Evaluating Metabolic Pathways and Biofilm Formation in Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia has recently arisen as a prominent nosocomial pathogen because of its high antimicrobial resistance and ability to cause chronic respiratory infections. Often the infections are worsened by biofilm formation which enhances antibiotic tolerance. We have previously found that mutation of the gpmA gene, encoding the glycolytic enzyme phosphoglycerate mutase, impacts the formation of this biofilm on biotic and abiotic surfaces at early time points. This finding, indicating an association between carbon source and biofilm formation, led us to hypothesize that metabolism would influence S. maltophilia biofilm formation and planktonic growth. In the present study, we tested the impact of various growth substrates on biofilm levels and growth kinetics to determine metabolic requirements for these processes. We found that S. maltophilia wild type preferred amino acids versus glucose for planktonic and biofilm growth and that gpmA deletion inhibited growth in amino acids. Furthermore, supplementation of the ΔgpmA strain by glucose or ribose phenotypically complemented growth defects. These results suggest that S. maltophilia shuttles amino acid carbon through gluconeogenesis to an undefined metabolic pathway supporting planktonic and biofilm growth. Further evaluation of these metabolic pathways might reveal novel metabolic activities of this pathogen. IMPORTANCE Stenotrophomonas maltophilia is a prominent opportunistic pathogen that often forms biofilms during infection. However, the molecular mechanisms of virulence and biofilm formation are poorly understood. The glycolytic enzyme phosphoglycerate mutase appears to play a role in biofilm formation, and we used a mutant in its gene (gpmA) to probe the metabolic circuitry potentially involved in biofilm development. The results of our study indicate that S. maltophilia displays unique metabolic activities, which could be exploited for inhibiting growth and biofilm formation of this pathogen.

Hydrodynamic Hunters.

The Gram-negative Bdellovibrio bacteriovorus (BV) is a model bacterial predator that hunts other bacteria and may serve as a living antibiotic. Despite over 50 years since its discovery, it is suggested that BV probably collides into its prey at random. It remains unclear to what degree, if any, BV uses chemical cues to target its prey. The targeted search problem by the predator for its prey in three dimensions is a difficult problem: it requires the predator to sensitively detect prey and forecast its mobile prey's future position on the basis of previously detected signal. Here instead we find that rather than chemically detecting prey, hydrodynamics forces BV into regions high in prey density, thereby improving its odds of a chance collision with prey and ultimately reducing BV's search space for prey. We do so by showing that BV's dynamics are strongly influenced by self-generated hydrodynamic flow fields forcing BV onto surfaces and, for large enough defects on surfaces, forcing BV in orbital motion around these defects. Key experimental controls and calculations recapitulate the hydrodynamic origin of these behaviors. While BV's prey (Escherichia coli) are too small to trap BV in hydrodynamic orbit, the prey are also susceptible to their own hydrodynamic fields, substantially confining them to surfaces and defects where mobile predator and prey density is now dramatically enhanced. Colocalization, driven by hydrodynamics, ultimately reduces BV's search space for prey from three to two dimensions (on surfaces) even down to a single dimension (around defects). We conclude that BV's search for individual prey remains random, as suggested in the literature, but confined, however-by generic hydrodynamic forces-to reduced dimensionality.

Pseudomonas aeruginosa Magnesium Transporter MgtE Inhibits Type III Secretion System Gene Expression by Stimulating rsmYZ Transcription.

Pseudomonas aeruginosa causes numerous acute and chronic opportunistic infections in humans. One of its most formidable weapons is a type III secretion system (T3SS), which injects powerful toxins directly into host cells. The toxins lead to cell dysfunction and, ultimately, cell death. Identification of regulatory pathways that control T3SS gene expression may lead to the discovery of novel therapeutics to treat P. aeruginosa infections. In a previous study, we found that expression of the magnesium transporter gene mgtE inhibits T3SS gene transcription. MgtE-dependent inhibition appeared to interfere with the synthesis or function of the master T3SS transcriptional activator ExsA, although the exact mechanism was unclear. We now demonstrate that mgtE expression acts through the GacAS two-component system to activate rsmY and rsmZ transcription. This event ultimately leads to inhibition of exsA translation. This inhibitory effect is specific to exsA as translation of other genes in the exsCEBA operon is not inhibited by mgtE Moreover, our data reveal that MgtE acts solely through this pathway to regulate T3SS gene transcription. Our study reveals an important mechanism that may allow P. aeruginosa to fine-tune T3SS activity in response to certain environmental stimuli.IMPORTANCE The type III secretion system (T3SS) is a critical virulence factor utilized by numerous Gram-negative bacteria, including Pseudomonas aeruginosa, to intoxicate and kill host cells. Elucidating T3SS regulatory mechanisms may uncover targets for novel anti-P. aeruginosa therapeutics and provide deeper understanding of bacterial pathogenesis. We previously found that the magnesium transporter MgtE inhibits T3SS gene transcription in P. aeruginosa In this study, we describe the mechanism of MgtE-dependent inhibition of the T3SS. Our report also illustrates how MgtE might respond to environmental cues, such as magnesium levels, to fine-tune T3SS gene expression.

Statistical signatures of a targeted search by bacteria.

Chemoattractant gradients are rarely well-controlled in nature and recent attention has turned to bacterial chemotaxis toward typical bacterial food sources such as food patches or even bacterial prey. In environments with localized food sources reminiscent of a bacterium's natural habitat, striking phenomena-such as the volcano effect or banding-have been predicted or expected to emerge from chemotactic models. However, in practice, from limited bacterial trajectory data it is difficult to distinguish targeted searches from an untargeted search strategy for food sources. Here we use a theoretical model to identify statistical signatures of a targeted search toward point food sources, such as prey. Our model is constructed on the basis that bacteria use temporal comparisons to bias their random walk, exhibit finite memory and are subject to random (Brownian) motion as well as signaling noise. The advantage with using a stochastic model-based approach is that a stochastic model may be parametrized from individual stochastic bacterial trajectories but may then be used to generate a very large number of simulated trajectories to explore average behaviors obtained from stochastic search strategies. For example, our model predicts that a bacterium's diffusion coefficient increases as it approaches the point source and that, in the presence of multiple sources, bacteria may take substantially longer to locate their first source giving the impression of an untargeted search strategy.

GroEL/ES inhibitors as potential antibiotics.

We recently reported results from a high-throughput screening effort that identified 235 inhibitors of the Escherichia coli GroEL/ES chaperonin system [Bioorg. Med. Chem. Lett.2014, 24, 786]. As the GroEL/ES chaperonin system is essential for growth under all conditions, we reasoned that targeting GroEL/ES with small molecule inhibitors could be a viable antibacterial strategy. Extending from our initial screen, we report here the antibacterial activities of 22 GroEL/ES inhibitors against a panel of Gram-positive and Gram-negative bacteria, including E. coli, Bacillus subtilis, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae. GroEL/ES inhibitors were more effective at blocking the proliferation of Gram-positive bacteria, in particular S. aureus, where lead compounds exhibited antibiotic effects from the low-µM to mid-nM range. While several compounds inhibited the human HSP60/10 refolding cycle, some were able to selectively target the bacterial GroEL/ES system. Despite inhibiting HSP60/10, many compounds exhibited low to no cytotoxicity against human liver and kidney cell lines. Two lead candidates emerged from the panel, compounds 8 and 18, that exhibit >50-fold selectivity for inhibiting S. aureus growth compared to liver or kidney cell cytotoxicity. Compounds 8 and 18 inhibited drug-sensitive and methicillin-resistant S. aureus strains with potencies comparable to vancomycin, daptomycin, and streptomycin, and are promising candidates to explore for validating the GroEL/ES chaperonin system as a viable antibiotic target.

Expression of the lipopolysaccharide biosynthesis gene lpxD affects biofilm formation of Pseudomonas aeruginosa.

Bacterial biofilms are an important cause of nosocomial infections. Microorganisms such as Pseudomonas aeruginosa colonize biotic and abiotic surfaces leading to chronic infections that are difficult to eradicate. To characterize novel genes involved in biofilm formation, we identified the lpxD gene from a transposon-mutant library of P. aeruginosa. This gene encodes a glucosamine-N acyltransferase, which is important for lipopolysaccharide biosynthesis. Our results showed that a loss-of-expression mutant of lpxD was defective for biofilm formation on biotic and abiotic surfaces. Additionally, this mutant strain exhibited significantly decreased bacterial attachment to cultured airway epithelial cells, as well as increased bacterial cytotoxicity toward airway cells. However, consistent with a defect in lipid A structure, airway cells incubated with the lpxD mutant or with mutant lipid A extracts exhibited decreased IL-8 production and necrosis, respectively. Overall, our data indicate that manipulating lpxD expression may influence P. aeruginosa's ability to establish biofilm infections.

Involvement of stress-related genes polB and PA14_46880 in biofilm formation of Pseudomonas aeruginosa.

Chronic infections of Pseudomonas aeruginosa are generally established through production of biofilm. During biofilm formation, production of an extracellular matrix and establishment of a distinct bacterial phenotype make these infections difficult to eradicate. However, biofilm studies have been hampered by the fact that most assays utilize nonliving surfaces as biofilm attachment substrates. In an attempt to better understand the mechanisms behind P. aeruginosa biofilm formation, we performed a genetic screen to identify novel factors involved in biofilm formation on biotic and abiotic surfaces. We found that deletion of genes polB and PA14_46880 reduced biofilm formation significantly compared to that in the wild-type strain PA14 in an abiotic biofilm system. In a biotic biofilm model, wherein biofilms form on cultured airway cells, the ΔpolB and ΔPA14_46880 strains showed increased cytotoxic killing of the airway cells independent of the total number of bacteria bound. Notably, deletion mutant strains were more resistant to ciprofloxacin treatment. This phenotype was linked to decreased expression of algR, an alginate transcriptional regulatory gene, under ciprofloxacin pressure. Moreover, we found that pyocyanin production was increased in planktonic cells of mutant strains. These results indicate that inactivation of polB and PA14_46880 may inhibit transition of P. aeruginosa from a more acute infection lifestyle to the biofilm phenotype. Future investigation of these genes may lead to a better understanding of P. aeruginosa biofilm formation and chronic biofilm infections.

Antibiotic treatment of Pseudomonas aeruginosa biofilms stimulates expression of the magnesium transporter gene mgtE.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen with the capacity to cause serious disease, including chronic biofilm infections in the lungs of cystic fibrosis (CF) patients. These infections are treated with high concentrations of antibiotics. Virulence modulation is an important tool utilized by P. aeruginosa to propagate infection and biofilm formation in the CF airway. Many different virulence modulatory pathways and proteins have been identified, including the magnesium transporter protein MgtE. We have recently found that isogenic deletion of mgtE leads to increased cytotoxicity through effects on the type III secretion system. To explore the role of the CF lung environment in MgtE activity, we investigated mgtE transcriptional regulation following antibiotic treatment. Utilizing quantitative real-time-PCR, we have demonstrated an increase in mgtE transcript levels following antibiotic treatment with most of the 12 antibiotics tested. To begin to determine the regulatory network governing mgtE expression, we screened a transposon-mutant library of P. aeruginosa to look for mutants with potentially altered mgtE activity, using cytotoxicity as a readout. In this screen, we observed that AlgR, which regulates production of the biofilm polysaccharide alginate, alters MgtE-mediated cytotoxicity. This cross-talk between MgtE and AlgR suggests that AlgR is involved in linking external inducing signals (e.g. antibiotics) to mgtE transcription and downstream virulence and biofilm activities. Analysing such interactions may lead to a better understanding of how the CF lung environment shapes P. aeruginosa biofilm infections.

MgtE is a dual-function protein in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa causes a wide range of infections, including chronic biofilm infections in the lungs of individuals with cystic fibrosis. We previously found that the inner-membrane protein MgtE can function both as a magnesium transporter and a virulence modulator, although the exact mechanism governing these activities is unclear. To address this issue, we carried out an experimental characterization of P. aeruginosa MgtE and generated a computer-rendered model. Our in silico analysis demonstrated the structural similarity of P. aeruginosa MgtE to that of the crystal structure of MgtE in Thermus thermophilus. Experimentally, we verified that MgtE is not essential for growth and found that it may not be involved directly in biofilm formation, even under low-magnesium conditions. We demonstrated both magnesium transport and cytotoxicity-regulating functions, and showed that magnesium-binding sites in the connecting helix region of MgtE are vital in coupling these two functions. Furthermore, limiting magnesium environments stimulated mgtE transcriptional responses. Our results suggested that MgtE might play an important role in linking magnesium availability to P. aeruginosa pathogenesis.

Thermoplastic polyurethane surface coated with polymer brushes for reduced protein and cell attachment.

The objective of this study was to coat negatively charged polymer brushes covalently onto the surface of thermoplastic polyurethane (TPU) using a simple conventional surface free-radical polymerization technique. The coated surfaces were assessed with contact angle, protein adsorption, cell adhesion and bacterial adhesion. Bovine serum albumin (BSA) and bovine fibrinogen (BFG) were used for protein adsorption evaluation. Mouse fibroblasts (NIH-3T3) and Pseudomonas aeruginosa (P. aeruginosa) were used to assess surface adhesion. Results show that the TPU surface modified with the attached polymer brushes exhibited significantly reduced contact angle, protein adsorption, and cell as well as bacterial adhesion, among which the negatively charged polymers showed the extremely low values in all the tests. Its contact angle is 5°, as compared to 70° for original TPU. Its BSA, BFG, 3T3 adhesion and P. aeruginosa adhesion were 93%, 84%, 92%, and 93% lower than original TPU. Furthermore, the TPU surface coated with negatively charged polymer brushes exhibited a hydrogel-like property. The results indicate that placing acrylic acids using a simple surface-initiated free-radical polymerization onto a TPU surface and then converting those to negative charges can be an effective and efficient route for fouling resistant applications.

Effect of simethicone on the bactericidal efficacy of a high-level disinfectant.

Introduction. Simethicone is an over-the-counter product that is frequently used by clinicians during endoscopic procedures to reduce foaming and improve visualization. Published studies have found simethicone residue on endoscopes after cleaning and disinfecting the devices as per the manufacturer's instructions. Some literature suggests that simethicone residue may reduce disinfection efficacy and increase the risk of patient infections.Gap Statement. However, there appears to be a lack of direct evidence in the literature to either disprove this or correlate simethicone presence with an increased microbial risk.Aim: Research was conducted to evaluate the in vitro impact of simethicone on disinfection efficacy.Methodology. Bacteria were grown in a microtitre plate assay in the presence of a range of simethicone concentrations and then treated with a disinfectant. Bacterial growth was assessed by spotting each microtitre well onto an agar plate.Results. The results demonstrated that, under the conditions tested, simethicone did not reduce the efficacy of Cidex ortho-phthalaldehyde disinfectant, which demonstrated at least a 6-log unit reduction in bacterial viability. Additional experiments showed that direct exposure to 66 mg ml-1 of simethicone reduced bacterial viability.Conclusion. These results indicate that simethicone may not reduce the bactericidal efficacy of disinfectant during reprocessing, under certain conditions.

A guide to Stenotrophomonas maltophilia virulence capabilities, as we currently understand them.

The Gram-negative pathogen Stenotrophomonas maltophilia causes a wide range of human infections. It causes particularly serious lung infections in individuals with cystic fibrosis, leading to high mortality rates. This pathogen is resistant to most known antibiotics and harbors a plethora of virulence factors, including lytic enzymes and serine proteases, that cause acute infection in host organisms. S. maltophilia also establishes chronic infections through biofilm formation. The biofilm environment protects the bacteria from external threats and harsh conditions and is therefore vital for the long-term pathogenesis of the microbe. While studies have identified several genes that mediate S. maltophilia's initial colonization and biofilm formation, the cascade of events initiated by these factors is poorly understood. Consequently, understanding these and other virulence factors can yield exciting new targets for novel therapeutics.

Impact of a Wellness Leadership Intervention on the Empathy, Burnout, and Resting Heart Rate of Medical Faculty.

Objective: To evaluate the efficacy of a wellness leadership intervention for improving the empathy, burnout, and physiological stress of medical faculty leaders. Participants and Methods: Participants were 49 medical faculty leaders (80% physicians, 20% basic scientists; 67% female). The 6-week course was evaluated with a 15-week longitudinal waitlist-control quasi-experiment from September 1, 2021, through December 20, 2021 (during the COVID-19 pandemic). We analyzed 3 pretest-posttest-posttest and 6 weekly survey measurements of affective empathy and burnout, and mean=85 (SD=31) aggregated daily resting heart rates per participant, using 2-level hierarchical linear modeling. Results: The course found a preventive effect for leaders' burnout escalation. As the control group awaited the course, their empathy decreased (coefficientTime=-1.27; P=.02) and their resting heart rates increased an average of 1.4 beats/min (coefficientTime=0.18; P<.001), reflecting the toll of the pandemic. Intervention group leaders reported no empathy decrements (coefficientTime=.33; P=.59) or escalated resting heart rate (coefficientTime=-0.05; P=.27) during the same period. Dose-response analysis revealed that both groups reduced their self-rated burnout over the 6 weeks of the course (coefficientTime=-0.28; P=.007), and those who attended more of the course showed less heart rate increase (coefficientTime∗Dosage=-0.05; P<.001). In addition, 12.73% of the within-person fluctuation in empathy was associated with burnout and resting heart rate. Conclusion: A wellness leadership intervention helped prevent burnout escalation and empathy decrement in medical faculty leaders during the COVID-19 pandemic, showing potential to improve the supportiveness and psychological safety of the medical training environment.

DNA alternate polymerase PolB mediates inhibition of type III secretion in Pseudomonas aeruginosa.

Opportunistic pathogen Pseudomonas aeruginosa uses a variety of virulence factors to cause acute and chronic infections. We previously found that alternate DNA polymerase gene polB inhibits P. aeruginosa pyocyanin production. We investigated whether polB also affects T3SS expression. polB overexpression significantly reduced T3SS transcription and repressed translation of the master T3SS regulator ExsA, while not affecting exsA mRNA transcript abundance. Further, polB does not act through previously described genetic pathways that post-transcriptionally regulate ExsA. Our results show a novel T3SS regulatory component which may lead to development of future drugs to target this mechanism.

The Pseudomonas aeruginosa magnesium transporter MgtE inhibits transcription of the type III secretion system.

Pseudomonas aeruginosa is an opportunistic pathogen that causes life-long pneumonia in individuals with cystic fibrosis (CF). These long-term infections are maintained by bacterial biofilm formation in the CF lung. We have recently developed a model of P. aeruginosa biofilm formation on cultured CF airway epithelial cells. Using this model, we discovered that mutation of a putative magnesium transporter gene, called mgtE, led to increased cytotoxicity of P. aeruginosa toward epithelial cells. This altered toxicity appeared to be dependent upon expression of the type III secretion system (T3SS). In this study, we found that mutation of mgtE results in increased T3SS gene transcription. Through epistasis analyses, we discovered that MgtE influences the ExsE-ExsC-ExsD-ExsA gene regulatory system of T3SS by either directly or indirectly inhibiting ExsA activity. While variations in calcium levels modulate T3SS gene expression in P. aeruginosa, we found that addition of exogenous magnesium did not inhibit T3SS activity. Furthermore, mgtE variants that were defective for magnesium transport could still complement the cytotoxicity effect. Thus, the magnesium transport function of MgtE does not fully explain the regulatory effects of MgtE on cytotoxicity. Overall, our results indicate that MgtE modulates expression of T3SS genes.

Polysaccharide capsule and sialic acid-mediated regulation promote biofilm-like intracellular bacterial communities during cystitis.

Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs). A murine UTI model has revealed an infection cascade whereby UPEC undergoes cycles of invasion of the bladder epithelium, intracellular proliferation in polysaccharide-containing biofilm-like masses called intracellular bacterial communities (IBC), and then dispersal into the bladder lumen to initiate further rounds of epithelial colonization and invasion. We predicted that the UPEC K1 polysaccharide capsule is a key constituent of the IBC matrix. Compared to prototypic E. coli K1 strain UTI89, a capsule assembly mutant had a fitness defect in functionally TLR4(+) and TLR4(-) mice, suggesting a protective role of capsule in inflamed and noninflamed hosts. K1 capsule assembly and synthesis mutants had dramatically reduced IBC formation, demonstrating the common requirement for K1 polysaccharide in IBC development. The capsule assembly mutant appeared dispersed in the cytoplasm of the bladder epithelial cells and failed to undergo high-density intracellular replication during later stages of infection, when the wild-type strain continued to form serial generations of IBC. Deletion of the sialic acid regulator gene nanR partially restored IBC formation in the capsule assembly mutant. These data suggest that capsule is necessary for efficient IBC formation and that aberrant sialic acid accumulation, resulting from disruption of K1 capsule assembly, produces a NanR-mediated defect in intracellular proliferation and IBC development. Together, these data demonstrate the complex but important roles of UPEC polysaccharide encapsulation and sialic acid signaling in multiple stages of UTI pathogenesis.

Phosphoglycerate mutase affects Stenotrophomonas maltophilia attachment to biotic and abiotic surfaces.

Stenotrophomonas maltophilia biofilm formation is of increasing medical concern, particularly for lung infections. However, the molecular mechanisms facilitating the biofilm lifestyle in S. maltophilia are poorly understood. We generated and screened a transposon mutant library for mutations that lead to altered biofilm formation compared to wild type. One of these mutations, in the gene for glycolytic enzyme phosphoglycerate mutase (gpmA), resulted in impaired attachment on abiotic and biotic surfaces. As adherence to a surface is the initial step in biofilm developmental processes, our results reveal a unique factor that could affect S. maltophilia biofilm initiation and, possibly, subsequent development.

An antibacterial dental light-cured glass-ionomer cement with improved hardness.

An antibacterial dental light-cured glass-ionomer cement has been developed and evaluated. An antibacterial furanone derivative was synthesized and covalently attached onto the surface of alumina filler particles. The formed antibacterial fillers were then mixed into a light-curable glass-ionomer cement formulation. Surface hardness and bacterial viability were used to evaluate the modified cements. Effects of coated furanone moiety content on the modified fillers, modified alumina filler particle size and loading, and total glass filler content were investigated. Results showed that increasing antibacterial furanone content, modified particle size and loading, and total glass filler content generally increased surface hardness. Increasing furanone moiety, filler loading and total filler content increased antibacterial activity. On the other hand, increasing particle size decreased antibacterial activity. The leaching tests indicate that the modified experimental cement showed no leachable antibacterial component to bacteria and cells.

A self-cured glass-ionomer cement with improved antibacterial function and hardness.

A novel antimicrobial dental self-cured glass-ionomer cement has been developed and evaluated. Alumina filler particles were covalently coated with an antibacterial polymer and blended into a self-cured glass-ionomer cement formulation. Surface hardness and bacterial viability were used to evaluate the modified cements. Results showed that the modified cements exhibited a significantly enhanced antibacterial activity along with improved surface hardness. Effects of antibacterial moiety content, alumina particle size and loading, and total filler content were investigated. It was found that increasing antibacterial moiety content, particle size and loading, and total filler content generally increased surface hardness. Increasing antibacterial moiety, filler loading and total filler content increased antibacterial activity. On the other hand, increasing particle size showed a negative impact on antibacterial activity. The leaching tests indicate no cytotoxicity produced from the modified cements to both bacteria and 3T3 mouse fibroblast cells.

An improved dental composite with potent antibacterial function.

A new BisGMA-based antibacterial dental composite has been formulated and evaluated. Compressive strength and bacterial viability were utilized to evaluate the formed composites. It was found that the new composite exhibited a significantly enhanced antibacterial function along with improved mechanical and physical properties. The bromine-containing derivative-modified composite was more potent in antibacterial activity than the chlorine-containing composite. The modified composites also exhibited an increase of 30-53% in compressive yield strength, 15-30% in compressive modulus, 15-33% in diametral tensile strength and 6-20% in flexural strength, and a decrease of 57-76% in bacterial viability, 23-37% in water sorption, 8-15% in shrinkage, 8-13% in compressive strength, and similar degree of conversion, than unmodified composite. It appears that this experimental composite may possibly be introduced to dental clinics as an attractive dental restorative due to its improved properties as well as enhanced antibacterial function.