Cranberry-Derived Proanthocyanidins Potentiate ß-Lactam Antibiotics against Resistant Bacteria.

The emergence and spread of extended-spectrum ß-lactamases (ESBLs), metallo-ß-lactamases (MBLs), or variant low-affinity penicillin-binding proteins (PBPs) pose a major threat to our ability to treat bacterial infection using ß-lactam antibiotics. Although combinations of ß-lactamase inhibitors with ß-lactam agents have been clinically successful, there are no MBL inhibitors in current therapeutic use. Furthermore, recent clinical use of new-generation cephalosporins targeting PBP2a, an altered PBP, has led to the emergence of resistance to these antimicrobial agents. Previous work shows that natural polyphenols such as cranberry-extracted proanthocyanidins (cPAC) can potentiate non-ß-lactam antibiotics against Gram-negative bacteria. This study extends beyond previous work by investigating the in vitro effect of cPAC in overcoming ESBL-, MBL-, and PBP2a-mediated ß-lactam resistance. The results show that cPAC exhibit variable potentiation of different ß-lactams against ß-lactam-resistant Enterobacteriaceae clinical isolates as well as ESBL- and MBL-producing E. coli We also discovered that cPAC have broad-spectrum inhibitory properties in vitro on the activity of different classes of ß-lactamases, including CTX-M3 ESBL and IMP-1 MBL. Furthermore, we observe that cPAC selectively potentiate oxacillin and carbenicillin against methicillin-resistant but not methicillin-sensitive staphylococci, suggesting that cPAC also interfere with PBP2a-mediated resistance. This study motivates the need for future work to identify the most bioactive compounds in cPAC and to evaluate their antibiotic-potentiating efficacy in vivoIMPORTANCE The emergence of ß-lactam-resistant Enterobacteriaceae and staphylococci compromises the effectiveness of ß-lactam-based therapy. By acquisition of ESBLs, MBLs, or PBPs, it is highly likely that bacteria may become completely resistant to the most effective ß-lactam agents in the near future. In this study, we described a natural extract rich in proanthocyanidins which exerts adjuvant properties by interfering with two different resistance mechanisms. By their broad-spectrum inhibitory ability, cranberry-extracted proanthocyanidins could have the potential to enhance the effectiveness of existing ß-lactam agents.

Plastic Teabags Release Billions of Microparticles and Nanoparticles into Tea.

The increasing presence of micro- and nano-sized plastics in the environment and food chain is of growing concern. Although mindful consumers are promoting the reduction of single-use plastics, some manufacturers are creating new plastic packaging to replace traditional paper uses, such as plastic teabags. The objective of this study was to determine whether plastic teabags could release microplastics and/or nanoplastics during a typical steeping process. We show that steeping a single plastic teabag at brewing temperature (95 °C) releases approximately 11.6 billion microplastics and 3.1 billion nanoplastics into a single cup of the beverage. The composition of the released particles is matched to the original teabags (nylon and polyethylene terephthalate) using Fourier-transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). The levels of nylon and polyethylene terephthalate particles released from the teabag packaging are several orders of magnitude higher than plastic loads previously reported in other foods. An initial acute invertebrate toxicity assessment shows that exposure to only the particles released from the teabags caused dose-dependent behavioral and developmental effects.

Proanthocyanidin Interferes with Intrinsic Antibiotic Resistance Mechanisms of Gram-Negative Bacteria.

Antibiotic resistance is spreading at an alarming rate among pathogenic bacteria in both medicine and agriculture. Interfering with the intrinsic resistance mechanisms displayed by pathogenic bacteria has the potential to make antibiotics more effective and decrease the spread of acquired antibiotic resistance. Here, it is demonstrated that cranberry proanthocyanidin (cPAC) prevents the evolution of resistance to tetracycline in Escherichia coli and Pseudomonas aeruginosa, rescues antibiotic efficacy against antibiotic-exposed cells, and represses biofilm formation. It is shown that cPAC has a potentiating effect, both in vitro and in vivo, on a broad range of antibiotic classes against pathogenic E. coli, Proteus mirabilis, and P. aeruginosa. Evidence that cPAC acts by repressing two antibiotic resistance mechanisms, selective membrane permeability and multidrug efflux pumps, is presented. Failure of cPAC to potentiate antibiotics against efflux pump-defective mutants demonstrates that efflux interference is essential for potentiation. The use of cPAC to potentiate antibiotics and mitigate the development of resistance could improve treatment outcomes and help combat the growing threat of antibiotic resistance.

Cranberry-derived proanthocyanidins impair virulence and inhibit quorum sensing of Pseudomonas aeruginosa.

Bacteria have evolved multiple strategies for causing infections that include producing virulence factors, undertaking motility, developing biofilms, and invading host cells. N-acylhomoserine lactone (AHL)-mediated quorum sensing (QS) tightly regulates the expression of multiple virulence factors in the opportunistic pathogenic bacterium Pseudomonas aeruginosa. Thus, inhibiting QS could lead to health benefits. In this study, we demonstrate an anti-virulence activity of a cranberry extract rich in proanthocyanidins (cerPAC) against P. aeruginosa in the model host Drosophila melanogaster and show this is mediated by QS interference. cerPAC reduced the production of QS-regulated virulence determinants and protected D. melanogaster from fatal infection by P. aeruginosa PA14. Quantification of AHL production using liquid chromatography-mass spectrometry confirmed that cerPAC effectively reduced the level of AHLs produced by the bacteria. Furthermore, monitoring QS signaling gene expression revealed that AHL synthases LasI/RhlI and QS transcriptional regulators LasR/RhlR genes were inhibited and antagonized, respectively, by cerPAC. Molecular docking studies suggest that cranberry-derived proanthocyanidin binds to QS transcriptional regulators, mainly interacting with their ligand binding sites. These findings provide insights into the underlying mechanisms of action of a cerPAC to restrict the virulence of P. aeruginosa and can have implications in the development of alternative approaches to control infections.

Interference of Quorum Sensing by Delftia sp. VM4 Depends on the Activity of a Novel N-Acylhomoserine Lactone-Acylase.

BACKGROUND: Turf soil bacterial isolate Delftia sp. VM4 can degrade exogenous N-acyl homoserine lactone (AHL), hence it effectively attenuates the virulence of bacterial soft rot pathogen Pectobacterium carotovorum subsp. carotovorum strain BR1 (Pcc BR1) as a consequence of quorum sensing inhibition. METHODOLOGY/PRINCIPAL FINDINGS: Isolated Delftia sp. VM4 can grow in minimal medium supplemented with AHL as a sole source of carbon and energy. It also possesses the ability to degrade various AHL molecules in a short time interval. Delftia sp. VM4 suppresses AHL accumulation and the production of virulence determinant enzymes by Pcc BR1 without interference of the growth during co-culture cultivation. The quorum quenching activity was lost after the treatment with trypsin and proteinase K. The protein with quorum quenching activity was purified by three step process. Matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) and Mass spectrometry (MS/MS) analysis revealed that the AHL degrading enzyme (82 kDa) demonstrates homology with the NCBI database hypothetical protein (Daci_4366) of D. acidovorans SPH-1. The purified AHL acylase of Delftia sp. VM4 demonstrated optimum activity at 20-40°C and pH 6.2 as well as AHL acylase type mode of action. It possesses similarity with an α/ß-hydrolase fold protein, which makes it unique among the known AHL acylases with domains of the N-terminal nucleophile (Ntn)-hydrolase superfamily. In addition, the kinetic and thermodynamic parameters for hydrolysis of the different AHL substrates by purified AHL-acylase were estimated. Here we present the studies that investigate the mode of action and kinetics of AHL-degradation by purified AHL acylase from Delftia sp. VM4. SIGNIFICANCE: We characterized an AHL-inactivating enzyme from Delftia sp. VM4, identified as AHL acylase showing distinctive similarity with α/ß-hydrolase fold protein, described its biochemical and thermodynamic properties for the first time and revealed its potential application as an anti-virulence agent against bacterial soft rot pathogen Pectobacterium carotovorum subsp. carotovorum based on quorum quenching mechanism.

Polyphenolic extract from maple syrup potentiates antibiotic susceptibility and reduces biofilm formation of pathogenic bacteria.

Phenolic compounds are believed to be promising candidates as complementary therapeutics. Maple syrup, prepared by concentrating the sap from the North American maple tree, is a rich source of natural and process-derived phenolic compounds. In this work, we report the antimicrobial activity of a phenolic-rich maple syrup extract (PRMSE). PRMSE exhibited antimicrobial activity as well as strong synergistic interaction with selected antibiotics against Gram-negative clinical strains of Escherichia coli, Proteus mirabilis, and Pseudomonas aeruginosa. Among the phenolic constituents of PRMSE, catechol exhibited strong synergy with antibiotics as well as with other phenolic components of PRMSE against bacterial growth. At sublethal concentrations, PRMSE and catechol efficiently reduced biofilm formation and increased the susceptibility of bacterial biofilms to antibiotics. In an effort to elucidate the mechanism for the observed synergy with antibiotics, PRMSE was found to increase outer membrane permeability of all bacterial strains and effectively inhibit efflux pump activity. Furthermore, transcriptome analysis revealed that PRMSE significantly repressed multiple-drug resistance genes as well as genes associated with motility, adhesion, biofilm formation, and virulence. Overall, this study provides a proof of concept and starting point for investigating the molecular mechanism of the reported increase in bacterial antibiotic susceptibility in the presence of PRMSE.

Biochemical and thermal stabilization parameters of polygalacturonase from Erwinia carotovora subsp. carotovora BR1.

With emphasis on thermal behavior in presence of different pH conditions and salts, the kinetic and thermodynamic parameters of purified polygalacturonase (PG) of E. carotovora subsp. carotovora (Ecc) BR1 were studied since characterization of an enzyme is significant in the context of burgeoning biotechnological applications. Thermodynamic parameters for polygalacturonic acid hydrolysis by purified PG were, deltaH* = 7.98 kJ/mol, deltaG* = 68.86 kJ/mol, deltaS*= -194.48 J/mol/K, deltaG(E-S) = -1.04 kJ/mol and deltaG(E-T) = -8.96 kJ/mol. Its turnover number (k(cat)) was 21/sec. Purified PG was stable in 20-50 degrees C temperature range and was deactivated at 60 degrees C and 70 degrees C. Thermodynamic parameters (deltaH*, deltaG*, deltaS*) for irreversible inactivation of PG at different temperatures (30-60 degrees C) were determined, where effectiveness of various salts and different pH (4-8) individually for thermal stability of PG were characterized. The efficacy of various salts for thermal stability of PG was in the following order: MgCl2 >BaCl2 >KCl >CaCl2 >NaCl. Present work projects biochemical, thermodynamics of substrate hydrolysis as well as thermal stabilization parameters of PG from Ecc.