations lead to synergism and could potentially improve clinical outcome for affected patients.

Fungicide-tolerant persister formation during cryptococcal pulmonary infection

Bacterial persisters, a subpopulation of genetically susceptible cells that are normally dormant and tolerant to bactericides, have been studied extensively because of their clinical importance. In comparison, much less is known about the determinants underlying fungicide-tolerant fungal persister formation invivo. Here, we report that during mouse lung infection, Cryptococcus neoformans forms persisters that are highly tolerant to amphotericin B (AmB), the standard of care for treating cryptococcosis. By exploring stationary-phase indicator molecules and developing single-cell tracking strategies, we show that in the lung, AmB persisters are enriched in cryptococcal cells that abundantly produce stationary-phase molecules. The antioxidant ergothioneine plays a specific and key role in AmB persistence, which is conserved in phylogenetically distant fungi. Furthermore, the antidepressant sertraline (SRT) shows potent activity specifically against cryptococcal AmB persisters. Our results provide evidence for and the determinant of AmB-tolerant persister formation in pulmonary cryptococcosis, which has potential clinical significance.

Streptococcus pneumoniae favors tolerance via metabolic adaptation over resistance to circumvent fluoroquinolones.

Streptococcus pneumoniae is a major human pathogen of global health concern and the rapid emergence of antibiotic resistance poses a serious public health problem worldwide. Fluoroquinolone resistance in S. pneumoniae is an intriguing case because the prevalence of fluoroquinolone resistance does not correlate with increasing usage and has remained rare. Our data indicate that deleterious fitness costs in the mammalian host constrain the emergence of fluoroquinolone resistance both by de novo mutation and recombination. S. pneumoniae was able to circumvent such deleterious fitness costs via the development of antibiotic tolerance through metabolic adaptation that reduced the production of reactive oxygen species, resulting in a fitness benefit during infection of mice treated with fluoroquinolones. These data suggest that the emergence of fluoroquinolone resistance is tightly constrained in S. pneumoniae by fitness tradeoffs and that mutational pathways involving metabolic networks to enable tolerance phenotypes are an important contributor to the evasion of antibiotic-mediated killing.IMPORTANCEThe increasing prevalence of antibiotic resistant bacteria is a major global health concern. While many species have the potential to develop antibiotic resistance, understanding the barriers to resistance emergence in the clinic remains poorly understood. A prime example of this is fluroquinolone resistance in Streptococcus pneumoniae, whereby, despite continued utilization, resistance to this class of antibiotic remains rare. In this study, we found that the predominant pathways for developing resistance to this antibiotic class severely compromised the infectious capacity of the pneumococcus, providing a key impediment for the emergence of resistance. Using in vivo models of experimental evolution, we found that S. pneumoniae responds to repeated fluoroquinolone exposure by modulating key metabolic pathways involved in the generation of redox molecules, which leads to antibiotic treatment failure in the absence of appreciable shifts in resistance levels. These data underscore the complex pathways available to pathogens to evade antibiotic mediating killing via antibiotic tolerance.

Rhizobium determinants of rhizosphere persistence and root colonization.

Bacterial persistence in the rhizosphere and colonization of root niches are critical for the establishment of many beneficial plant-bacteria interactions including those between Rhizobium leguminosarum and its host legumes. Despite this, most studies on R. leguminosarum have focused on its symbiotic lifestyle as an endosymbiont in root nodules. Here, we use random barcode transposon sequencing to assay gene contributions of R. leguminosarum during competitive growth in the rhizosphere and colonization of various plant species. This facilitated the identification of 189 genes commonly required for growth in diverse plant rhizospheres, mutation of 111 of which also affected subsequent root colonization (rhizosphere progressive), and a further 119 genes necessary for colonization. Common determinants reveal a need to synthesize essential compounds (amino acids, ribonucleotides, and cofactors), adapt metabolic function, respond to external stimuli, and withstand various stresses (such as changes in osmolarity). Additionally, chemotaxis and flagella-mediated motility are prerequisites for root colonization. Many genes showed plant-specific dependencies highlighting significant adaptation to different plant species. This work provides a greater understanding of factors promoting rhizosphere fitness and root colonization in plant-beneficial bacteria, facilitating their exploitation for agricultural benefit.

In-patient evolution of a high-persister Escherichia coli strain with reduced in vivo antibiotic susceptibility.

Gram-negative bacterial bloodstream infections (GNB-BSI) are common and frequently lethal. Despite appropriate antibiotic treatment, relapse of GNB-BSI with the same bacterial strain is common and associated with poor clinical outcomes and high healthcare costs. The role of persister cells, which are sub-populations of bacteria that survive for prolonged periods in the presence of bactericidal antibiotics, in relapse of GNB-BSI is unclear. Using a cohort of patients with relapsed GNB-BSI, we aimed to determine how the pathogen evolves within the patient between the initial and subsequent episodes of GNB-BSI and how these changes impact persistence. Using Escherichia coli clinical bloodstream isolate pairs (initial and relapse isolates) from patients with relapsed GNB-BSI, we found that 4/11 (36%) of the relapse isolates displayed a significant increase in persisters cells relative to the initial bloodstream infection isolate. In the relapsed E. coli strain with the greatest increase in persisters (100-fold relative to initial isolate), we determined that the increase was due to a loss-of-function mutation in the ptsI gene encoding Enzyme I of the phosphoenolpyruvate phosphotransferase system. The ptsI mutant was equally virulent in a murine bacteremia infection model but exhibited 10-fold increased survival to antibiotic treatment. This work addresses the controversy regarding the clinical relevance of persister formation by providing compelling data that not only do high-persister mutations arise during bloodstream infection in humans but also that these mutants display increased survival to antibiotic challenge in vivo.

Protozoan predation enhances stress resistance and antibiotic tolerance in Burkholderia cenocepacia by triggering the SOS response.

Bacterivorous protists are thought to serve as training grounds for bacterial pathogens by subjecting them to the same hostile conditions that they will encounter in the human host. Bacteria that survive intracellular digestion exhibit enhanced virulence and stress resistance after successful passage through protozoa but the underlying mechanisms are unknown. Here we show that the opportunistic pathogen Burkholderia cenocepacia survives phagocytosis by ciliates found in domestic and hospital sink drains, and viable bacteria are expelled packaged in respirable membrane vesicles with enhanced resistance to oxidative stress, desiccation, and antibiotics, thereby contributing to pathogen dissemination in the environment. Reactive oxygen species generated within the protozoan phagosome promote the formation of persisters tolerant to ciprofloxacin by activating the bacterial SOS response. In addition, we show that genes encoding antioxidant enzymes are upregulated during passage through ciliates increasing bacterial resistance to oxidative radicals. We prove that suppression of the SOS response impairs bacterial intracellular survival and persister formation within protists. This study highlights the significance of protozoan food vacuoles as niches that foster bacterial adaptation in natural and built environments and suggests that persister switch within phagosomes may be a widespread phenomenon in bacteria surviving intracellular digestion.

Drug repurposing: insights into the antimicrobial effects of AKBA against MRSA

Staphylococcus aureus is a major threat in infectious diseases due to its varied infection types and increased resistance. S. aureus could form persister cells under certain condition and could also attach on medical apparatus to form biofilms, which exhibited extremely high resistance to antibiotics. 3-Acetyl-11-keto-beta-boswellic acid (AKBA) is a well-studied anti-tumor and antioxidant drug. This study is aimed to determine the antimicrobial effects of AKBA against S. aureus and its persister cells and biofilms. The in vitro antimicrobial susceptibility of AKBA was assessed by micro-dilution assay, disc diffusion assay and time-killing assay. Drug combination between AKBA and conventional antibiotics was detected by checkerboard assay. And the antibiofilm effects of AKBA against S. aureus were explored by crystal violet staining combined with SYTO/PI probes staining. Next, RBC lysis activity and CCK-8 kit were used to determine the cytotoxicity of AKBA. In addition, murine subcutaneous abscess model was used to assess the antimicrobial effects of AKBA in vivo. Our results revealed that AKBA was found to show effective antimicrobial activity against methicillin-resistant S. aureus (MRSA) with the minimal inhibitory concentration of 4–8 µg/mL with undetectable cytotoxicity. And no resistant mutation was induced by AKBA after 20 days of consecutive passage. Further, we found that AKBA could be synergy with gentamycin or amikacin against S. aureus and its clinical isolates. By crystal violet and SYTO9/PI staining, AKBA exhibited strong biofilm inhibitory and eradication effects at the concentration of 1 ~ 4 µg/mL. In addition, the effective antimicrobial effect was verified in vivo in a mouse model. And no detectable in vivo toxicity was found. These results indicated that AKBA has great potential to development as an alternative treatment for the refractory S. aureus infections.

Experimental measurement and computational prediction of bacterial Hanks-type Ser/Thr signaling system regulatory targets.

Bacteria possess diverse classes of signaling systems that they use to sense and respond to their environments and execute properly timed developmental transitions. One widespread and evolutionarily ancient class of signaling systems are the Hanks-type Ser/Thr kinases, also sometimes termed "eukaryotic-like" due to their homology with eukaryotic kinases. In diverse bacterial species, these signaling systems function as critical regulators of general cellular processes such as metabolism, growth and division, developmental transitions such as sporulation, biofilm formation, and virulence, as well as antibiotic tolerance. This multifaceted regulation is due to the ability of a single Hanks-type Ser/Thr kinase to post-translationally modify the activity of multiple proteins, resulting in the coordinated regulation of diverse cellular pathways. However, in part due to their deep integration with cellular physiology, to date, we have a relatively limited understanding of the timing, regulatory hierarchy, the complete list of targets of a given kinase, as well as the potential regulatory overlap between the often multiple kinases present in a single organism. In this review, we discuss experimental methods and curated datasets aimed at elucidating the targets of these signaling pathways and approaches for using these datasets to develop computational models for quantitative predictions of target motifs. We emphasize novel approaches and opportunities for collecting data suitable for the creation of new predictive computational models applicable to diverse species.

Proteomic characterization of persisters in Enterococcus faecium

BackgroundEnterococcus faecium is a Gram-positive bacterium, naturally present in the human intestinal microbiota, but is also an opportunistic pathogen responsible for healthcare-associated infections. Persisters are individuals of a subpopulation able to survive by arrest of growth coping with conditions that are lethal for the rest of the population. These persistent cells can grow again when the stress disappears from their environment and can cause relapses.ResultsIn this study, we highlighted that ciprofloxacin (10-fold the MIC) led to the formation of persister cells of E. faecium. The kill curve was typically biphasic with an initial drop of survival (more than 2 orders of magnitude reduction) followed by a constant bacterial count. Growth curves and antimicrobial susceptibility tests of these persisters were similar to those of the original cells. In addition, by genomic analyses, we confirmed that the persisters were genotypically identical to the wild type. Comparative proteomic analysis revealed that 56 proteins have significantly different abundances in persisters compared to cells harvested before the addition of stressing agent. Most of them were related to energetic metabolisms, some polypeptides were involved in transcription regulation, and seven were stress proteins like CspA, PrsA, ClpX and particularly enzymes linked to the oxidative stress response.ConclusionsThis work provided evidences that the pathogen E. faecium was able to enter a state of persister that may have an impact in chronic infections and relapses. Moreover, putative key effectors of this phenotypical behavior were identified by proteomic approach.

TooManyCellsInteractive: A visualization tool for dynamic exploration of single-cell data.

As single-cell sequencing technologies continue to advance, the growing volume and complexity of the ensuing data present new analytical challenges. Large cellular populations from single-cell atlases are more difficult to visualize and require extensive processing to identify biologically relevant subpopulations. Managing these workflows is also laborious for technical users and unintuitive for nontechnical users. We present TooManyCellsInteractive (TMCI), a browser-based JavaScript application for interactive exploration of cell populations. TMCI provides an intuitive interface to visualize and manipulate a radial tree representation of hierarchical cell subpopulations and allows users to easily overlay, filter, and compare biological features at multiple resolutions. Here we describe the software architecture and demonstrate how we used TMCI in a pan-cancer analysis to identify unique survival pathways among drug-tolerant persister cells. TMCI will facilitate exploration and visualization of large-scale sequencing data in a user-friendly way. TMCI is freely available at https://github.com/schwartzlab-methods/too-many-cells-interactive. An example tree from data within this article is available at https://tmci.schwartzlab.ca/.

The (p)ppGpp synthetase Rsh promotes rifampicin tolerant persister cell formation in Brucella abortus by regulating the type II toxin-antitoxin module mbcTA.

Persister cells are transiently tolerant to antibiotics and are associated with recalcitrant chronic infections due to recolonization of host cells after antibiotic removal. Brucella spp. are facultative pathogens that establish intracellular infection cycles in host cells which results in chronic persistent infections. Brucella abortus forms multi-drug persister cells which are promoted by the (p)ppGpp synthetase Rsh during rifampicin exposure. Here, we confirmed that Rsh promoted persister cells formation in B. abortus stationary phase treated with rifampicin and enrofloxacin. Deletion of the gene for Rsh decreased persister cells level in the presence of these drugs in different growth phases. However, persister cells formation by deletion strain varied in different growth phases in the presence of other antibiotics. Rsh also was involved in persister cells formation during rifampicin treatment under certain stress conditions, including acidic conditions, exposure to PBS, and heat stress. Moreover, Rsh impacted persister cell levels during rifampicin or enrofloxacin treatment in RAW264.7 macrophages. Certain typeIItoxin-antitoxin modules were upregulated under various stress conditions in B. abortus. We established that Rsh positively regulated the type II toxin-antitoxin mbcTA. Moreover, rifampicin-tolerant persister cells formation was elevated and ATP levels were decreased when mbcTA promoter was overexpressed in Rsh deletion background in stationary phase. Our results establish that (p)ppGpp synthetase Rsh plays a key role in B. abortus persistence and may serve as a potent novel target in combination with rifampicin in the development of new therapeutic approaches and prevention strategies to treat chronic infections of Brucella.