A Simple Fragment of Cyclic Acyldepsipeptides Is Necessary and Sufficient for ClpP Activation and Antibacterial Activity

The development of new antibacterial agents, particularly those with unique biological targets, is essential to keep pace with the inevitable emergence of drug resistance in pathogenic bacteria. We identified the minimal structural component of the cyclic acyldepsipeptide (ADEP) antibiotics that exhibits antibacterial activity. We found that N‐acyldifluorophenylalanine fragments function via the same mechanism of action as ADEPs, as evidenced by the requirement of ClpP for the fragments' antibacterial activity, the ability of fragments to activate Bacillus subtilis ClpP in vitro, and the capacity of an N‐acyldifluorophenylalanine affinity matrix to capture ClpP from B. subtilis cell lysates. N‐acyldifluorophenylalanine fragments are much simpler in structure than the full ADEPs and are also highly amenable to structural diversification. Thus, the stage has been set for the development of non‐peptide activators of ClpP that can be used as antibacterial agents.     

Acyldepsipeptide Probes Facilitate Specific Detection of Caseinolytic Protease P Independent of Its Oligomeric and Activity State

Caseinolytic protease P (ClpP) is a tetradecameric peptidase that assembles with chaperones such as ClpX to gain proteolytic activity. Acyldepsipeptides (ADEPs) are small-molecule mimics of ClpX that bind into hydrophobic pockets on the apical site of the complex, thereby activating ClpP. Detection of ClpP has so far been facilitated with active-site-directed probes which depend on the activity and oligomeric state of the complex. To expand the scope of ClpP labeling, we took a stepwise synthetic approach toward customized ADEP photoprobes. Structure–activity relationship studies with small fragments and ADEP derivatives paired with modeling studies revealed the design principles for suitable probe molecules. The derivatives were tested for activation of ClpP and subsequently applied in labeling studies of the wild-type peptidase as well as enzymes bearing mutations at the active site and an oligomerization sensor. Satisfyingly, the ADEP photoprobes provided a labeling readout of ClpP independent of its activity and oligomeric state.     

Generation of Lasso Peptide-Based ClpP Binders

The Clp protease system fulfills a plethora of important functions in bacteria. It consists of a tetradecameric ClpP barrel holding the proteolytic centers and two hexameric Clp-ATPase rings, which recognize, unfold, and then feed substrate proteins into the ClpP barrel for proteolytic degradation. Flexible loops carrying conserved tripeptide motifs protrude from the Clp-ATPases and bind into hydrophobic pockets (H-pockets) on ClpP. Here, we set out to engineer microcin J25 (MccJ25), a ribosomally synthesized and post-translationally modified peptide (RiPP) of the lasso peptide subfamily, by introducing the conserved tripeptide motifs into the lasso peptide loop region to mimic the Clp-ATPase loops. We studied the capacity of the resulting lasso peptide variants to bind to ClpP and affect its activity. From the nine variants generated, one in particular (12IGF) was able to activate ClpP from Staphylococcus aureus and Bacillus subtilis. While 12IGF conferred stability to ClpP tetradecamers and stimulated peptide degradation, it did not trigger unregulated protein degradation, in contrast to the H-pocket-binding acyldepsipeptide antibiotics (ADEPs). Interestingly, synergistic interactions between 12IGF and ADEP were observed.     

Examination of a Structural Model of Peptidomimicry by Cyclic Acyldepsipeptide Antibiotics in Their Interaction with the ClpP Peptidase

The cyclic acyldepsipeptide (ADEP) antibiotics act by binding the ClpP peptidase and dysregulating its activity. Their exocyclic N‐acylphenylalanine is thought to structurally mimic the ClpP‐binding, (I/L)GF tripeptide loop of the peptidase's accessory ATPases. We found that ADEP analogues with exocyclic N‐acyl tripeptides or dipeptides resembling the (I/L)GF motif were weak ClpP activators and had no bioactivity. In contrast, ADEP analogues possessing difluorophenylalanine N‐capped with methyl‐branched acyl groups—like the side chains of residues in the (I/L)GF motifs—were superior to the parent ADEP with respect to both ClpP activation and bioactivity. We contend that the ADEP's N‐acylphenylalanine moiety is not simply a stand‐in for the ATPases' (I/L)GF motif; it likely has physicochemical properties that are better suited for ClpP binding. Further, our finding that the methyl‐branching on the acyl group of the ADEPs improves activity opens new avenues for optimization.     

Antibacterial Small Molecules That Potently Inhibit Staphylococcus aureus Lipoteichoic Acid Biosynthesis

The rise of antibiotic resistance, especially in Staphylococcus aureus, and the increasing death rate due to multiresistant bacteria have been well documented. The need for new chemical entities and/or the identification of novel targets for antibacterial drug development is high. Lipoteichoic acid (LTA), a membrane-attached anionic polymer, is important for the growth and virulence of many Gram-positive bacteria, and interest has been high in the discovery of LTA biosynthesis inhibitors. Thus far, only a handful of LTA biosynthesis inhibitors have been described with moderate (MIC=5.34 μg mL⁻¹) to low (MIC=1024 μg mL⁻¹) activities against S. aureus. Herein we describe the identification of novel compounds that potently inhibit LTA biosynthesis in S. aureus, displaying impressive antibacterial activities (MIC as low as 0.25 μg mL⁻¹) against methicillin-resistant S. aureus (MRSA). Under similar in vitro assay conditions, these compounds are 4-fold more potent than vancomycin and 8-fold more potent than linezolid against MRSA.     

Functional Identification of Serine Hydroxymethyltransferase as a Key Gene Involved in Lysostaphin Resistance and Virulence Potential of Staphylococcus aureus Strains

Gaining an insight into the mechanism underlying antimicrobial-resistance development in Staphylococcus aureus is crucial for identifying effective antimicrobials. We isolated S. aureus sequence type 72 from a patient in whom the S. aureus infection was highly resistant to various antibiotics and lysostaphin, but no known resistance mechanisms could explain the mechanism of lysostaphin resistance. Genome-sequencing followed by subtractive and functional genomics revealed that serine hydroxymethyltransferase (glyA or shmT gene) plays a key role in lysostaphin resistance. Serine hydroxymethyltransferase (SHMT) is indispensable for the one-carbon metabolism of serine/glycine interconversion and is linked to folate metabolism. Functional studies revealed the involvement of SHMT in lysostaphin resistance, as ΔshmT was susceptible to the lysostaphin, while complementation of the knockout expressing shmT restored resistance against lysostaphin. In addition, the ΔshmT showed reduced virulence under in vitro (mammalian cell lines infection) and in vivo (wax-worm infection) models. The SHMT inhibitor, serine hydroxymethyltransferase inhibitor 1 (SHIN1), protected the 50% of the wax-worm infected with wild type S. aureus. These results suggest SHMT is relevant to the extreme susceptibility to lysostaphin and the host immune system. Thus, the current study established that SHMT plays a key role in lysostaphin resistance development and in determining the virulence potential of multiple drug-resistant S. aureus.     

Optimization of Propidium Monoazide Quantitative PCR for Evaluating Performances of Bioaerosol Samplers for Sampling Airborne Staphylococcus aureus

Airborne Staphylococcus aureus causes a significant proportion of nosocomial infections. The purpose of this study was to combine real-time quantitative polymerase chain reaction with the DNA-binding agent propidium monoazide (PMA-qPCR) to assess exposures to airborne S. aureus. In this work, we generated a S. aureus aerosol and used our assay to detect viable, airborne S. aureus in a study chamber. The biological collection efficiencies of three samplers (the AGI-30 impinger, BioSampler, and Nuclepore filter sampler) were evaluated using the S. aureus aerosols. The effects of storage in collection fluid on S. aureus sampled by the AGI-30 impinger and BioSampler were evaluated. Furthermore, air samples from an intensive care unit (ICU) and a gymnasium (GYM) were subsequently used to test the performance of a BioSampler combined with our PMA-qPCR technique. The BioSampler was more effective than the AGI-30 and Nuclepore filter samplers for preserving the culturability and viability of S. aureus aerosol samples. After sampling by impingement, the loss of viable S. aureus was minimized by treating the cells with PMA prior to storage at ?20°C and analyzing the samples by qPCR within 3 weeks. In field applications, we noted that traditional culture assays tended to underestimate the viable concentrations of S. aureus by approximately one order of magnitude. Overall, combining qPCR with and without PMA staining may be useful for assessing exposure to airborne S. aureus. However, a complex set of parameters that may affect the efficiency of PMA-qPCR must be taken into account before applying PMA-qPCR to bioaerosol detection.

Copyright 2014 American Association for Aerosol Research     

Imidazolium-based ionic thermoplastic polyurethane with low surface energy and antibacterial activities

A series of novel thermoplastic polyurethane elastomers with both low surface energy and antibacterial activity were synthesized in this paper. Firstly, four imidazole salt diols with different alkyl chain lengths at the N-position of imidazolium cation ([CnIm⁺][Cl⁻]) were prepared. Then, imidazolium-based ionic thermoplastic polyurethane elastomers (Si-ITPUs) were formulated by incorporating polydimethylsiloxane and polytetramethylene glycol as a mixed soft segment, 1,4-butanediol and [CnIm⁺][Cl⁻] as a chain extender, and 4,4′-dicyclohexylmethane diisocyanate. The structure, thermal stability, mechanical properties, physical crosslinking density, and the anti-graffiti properties of Si-ITPUs were evaluated. The surface energy and water absorption were evaluated through static contact angle and water resistance tests, respectively. Furthermore, the antibacterial activities against both Staphylococcus.aureus and Escherichia coli were characterized by the plate colony counting method. The results demonstrated that Si-ITPUs possessed surface energy of approximately 23 mN/m, which conferred excellent anti-graffiti and self-cleaning performance. Furthermore, the increase of the alkyl chain length of substitutions resulted in a decrease in the tensile strength but an increase in the elongation at break. Si-ITPU-C₄ exhibited optimized antibacterial activities with an antibacterial rate of more than 99.9% against both S. aureus and E. coli. This novel polyurethane is anticipated to find applications in the medical device industry and food processing sector.     

Green cinnamaldehyde and thymol modified zinc oxide with double synergistic antibacterial effects in polypropylene

The green cinnamaldehyde (CA) and thymol (THY) separately or successively modified O-ZnO (O-ZnO-CA, O-ZnO-THY, and O-ZnO-CA-THY) were prepared after (3-aminopropyl) triethoxysilane (KH550) was grafted to the surface of ZnO (O-ZnO) and characterized by FTIR, TGA, and SEM. Polypropylene composites (PP/O-ZnO-CA, PP/O-ZnO-THY, and PP/O-ZnO-CA-THY) were prepared by melting the modified O-ZnO and PP. The antibacterial tests showed that the antibacterial rate of PP/O-ZnO-CA and PP/O-ZnO-THY against S. aureus and E. coli was obviously enhanced. It indicated an excellent synergetic antibacterial effect of O-ZnO and the little CA or THY grafted on the surface of O-ZnO. The antibacterial effect of O-ZnO-CA and O-ZnO-THY was related to the hydrophobic and hydrophilic groups contained in CA and THY. Importantly, CA and THY in O-ZnO-CA-THY exhibited another excellent synergetic antibacterial effect against both S. aureus and E. coli. The antibacterial rate of three PP composites containing 4 phr O-ZnO-CA-THY against S. aureus and E. coli reached 95% and 90%, respectively. The mechanism was regarded as that CA and THY grafted onto the same O-ZnO improved the contact probability between antibacterial agents and bacteria. Moreover, O-ZnO-CA, O-ZnO-THY, and O-ZnO-CA-THY had excellent migration resistance in the PP matrix.     

Supercritical CO2 extraction of ucuúba (Virola surinamensis) seed oil: global yield,kinetic data,fatty acid profile,and antimicrobial activities

Virola surinamensis is an abundant floodplain tree, popularly known as ucuúba, that grows in the Amazon. In this study, ucuúba seed oil was obtained by supercritical fluid extraction under different operating conditions, as well as Soxhlet extraction. The operating conditions for supercritical extraction were an extraction temperature of 40, 60, or 80?°C, a pressure of 350?bar, and a CO₂ mass flow of 7.9?×?10^?5 kg/s. The supercritical extraction curves were fitted to mass transfer models, and the fatty acid profiles of the extracts were determined by gas chromatography. The antimicrobial activity was assessed against Candida albicans, Staphylococcus aureus, and Escherichia coli. The highest yield obtained using supercritical CO₂ was 64.39% and the lowest was 59.21%. The phytochemical analysis showed the presence of steroids, terpenes, coumarins, and phenolic compounds. All ucuúba oil samples showed antioxidant activity. Regarding the antimicrobial activity, ucuúba oil only showed activity against S. aureus.     

Preparation of antibacterial and antifungal breathable polyether block amide/chloropropane diol membranes via solution casting

A highly hydrophilic block copolymer polyether block amide (PEBA) is modified with chloropropane diol (CPD) to impart antibacterial and antifungal properties to it without compromising with its breathability. The antibacterial properties of modified membranes are evaluated against Staphylococcus aureus (S. aureus, Gram positive) and Escherichia coli (E. coli, Gram negative) bacteria by membrane culture method. CPD plays an important role in the antibacterial property with the inhibition rate reaching 99.99% for CPD modified membranes which was 27.55% and 16.82% for pristine membrane (against S. aureus and E. coli respectively). The antifungal properties studied against Aspergllus niger, Penicillium pinophilum, Aureobasidium pullulans, Chaetomium globosum, and Trichoderma virens show heavy‐growth of fungi for pristine PEBA membrane while no growth was observed in case of CPD modified membranes. Breathability of membrane is determined in terms of water vapor transmission rate (WVTR) and it increase from 1496 g/m²/day to 2354 g/m²/day after modification. The membranes are characterized by FTIR‐ATR, SEM‐EDX, DSC, and TGA. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018 , 135, 46097.     

Treatment of Air Filters Using the Antimicrobial Natural Products Propolis and Grapefruit Seed Extract for Deactivation of Bioaerosols

Two natural products; i.e., grapefruit seed extract (GSE) and propolis, were investigated for use in antimicrobial air filters. Staphylococcus aureus was investigated as a test bioaerosol, and was deposited on the antimicrobial filters, which were treated by spraying with various areal densities of GSE and propolis. The pressure drop and particle penetration were investigated to assess the filtration performance of the bioaerosol, and the bacterial-inactivation performance of the filters was evaluated by quantifying S. aureus. There was little change in the pressure drop as a function of the areal density of GSE up to 185.9 μg/cm²; however, a significant change in the pressure drop was found for the air filter coated with propolis at an areal density of 98.4 μg/cm². The penetration levels of bioaerosols in both filters were uniform and in the range 1.4–2.0% (based on particle number), regardless of the areal density of the deposited GSE or propolis. The inactivation rates of the filters with identical deposition masses of GSE and propolis were similar in the ranges of 92.1–100%, 75.2–89.1%, and 54.4–75.5% at the control filters with colony numbers of 10³, 10⁴, and 10⁵ CFU/mL, respectively. The bacterial inactivation rate could be described by an exponential function of the areal density of GSE/propolis per number of colonies.

Copyright 2015 American Association for Aerosol Research     

Preparation of cetylpyridinium montmorillonite for antibacterial applications

The antibacterial activities of cetylpyridinium-montmorillonites (CP⁺-Mt) were tested on Staphylococcus aureus and Pseudomonas aeruginosa. The Mt were prepared by using the five different CP⁺ amounts of 0.5, 0.7, 1.0, 1.5, and 2.0 times of cation exchange capacities (CEC) of Na⁺-Mt. Desorption of CP⁺ from the surface was also determined by successive adsorption–desorption experiments. The antibacterial activity tests were conducted by using Na⁺-Mt and CP⁺-Mt through the disk diffusion (Kirby–Bauer) method against the P. aeruginosa ATCC 27853, and S. aureus ATCC 29213 strains. XRD analyses of the CP⁺-Mt showed that basal spacing regularly increased by increasing the amount of CP⁺ cations. Adsorption/desorption studies revealed that desorption occurred only in 2.0 CEC CP⁺-Mt by dilution with water and in 1.0 CEC CP⁺-Mt at a pH of 2.0. Na⁺-Mt exhibited no antibacterial activity against both bacteria. All of the CP⁺-Mt samples prepared were active against S. aureus, whereas they had no antibacterial activity against P. aeruginosa. Minimum inhibitory concentration (MIC) was found to be 1 mg/plate against S. aureus, determined with 0.5 CEC CP⁺-Mt. Because nearly no desorption of CP⁺ was observed, the antibacterial activity was attributed to the CP⁺ bound to the Mt surface.     

An investigation of heat transfer effects in isothermal crystallization studies of low‐density polyethylene

Isothermal crystallization kinetics of low‐density polyethylene (LDPE) were measured from 96°C‐103°C using a power‐compensating differential scanning calorimetry (DSC). Crystallization kinetics were measured using different sample thicknesses and on samples compounded with nickel, a filler with high thermal conductivity. For the unfilled material, sample thickness and temperature had a significant effect on the rate of crystallization as measured by the Avrami rate constant K, but had no effect on the nucleation mechanism and dimensionality of growth, as measured by the Avrami constant n. The crystallization growth rate as expressed by K^1/n scaled approximately with the thickness of the sample. For the filled material, K was much higher and independent of nickel content, suggesting a limiting growth rate for polyethylene at a given temperature in this equipment. The dependence of crystallization rale on sample thickness indicates that barriers to heat transfer can be important. This work shows that for most crystallization rates, thermal conductivity, rather than interfacial resistance between sample and pan, limits heat transfer. Even though thermal conductivity typically dominates heat‐transfer resistance, sample‐pan thermal contact is still important, and some guidelines are given to determine whether good contact is being made.     

Specific recognition of staphylococcus aureus by staphylococcus aureus protein A-imprinted polymers

Staphylococcus aureus protein A-imprinted polyacrylamide gel beads (SpA-IPGB) that target Staphylococcus aureus (S. aureus) were synthesized by inverse-phase suspension polymerization and using SpA as template. Adsorption experiments of different proteins onto SpA-IPGB were conducted to determine the protein binding capacity and selectivity. Recognition specificity studies of the prepared SpA-IPGB for S. aureus were conducted to determine the recognition specificity for the bacteria. The adsorption test results showed that imprinted gel beads exhibited a good recognition for template proteins, as compared to control proteins. The recognition specificity study indicated that the adsorbed quantity of S. aureus onto the SpA-IPGB was much higher than for other types of bacteria. The adsorbed quantity of S. aureus onto the SPA-IPGB was 10³–10⁴ CFU/g of gel beads.     

Efficiency of silver‐based antibacterial additives and its influence in thermoplastic elastomers

Styrene‐butylene/ethylene‐styrene‐based thermoplastic elastomers (TPE) are polymers with soft touch properties that are widely used for manufacturing devices that involve hand contact. However, when contaminated with microorganisms these products can contribute to spreading diseases. The incorporation of antibacterial additives can help maintain low bacteria counts. This work evaluated the antibacterial action of TPE loaded with silver ions and silver nanoparticles. The additives nanosilver on fumed silica (NpAg_silica), silver phosphate glass (Ag⁺_phosphate), and bentonite organomodified with silver (Ag⁺_bentonite) were added to the TPE formulation. The compounds were evaluated for tensile and thermal properties and antimicrobial activity against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). All the additives eliminated over 90% of E. coli, but only NpAg_silica killed more than 80% of S. aureus population. The better effect of NpAg_silica was attributed to the additive's high specific surface area, which promoted greater contact with bacteria cells. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43956.     

Head‐to‐Head Prenyl Synthases in Pathogenic Bacteria

Many organisms contain head‐to‐head isoprenoid synthases; we investigated three such types of enzymes from the pathogens Neisseria meningitidis, Neisseria gonorrhoeae, and Enterococcus hirae. The E. hirae enzyme was found to produce dehydrosqualene, and we solved an inhibitor‐bound structure that revealed a fold similar to that of CrtM from Staphylococcus aureus. In contrast, the homologous proteins from Neisseria spp. carried out only the first half of the reaction, yielding presqualene diphosphate (PSPP). Based on product analyses, bioinformatics, and mutagenesis, we concluded that the Neisseria proteins were HpnDs (PSPP synthases). The differences in chemical reactivity to CrtM were due, at least in part, to the presence of a PSPP‐stabilizing arginine in the HpnDs, decreasing the rate of dehydrosqualene biosynthesis. These results show that not only S. aureus but also other bacterial pathogens contain head‐to‐head prenyl synthases, although their biological functions remain to be elucidated.     

Recognition property and preparation of Staphylococcus aureus protein A-imprinted polyacrylamide polymers by inverse-phase suspension and bulk polymerization

Staphylococcus aureus protein A-imprinted polyacrylamide gel beads (SpA-IPGB) and imprinted polyacrylamide particles (SpA-IPGP) were synthesized by inverse-phase suspension polymerization and bulk polymerization, using SpA as template, acrylamide (AM) as functional monomers. Recognition capacity studies of the prepared SpA-IPGB and SpA-IPGP on SpA and S. aureus were conducted to determine recognition specificity. Computer imitation docking studies were conducted between template proteins and acrylamide monomer to reveal the recognition mechanism of SpA molecularly imprinted polymer. The results showed that the imprinted gel beads had high adsorption capacity and specificity to the SpA and S. aureus, and the adsorption quantity could reach 6.85 × 10⁻³ μmol/g and 10³-10⁴ CFU/g. The adsorption capacities of SpA-IPGB on SpA and S. aureus were higher than that of SpA-IPGP and Non-imprinted polyacrylamide gel beads (Non-IPGB). The recognition specificity of SpA-IPGB and SpA-IPGP on S. aureus was much higher than on Escherichia coli and Streptococcus thermophilus. The formation of complementary shape, and hydrogen bonding, electrostatic, and hydrophobic interactions between the imprinting cavities and the template proteins is the driving force for effective and specific recognition of protein imprinted polymer.     

Antibacterial Thin Film Composite Polyamide Membranes Prepared by Sequential Interfacial Polymerization

In this work, thin film composite polyamide (PA) membranes are modified by polyethyleneimine (PEI) and 2,6‐diaminopyridine (DAP) through sequential interfacial polymerization to fabricate contact active antibacterial membranes. The modified membranes show improved hydrophilicity and enhancement of zeta potential. Upon tethering with PEI and DAP onto the PA membranes, the membrane flux increases from 35.7 to 46.7 and 50.0 L m^?2 h^?1, respectively. Further the salt rejection rate improves from 96.6% to 98.0% and 98.8%, respectively. The PA‐PEI membranes have a better antibacterial performance than PA‐DAP, with a bacteria killing ratio for both Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) over 96.7%, while a commercial LC LE‐4040 membrane presents bacteria killing ratio of 13.3% for E. coli and 8.4% for S. aureus, respectively.     

Comparative study of the elimination of toluene vapours in twin biotrickling filters using two microorganisms Bacillus cereus S1 and S2

BACKGROUND: To investigate the microbial degradation performance of organic pollutants in the atmosphere using a biotrickling filter, two microorganism strains, Bacillus cereus S1 and Bacillus cereus S2, were selected, identified and inoculated into a twin biotrickling filter for comparison. RESULTS: Both strains showed good performance towards the degradation of model organic pollutants when gas flow rates ranged from 100 to 600 L h⁻¹. For S1, the total maximum removal efficiency (RE) of toluene was maintained nearly 100% not only at gas flow rates of 100 L h⁻¹ corresponding to empty bed residence time (EBRT) 199.44 s, but also at gas flow rates of 200 L h⁻¹ (EBRT = 99.72 s) and 300 L h⁻¹ (EBRT = 66.48 s). However, S2 had a much lower degradation capability; near 100% removal efficiency was obtained only at the gas flow rate of 100 L h⁻¹ although both bacteria belong to the same Bacillus cereus. With further increase in gas flow rate, the total REs for both S1 and S2 decreased slightly at first and then dropped sharply to 46% and 35%, respectively, at an EBRT of 33.24 s, corresponding to a gas flow rate of 600 L h⁻¹. Starvation for between 2 and 10 days resulted in the re‐acclimation times of both strains ranging between 1.0 and 15.5 h. CONCLUSION: Strain S1 would be a better choice for inoculation into a biotrickling filter than strain S2, because of its much higher toluene removal capacity and rapid recovery to full performance. Copyright © 2008 Society of Chemical Industry