Static Growth Conditions Research Articles

The anti-biofilm efficacy of copper and zinc doped borate bioactive glasses.

Aim: Healthcare-acquired infections (HAIs) pose significant challenges in medical settings due to their resistance to conventional treatment methods. The role of bacterial biofilms in exacerbating these infections is well-documented, making HAIs particularly difficult to eradicate. Despite numerous research efforts, an effective solution to combat these infections remains elusive. This study aims to explore the potential of metal-ion (copper and zinc) doped borate bioactive glasses (BBGs) as a novel treatment modality to inhibit bacterial species commonly implicated in HAIs: Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa.Methods: The study analyzed the efficacy of both direct and indirect applications of BBGs on severe biofilms pre-formed under static and dynamic growth conditions; a comprehensive predictive modeling was developed, simulating diverse clinically relevant conditions.Results: Results demonstrate more than 4 log reduction in bacterial growth within 2days for direct application and 3days for indirect application of copper and zinc-doped BBGs. These findings were consistent across the three bacterial species, in both static and dynamic conditions.Conclusion: Copper and zinc-doped BBGs can be an effective approach in combating HAIs complicated by biofilms.

Synergy of lytic phage pB23 and meropenem combination against carbapenem-resistant Acinetobacter baumannii.

Phage-antibiotic combination treatment is a novel noteworthy drug delivery method in anti-infection. In the current study, we have isolated a new phage, pB23, against carbapenem-resistant Acinetobacter baumannii 2023. Synergistic antibacterial effect between phage pB23 and meropenem combination could be more stable, using moderate doses of phage (multiplicity of infection ranging from 0.1 to 1,000) based on results of in vitro antibacterial activity. Phage pB23 and meropenem combination could effectively clear mature biofilms and prevent biofilm formation of carbapenem-resistant Acinetobacter baumannii in vitro. Phage pB23 and meropenem combination also has good synergistic antibacterial effects against carbapenem-resistant Acinetobacter baumannii in different growth phases under static culture conditions. The pig skin explant model shows that phage pB23 and meropenem combination has a synergistic effect to remove bacteria from wounds ex vivo. Phage pB23 and meropenem combination also exhibited a synergistic antibacterial effect in vivo using a zebrafish infection mode. The potential promotion of phage proliferation by meropenem and the sensitivity recovery of phage-resistant bacteria to meropenem might elucidate the mechanism of the synergistic antimicrobial activity. In summary, our study illustrates that phage pB23 and meropenem combination could produce synergistic antibacterial effects against carbapenem-resistant Acinetobacter baumannii under static growth conditions. This study also demonstrates that phage-antibiotic combination will become an effective strategy to enhance antibacterial activity of individual drug and provide a new idea of the drug development for the treatment of infections due to carbapenem-resistant Acinetobacter baumannii and other multidrug-resistant bacteria.

Phage and Antibiotic Combinations Reduce Staphylococcus aureus in Static and Dynamic Biofilms Grown on an Implant Material.

Staphylococcus aureus causes the majority of implant-related infections. These infections present as biofilms, in which bacteria adhere to the surface of foreign materials and form robust communities that are resilient to the human immune system and antibiotic drugs. The heavy use of broad-spectrum antibiotics against these pathogens disturbs the host's microbiome and contributes to the growing problem of antibiotic-resistant infections. The use of bacteriophages as antibacterial agents is a potential alternative therapy. In this study, bioluminescent strains of S. aureus were grown to form 48-h biofilms on polyether ether ketone (PEEK), a material used to manufacture orthopaedic implants, in either static or dynamic growth conditions. Biofilms were treated with vancomycin, staphylococcal phage, or a combination of the two. We showed that vancomycin and staph phages were able to independently reduce the total bacterial load. Most phage-antibiotic combinations produced greater log reductions in surviving bacteria compared to single-agent treatments, suggesting antimicrobial synergism. In addition to demonstrating the efficacy of combining vancomycin and staph phage, our results demonstrate the importance of growth conditions in phage-antibiotic combination studies. Dynamic biofilms were found to have a substantial impact on apparent treatment efficacy, as they were more resilient to combination treatments than static biofilms.

Auxotrophic and prototrophic conditional genetic networks reveal the rewiring of transcription factors in Escherichia coli

Bacterial transcription factors (TFs) are widely studied in Escherichia coli. Yet it remains unclear how individual genes in the underlying pathways of TF machinery operate together during environmental challenge. Here, we address this by applying an unbiased, quantitative synthetic genetic interaction (GI) approach to measure pairwise GIs among all TF genes in E. coli under auxotrophic (rich medium) and prototrophic (minimal medium) static growth conditions. The resulting static and differential GI networks reveal condition-dependent GIs, widespread changes among TF genes in metabolism, and new roles for uncharacterized TFs (yjdC, yneJ, ydiP) as regulators of cell division, putrescine utilization pathway, and cold shock adaptation. Pan-bacterial conservation suggests TF genes with GIs are co-conserved in evolution. Together, our results illuminate the global organization of E. coli TFs, and remodeling of genetic backup systems for TFs under environmental change, which is essential for controlling the bacterial transcriptional regulatory circuits.

A Simple 96-Well Plate-Based Method for Development of Candida Biofilms Under Static Conditions.

We describe a rapid and simple in vitro method for development of Candida biofilms under static growth conditions. This 96-well microtiter-based method measures metabolic activity of sessile cells and can also be easily adapted for antifungal susceptibility testing. The entire procedure takes 2-3days to complete, reliably quantifies biofilms, and provides reproducible results that are imperative toward the standardization of antifungal susceptibility testing of biofilms.

Biomechanical Forces Regulate Gene Transcription During Stretch-Mediated Growth of Mammalian Neurons.

At birth, there are 100 billion neurons in the human brain, with functional neural circuits extending through the spine to the epidermis of the feet and toes. Following birth, limbs and vertebrae continue to grow by several orders of magnitude, forcing established axons to grow by up to 200 cm in length without motile growth cones. The leading regulatory paradigm suggests that biomechanical expansion of mitotic tissue exerts tensile force on integrated nervous tissue, which synchronizes ongoing growth of spanning axons. Here, we identify unique transcriptional changes in embryonic rat DRG and cortical neurons while the corresponding axons undergo physiological levels of controlled mechanical stretch in vitro. Using bioreactors containing cultured neurons, we recapitulated the peak biomechanical increase in embryonic rat crown-rump-length. Biologically paired sham and “stretch-grown” DRG neurons spanned 4.6- and 17.2-mm in length following static or stretch-induced growth conditions, respectively, which was associated with 456 significant changes in gene transcription identified by genome-wide cDNA microarrays. Eight significant genes found in DRG were cross-validated in stretch-grown cortical neurons by qRT-PCR, which included upregulation of Gpat3, Crem, Hmox1, Hpse, Mt1a, Nefm, Sprr1b, and downregulation of Nrep. The results herein establish a link between biomechanics and gene transcription in mammalian neurons, which elucidates the mechanism underlying long-term growth of axons, and provides a basis for new research in therapeutic axon regeneration.

The differences in the corrosion product compositions of Methanogen-induced microbiologically influenced corrosion (Mi-MIC) between static and dynamic growth conditions

Currently, corrosion rates (CR) and/or corrosion products (CP) obtained for methanogen-induced microbiologically influenced corrosion (Mi-MIC) on carbon steel are mainly analyzed from static-incubations. By using a multiport-flow-column, much higher CRs (0.72 mm/yr) were observed, indicating static-incubations are not suitable for determining the corrosive potential of Mi-MIC. With the combination of various analytical methods (ToF-SIMS/SEM-EDS/SEM-FIB) and contrary to previously published data, we observed that CPs contained phosphorus, oxygen, magnesium, calcium and iron but lacked carbon-related species (e.g. siderite). Overall, siderite nucleation is disrupted by methanogens, as they convert aqueous bicarbonate into carbon dioxide for methanogenesis resulting in increased localized corrosion.

Identification of a reliable fixative solution to preserve the complex architecture of bacterial biofilms for scanning electron microscopy evaluation.

Bacterial biofilms are organized sessile communities of bacteria enclosed in extracellular polymeric substances (EPS). To analyze organization of bacteria and EPS in high resolution and high magnification by scanning electron microscopy (SEM), it is important to preserve the complex architecture of biofilms. Therefore, fixation abilities of formalin, glutaraldehyde, and Methacarn (methanol/chloroform/acetic acid-6:3:1) fixatives were evaluated to identify which fixative would best preserve the complex structure of bacterial biofilms. Economically important Gram-negative Mannheimia haemolytica, the major pathogen associated with bovine respiratory disease complex, and Gram-positive Staphylococcus aureus, the major cause of chronic mastitis in cattle, bacteria were selected since both form biofilms on solid-liquid interface. For SEM analysis, round glass coverslips were placed into the wells of 24-well plates and diluted M. haemolytica or S. aureus cultures were added, and incubated at 37°C for 48-72 h under static growth conditions. Culture media were aspirated and biofilms were fixed with an individual fixative for 48 h. SEM examination revealed that all three fixatives were effective preserving the bacterial cell morphology, however only Methacarn fixative could consistently preserve the complex structure of biofilms. EPS layers were clearly visible on the top, in the middle, and in the bottom of the biofilms with Methacarn fixative. Biomass and three-dimensional structure of the biofilms were further confirmed spectrophotometrically following crystal violet staining and by confocal microscopy after viability staining. These findings demonstrate that Methacarn fixative solution is superior to the other fixatives evaluated to preserve the complex architecture of biofilms grown on glass coverslips for SEM evaluation.

Impedimetric detection of Pseudomonas aeruginosa attachment on flexible ITO-coated polyethylene terephthalate substrates

Biofilm monitoring in environmental and biomedical applications remains a challenge. Currently, conventional biochemical methods do not provide a quick quantitative measure of attached biomass. Thus, there is a need for rapid in situ detection tools for routine biofilm characterization. Electrochemical impedance spectroscopy (EIS) characterizes the electroactivity of bacteria within a biofilm and has been extensively used to monitor strong electroactive biofilms. Yet, studies on weak electricigens such as Pseudomonas aeruginosa remain underrepresented. Here, conductive indium tin oxide coated polyethylene terephthalate (ITO:PET) sheets were used as flexible growth substrates instead of more conventional carbonaceous or gold materials. EIS was compared with standard optical methods for the detection of P. aeruginosa biofilms formed on ITO:PET under static growth conditions. Relaxation time analysis showed a dominant time constant at approximately 1 s and confirmed the validity of a two time constant equivalent circuit model for the biofilm impedance. The interfacial resistance calculated from the equivalent circuit analysis showed a rapid drop after bacterial attachment whereas capacitance of the biofilm was masked by the capacitance of ITO:PET. The trends for interfacial resistance and capacitance were independent to the geometry of the ITO:PET working electrode. Moreover, most robust behaviour was observed for rectangular electrodes. EIS across a broad range of potentials with and without inhibitors showed a marked difference between the interfacial resistance of viable and energy inhibited biofilms. Moreover, EIS of exopolysaccharide Δpsl mutant showed a substantial drop in current. Overall, our results indicated that EIS enabled the detection of biofilm formation across large surface areas as early as 24 h after inoculation for the weak electroactive species P. aeruginosa using a flexible polymeric substrate.

Growth conditions, physiological properties, and selection of optimal parameters of biodegradation of anticancer drug daunomycin in industrial effluents by Bjerkandera adusta CCBAS930

The study characterizes the anamorphic Bjerkandera adusta strain CCBAS 930, including growth conditions, physiological properties, and enzymatic activities related to basic metabolism and specific properties coupled with the fungal secondary metabolism. It was established that the fungus grows in a wide pH range (3.5–7.5), up to 3% of salt concentration and a temperature of 5–30 °C. Media rich in natural organic components (potato, maize extracts, whey) are optimal for biomass propagation. Minimal media, containing mineral salts and glucose as well as static growth conditions, are required to obtain idiophasic mycelium, equivalent to the secondary metabolism of the fungus. Of the 7 complex C, N, and energy sources tested, the strain did not utilize only fibrous cellulose. Lipolytic activity reached the highest values of the enzymatic activities corresponding to those capabilities. The specific properties of strain B. adusta CCBAS 930 determined by the production of HRP-like peroxidase were related to the decolorization and biodegradation of anthraquinone derivative daunomycin. The decolorization of 30% of daunomycin effluents occurred most rapidly in iso-osmotic medium and non-enriched with nitrogen, containing 0.25% glucose, pH = 5.0–6.0, and 25–30 °C. In agitated cultures, the strain decolorized solutions of daunomycin by biosorption, which coincided with the inhibition of aerial mycelium production and HRP-like biosynthesis. Based on knowledge, potential and real possibilities of using the strain in bioremediation of colored industrial sewage were discussed.

The Yeast AIF Homolog Nde1 Integrates Signals from Metabolism and Proteostasis on the Mitochondrial Surface and Executes Cell Death

Little is known about the proteolytic turnover of mitochondrial proteins under static growth conditions. Combining dynamic isotope labeling and mass spectrometry in yeast cells, we found an exceptionally high turnover for the NADH dehydrogenase Nde1. This homolog of the mammalian apoptosis inducing factor AIF forms two distinct topomers in mitochondria, one residing in the intermembrane space and one cytosol-exposed form that spans the outer membrane. The latter serves as trigger of cell death with the potential to kill yeast cells in response to pro-apoptotic stimuli. The surface-exposed topomer is degraded by the cytosolic proteasome/Cdc48 system and the mitochondrial protease Yme1 in wild type cells, however, it is strongly enriched in respiratory deficient cells. Our data suggest that in addition to their role in electron transfer, mitochondrial NADH dehydrogenases such as Nde1 or AIF integrate signals from energy metabolism and cytosolic proteostasis to eliminate compromised individuals from growing cell populations.

Effects of benzalkonium chloride on cell viability, inflammatory response, and oxidative stress of human alveolar epithelial cells cultured in a dynamic culture condition

Recently, the importance of inhalation toxicity assessment increased due to recent humidifier disinfectant-associated deaths in children. Benzalkonium chloride (BAC) is currently used as a cationic surfactant and germicide in food industry processing lines and as a hand sanitizer. Animal models are mainly used as a method of evaluating the inhalation toxicity of a hazardous substance, but that approach requires considerable amounts of time and cost. As a replacement for animal experiments, in vitro cell culture can be used to assess toxicity. However, such culture does not reflect the natural microenvironment of the lung, particularly its dynamic nature. In this study, we simulated normal breathing levels (tidal volume 10%, 0.2 Hz) through surface elongation of an elastic membrane in a dynamic culture system. The low-cost dynamic system provided easy control of breathing rate during lung cell culture. We assessed the toxicity using different concentrations of BAC (0, 2, 5, 10, 20, and 40 μg/mL) under static and dynamic culture conditions. Following 24 h of exposure to BAC, cellular metabolic activity, cell membrane integrity, interleukin-8 (IL-8) and reactive oxygen species (ROS) levels, and the total amount of protein in cells were analyzed. Our results showed that significant differences in cellular metabolic activity, as well as IL-8 and ROS profiles, between static and dynamic cell growth conditions, following BAC exposure.

Biofilm formation by Prototheca zopfii isolated from clinical and subclinical bovine mastitis in distinct growth conditions under different dyes

ABSTRACT: Prototheca spp. have been reported as an emergent environmental mastitis pathogen in several countries. Biofilm formation is a significant factor associated with different degrees of virulence developed by many microorganisms, including Prototheca spp. The present study aimed to compare two growth conditions and two staining dyes to determine which combination was more appropriate to evaluate qualitatively and quantitatively the production of biofilm by P. zopfii. Biofilm formation was evaluated in polystyrene microplates under static and dynamic growth conditions and staining with crystal violet or cotton blue dye. All P. zopfii isolates from cows with mastitis were classified as biofilm-producers in all growth conditions and staining. The cotton blue dye proved to be more appropriate method to classify the intensity of P. zopfii biofilm production.

Reduction in Pseudomonas aeruginosa and Staphylococcus aureus biofilms from implant materials in a diffusion dominated environment.

Antibiotic-loaded calcium sulfate beads (CS-B) are used to treat biofilm-related periprosthetic joint infections (PJI). A previous study has shown that such beads are effective in reducing lawns biofilms grown on agar plates; however, the ability of CS-B to eradicate biofilms grown on solid orthopedic material surfaces has not been investigated. We grew biofilms of bioluminescent strains of Pseudomonas aeruginosa Xen41 and a USA300 MRSA Staphylococcus aureus SAP231 on ultra-high molecular weight polyethylene (PE), hydroxyapatite (HA), and 316L stainless steel (SS) coupons for three days under static growth conditions, with daily nutrient exchange. The coupons were rinsed with sterile phosphate buffered saline (PBS) to remove planktonic bacteria and placed in a petri dish, surrounded by four either antibiotic vancomycin and tobramycin loaded (CS-BV+T ) or unloaded beads (CS-BU ). A thin layer of agar was overlaid to simulate a periprosthetic infection where an implant abuts soft tissue and then incubated for 72 h. The amount of biofilm was measured by bioluminescence imaging (BLI) for activity and viable cell count (CFUs). Coupons exposed to CS-BV+T showed a significant reduction in the amount of biofilm within 24 h, regardless of the bacterial strain or material type. The coupons exposed to control CS-BU had no effect on bacteria over 72 h. Statement of Clinical Significance: Antibiotic-loaded calcium sulfate beads (CS-B) were effective in significantly reducing mature biofilms of P. aeruginosa and S. aureus from orthopedic relevant surfaces in our in vitro agar model. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:3081-3085, 2018.

Isolation and characterization of HepP: a virulence-related Pseudomonas aeruginosa heparinase

BackgroundPseudomonas aeruginosa is an opportunistic pathogen that causes serious infections in immunocompromised hosts including severely burned patients. In burn patients, P. aeruginosa infection often leads to septic shock and death. Despite numerous studies, the influence of severe thermal injuries on the pathogenesis of P. aeruginosa during systemic infection is not known. Through RNA-seq analysis, we recently showed that the growth of P. aeruginosa strain UCBPP-PA14 (PA14) in whole blood obtained from severely burned patients significantly altered the expression of the PA14 transcriptome when compared with its growth in blood from healthy volunteers. The expression of PA14_23430 and the adjacent gene, PA14_23420, was enhanced by seven- to eightfold under these conditions.ResultsQuantitative real-time PCR analysis confirmed the enhancement of expression of both PA14_23420 and PA14_23430 by growth of PA14 in blood from severely burned patients. Computer analysis revealed that PA14_23430 (hepP) encodes a potential heparinase while PA14_23420 (zbdP) codes for a putative zinc-binding dehydrogenase. This analysis further suggested that the two genes form an operon with zbdP first. Presence of the operon was confirmed by RT-PCR experiments.We characterized hepP and its protein product HepP. hepP was cloned from PA14 by PCR and overexpressed in E. coli. The recombinant protein (rHepP) was purified using nickel column chromatography. Heparinase assays using commercially available heparinase as a positive control, revealed that rHepP exhibits heparinase activity. Mutation of hepP resulted in delay of pellicle formation at the air-liquid interface by PA14 under static growth conditions. Biofilm formation by PA14ΔhepP was also significantly reduced. In the Caenorhabditis elegans model of slow killing, mutation of hepP resulted in a significantly lower rate of killing than that of the parent strain PA14.ConclusionsChanges within the blood of severely burned patients significantly induced expression of hepP in PA14. The heparinase encoded by hepP is a potential virulence factor for PA14 as HepP influences pellicle formation as well as biofilm development by PA14 and the protein is required for full virulence in the C. elegans model of slow killing.

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Distinct Expression of Immunoglobulin-Binding Proteins in Shiga Toxin-Producing Escherichia coli Implicates High Protein Stability and a Characteristic Phenotype.

Several immunoglobulin-binding proteins of Escherichia coli (Eib) have been isolated from both non-pathogenic and pathogenic E. coli strains. Shiga toxin (Stx)-producing E. coli (STEC) contain eibG either as a single gene or in combination with eibC, while other E. coli strains harbour single or multiple eib genes. The Eib proteins bind human immunoglobulins in a non-immune manner and contribute to bacterial chain-like adherence to human epithelial cells. In this study, the EibG expression in several STEC strains was analysed under different environmental conditions. STEC produced high levels of EibG in complex media and lower levels in low-grade and minimal media under static growth conditions. This characteristic was independent on the Eib subtypes. Microscopically, EibG-expressing STEC exhibited chain formation and aggregation in all employed media, while aggregates were only visible after growth in complex medium. Once expressed, EibG proteins demonstrate high stability during prolonged incubation. Our findings indicate that the regulation of the expression of Eib proteins is highly complex, although the protein levels vary among STEC strains. However, positive upregulation conditions generally result in distinct phenotypes of the isolates.

Bacterial Interactions with Immobilized Liquid Layers.

Bacterial interactions with surfaces are at the heart of many infection-related problems in healthcare. In this work, the interactions of clinically relevant bacteria with immobilized liquid (IL) layers on oil-infused polymers are investigated. Although oil-infused polymers reduce bacterial adhesion in all cases, complex interactions of the bacteria and liquid layer under orbital flow conditions are uncovered. The number of adherent Escherichia coli cells over multiple removal cycles increases in flow compared to static growth conditions, likely due to a disruption of the liquid layer continuity. Surprisingly, however, biofilm formation appears to remain low regardless of growth conditions. No incorporation of the bacteria into the layer is observed. Bacterial type is also found to affect the number of adherent cells, with more E. coli remaining attached under dynamic orbital flow than Staphylococcus aureus, Pseudomonas aeruginosa under identical conditions. Tests with mutant E. coli lacking flagella confirm that flagella play an important role in adhesion to these surfaces. The results presented here shed new light on the interaction of bacteria with IL layers, highlighting the fundamental differences between oil-infused and traditional solid interfaces, as well as providing important information for their eventual translation into materials that reduce bacterial adhesion in medical applications.

Characterizing the in vitro biofilm phenotype of Staphylococcus epidermidis isolates from central venous catheters

Central venous catheter (CVC)-related infections are commonly caused by Staphylococcus epidermidis that is able to form a biofilm on the catheter surface. Many studies involving biofilm formation by Staphylococcus have been published each adopting an own in vitro model. Since the capacity to form a biofilm depends on multiple environmental factors, direct comparison of results obtained in different studies remains challenging. This study characterized the phenotype (strong versus weak biofilm-producers) of S. epidermidis from CVCs in four different in vitro biofilm models, covering differences in material type (glass versus polymer) and nutrient presentation (static versus continuous flow). A good correlation in phenotype was obtained between glass and polymeric surfaces independent of nutrient flow, with 85% correspondence under static growth conditions and 80% under dynamic conditions. A 80% correspondence between static and dynamic conditions on polymeric surfaces could be demonstrated as well. Incubation time had a significant influence on the biofilm phenotype with only 55% correspondence between the dynamic models at different incubation times (48h versus 17h). Screening for the presence of biofilm-related genes only revealed that ica A was correlated with biofilm formation under static but not under dynamic conditions. In conclusion, this study highlights that a high level of standardization is necessary to interpret and compare results of different in vitro biofilm models.

Agitation down-regulates immunoglobulin binding protein EibG expression in Shiga toxin-producing Escherichia coli (STEC).

Shiga toxin (Stx)-producing Escherichia coli (STEC) carrying eibG synthesize Escherichia coli immunoglobulin binding protein (EibG). EibG nonspecifically binds to immunoglobulins and tends to aggregate in multimers but is poorly expressed in wild-type strains. To study synthesis of the proteins and their regulation in the pathogens, we identified natural growth conditions that increased EibG synthesis. EibG proteins as well as corresponding mRNA were highly expressed under static growth conditions while shearing stress created by agitation during growth repressed protein synthesis. Further regulation effects were driven by reduced oxygen tension, and pH up-regulated EibG expression, but to a lesser extent than growth conditions while decreased temperature down-regulated EibG. Bacteria with increased EibG expression during static growth conditions showed a distinct phenotype with chain formation and biofilm generation, which disappeared with motion. High and low EibG expression was reversible indicating a process with up- and down-regulation of the protein expression. Our findings indicate that shear stress represses EibG expression and might reduce bacterial attachments to cells and surfaces.