Combination of a pH-responsive peptide amphiphile and a conventional antibiotic in treating Gram-negative bacteria.

BACKGROUND: Clinical treatments ofgastric infections using antibiotics suffer from the undesired killing of commensal bacteria and emergence of antibiotic resistance. It is desirable to develop pH-responsive antimicrobial peptides (AMPs) that kill pathogenic bacteria such as H. pyloriand resistant E. coli under acidic environment with minimal impact to commensal bacteria whilst not causing antibiotic resistance. EXPERIMENTS: Using a combined approach of cell assays, molecular dynamics (MD) simulations and membrane models facilitating biophysical and biochemical measurements including small angle neutron scattering (SANS), we have characterized the pH-responsive physiochemical properties and antimicrobial performance of two amphiphilic AMPs, GIIKDIIKDIIKDI-NH2 and GIIKKIIDDIIKKI-NH2 (denoted as 3D and 2D, respectively), that were designed by selective substitutions of cationic residues of Lys (K) in the extensively studied AMP G(IIKK)3I-NH2 with anionic residue Asp (D). FINDINGS: Whilst 2D kept non-ordered coils across the entire pH range studied, 3D displayed a range of secondary structures when pH was shifted from basic to acidic, with distinct self-assembly into nanofibers in aqueous environment. Further experimental and modeling studies revealed that the AMPs interacted differently with the inner and outer membranes of Gram-negative bacteria in a pH-responsive manner and that the structural features characterized by membrane leakage and intramembrane nanoaggregates revealed from fluorescence spectroscopy and SANS were well linked to antimicrobial actions. Different antimicrobial efficacies of 2D and 3D were underlined by the interplay between their ability to bind to the outer membrane lipid LPS (lipopolysaccharide), outer membrane permeability change and inner membrane depolarization and leakage. Furthermore, AMP's binding with the inner membrane under acidic condition caused both the dissipation of membrane potential (Δψ) and the continuous dissipation of transmembrane ΔpH, with Δψ and ΔpH being the key components of the proton motive force. Combinations of antibiotic (Minocycline) with the pH-responsive AMP generated the synergistic effects against Gram-negative bacteria only under acidic condition. These features are crucial to target applications to gastric infections, anti-acne and wound healing.

Altered Oral Nitrate Reduction and Bacterial Profiles in Hypertensive Women Predict Blood Pressure Lowering Following Acute Dietary Nitrate Supplementation.

BACKGROUND: The efficacy of dietary nitrate supplementation to lower blood pressure (BP) in pregnant women is highly variable. We aimed to investigate whether differences in oral microbiota profiles and oral nitrate-reducing capacity may explain interindividual differences in BP lowering following nitrate supplementation. METHODS: Participants recruited for this study were both pregnant and nonpregnant women, with or without hypertension (n=55). Following an overnight fast, plasma, saliva, and tongue scraping samples were collected for measurement of nitrate/nitrite concentrations, oral NaR (nitrate reductase) activity, and microbiota profiling using 16S rRNA gene sequencing. Baseline BP was measured, followed by the administration of a single dose of dietary nitrate (400 mg nitrate in 70 mL beetroot juice). Post-nitrate intervention, plasma and salivary nitrate/nitrite concentrations and BP were determined 2.5 hours later. RESULTS: Women with hypertension had significantly lower salivary nitrite concentrations (P=0.006) and reduced abundance of the nitrate-reducing taxa Veillonella(P=0.007) compared with normotensive women. Oral NaR activity was not significantly different in pregnant versus nonpregnant women (P=0.991) but tended to be lower in hypertensive compared with normotensive women (P=0.099). Oral NaR activity was associated with both baseline diastolic BP (P=0.050) and change in diastolic BP following acute nitrate intake (P=0.01, adjusted for baseline BP). CONCLUSIONS: The abundance and activity of oral nitrate-reducing bacteria impact both baseline BP as well as the ability of dietary nitrate supplementation to lower BP. Strategies to increase oral nitrate-reducing capacity could lower BP and enhance the efficacy of dietary nitrate supplementation, in pregnancy as well as in nonpregnant adults. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT03930693.

Antibacterial effects in blood irradiated with a polychromatic device mediated through reactive oxygen species: possible involvement of haem.

The antibacterial effects of a polychromatic light device designed for intravenous application were assessed in vitro. Staphylococcus aureus, Klebsiella pneumoniae, or Escherichia coli were exposed to a 60-min sequential light cycle comprising 365, 530, and 630 nm wavelengths in circulated sheep blood. Bacteria were quantified by viable counting. The potential involvement of reactive oxygen species in the antibacterial effect was assessed using the antioxidant N-acetylcysteine-amide. A modified device was then used to determine the effects of the individual wavelengths. Exposure of blood to the standard wavelength sequence caused small (c. 0.5 Log 10 CFU) but statistically significant reductions in viable counts for all three bacteria, which were prevented by the addition of N-acetylcysteine-amide. Bacterial inactivation did not occur in blood-free medium, but supplementation with haem restored the moderate bactericidal effect. In single-wavelength experiments, bacterial inactivation occurred only with red (630 nm) light. Concentrations of reactive oxygen species were significantly higher under light stimulation than in unstimulated controls. In summary, exposure of bacteria within blood to a cycle of visible light wavelengths resulted in small but statistically significant bacterial inactivation apparently mediated by a 630 nm wavelength only, via reactive oxygen species possibly generated by excitation of haem groups.

Antimicrobial mouthwashes (gargling) and nasal sprays administered to patients with suspected or confirmed COVID-19 infection to improve patient outcomes and to protect healthcare workers treating them.

BACKGROUND: COVID-19 infection poses a serious risk to patients and - due to its contagious nature - to those healthcare workers (HCWs) treating them. If the mouth and nose of patients with infection are irrigated with antimicrobial solutions, this may help the patients by killing any coronavirus present at those sites. It may also reduce the risk of the active infection being passed to HCWs through droplet transmission or direct contact. However, the use of such antimicrobial solutions may be associated with harms related to the toxicity of the solutions themselves or alterations in the natural microbial flora of the mouth or nose. OBJECTIVES: To assess the benefits and harms of antimicrobial mouthwashes and nasal sprays administered to patients with suspected or confirmed COVID-19 infection to both the patients and the HCWs caring for them. SEARCH METHODS: Information Specialists from Cochrane ENT and Cochrane Oral Health searched the Central Register of Controlled Trials (CENTRAL 2020, Issue 6); Ovid MEDLINE; Ovid Embase and additional sources for published and unpublished trials. The date of the search was 1 June 2020.  SELECTION CRITERIA: This is a question that urgently requires evidence, however at the present time we did not anticipate finding many completed RCTs. We therefore planned to include the following types of studies: randomised controlled trials (RCTs); quasi-RCTs; non-randomised controlled trials; prospective cohort studies; retrospective cohort studies; cross-sectional studies; controlled before-and-after studies. We set no minimum duration for the studies.   We sought studies comparing antimicrobial mouthwash and/or nasal spray (alone or in combination) at any concentration, delivered with any frequency or dosage to suspected/confirmed COVID-19 patients. DATA COLLECTION AND ANALYSIS: We used standard Cochrane methodological procedures. Our primary outcomes were: 1) RECOVERY* (www.recoverytrial.net) outcomes in patients (mortality; hospitalisation status; use of ventilation; use of renal dialysis or haemofiltration); 2) incidence of symptomatic or test-positive COVID-19 infection in HCWs; 3) significant adverse event: anosmia (or disturbance in sense of smell). Our secondary outcomes were: 4) change in COVID-19 viral load in patients; 5) COVID-19 viral content of aerosol (when present); 6) other adverse events: changes in microbiome in oral cavity, nasal cavity, oro- or nasopharynx; 7) other adverse events: allergy, irritation/burning of nasal, oral or oropharyngeal mucosa (e.g. erosions, ulcers, bleeding), long-term staining of mucous membranes or teeth, accidental ingestion. We planned to use GRADE to assess the certainty of the evidence for each outcome. MAIN RESULTS: We found no completed studies to include in this review. We identified 16 ongoing studies (including 14 RCTs), which aim to enrol nearly 1250 participants. The interventions included in these trials are ArtemiC (artemisinin, curcumin, frankincense and vitamin C), Citrox (a bioflavonoid), cetylpyridinium chloride, chlorhexidine, chlorine dioxide, essential oils, hydrogen peroxide, hypertonic saline, Kerecis spray (omega 3 viruxide - containing neem oil and St John's wort), neem extract, nitric oxide releasing solution, povidone iodine and saline with baby shampoo.  AUTHORS' CONCLUSIONS: We identified no studies for inclusion in this review. This is not surprising given the relatively recent emergence of COVID-19 infection. It is promising that the question posed in this review is being addressed by a number of RCTs and other studies. We are concerned that few of the ongoing studies specifically state that they will evaluate adverse events such as changes in the sense of smell or to the oral and nasal microbiota, and any consequences thereof. Very few interventions have large and dramatic effect sizes. If a positive treatment effect is demonstrated when studies are available for inclusion in this review, it may not be large. In these circumstances in particular it may be a challenge to weigh up the benefits against the harms if the latter are of uncertain frequency and severity.

Does the Oral Microbiome Play a Role in Hypertensive Pregnancies?

Chronic hypertension during gestation is associated with an increased risk of adverse pregnancy outcomes including pre-eclampsia, fetal growth restriction and preterm birth. Research into new chemotherapeutic regimes for the treatment of hypertension in pregnancy is limited due to concerns about fetal toxicity and teratogenicity, and new therapeutic avenues are being sought in alternative physiological pathways. Historically, generation of the vasodilator nitric oxide was believed to be solely from L-arginine by means of nitric oxide synthase enzymes. Recently, a novel pathway for the reduction of dietary inorganic nitrate to nitrite by the bacteria in the oral cavity and subsequently to vasodilatory nitric oxide within the body has been uncovered. Dietary nitrate is abundant in green leafy vegetables, including beetroot and spinach, and reduction of exogenous nitrate to nitrite by oral bacteria can increase nitric oxide in the vasculature, lessening hypertension. Supplements rich in nitrate may be an attractive choice for treatment due to fewer side effects than drugs that are currently used to treat hypertensive pregnancy disorders. Additionally, manipulation of the composition of the oral microbiota using pro- and prebiotics in tandem with additional dietary interventions to promote cardiovascular health during gestation may offer a safe and effective means of treating hypertensive pregnancy disorders including gestational hypertension and pre-eclampsia. The use of dietary inorganic nitrate as a supplement during pregnancy requires further exploration and large scale studies before it may be considered as part of a treatment regime. The aim of this article is to review the current evidence that oral microbiota plays a role in hypertensive pregnancies and whether it could be manipulated to improve patient outcomes.

Metal ions and graphene-based compounds as alternative treatment options for burn wounds infected by antibiotic-resistant Pseudomonas aeruginosa.

Burn infections caused by Pseudomonas aeruginosa pose a major complication in wound healing. This study aimed to determine the antimicrobial effect of metal ions, graphene (Gr), and graphene oxide (GO), individually and in combination, against the planktonic and biofilm states of two antimicrobially resistant clinical strains of P. aeruginosa each with different antibiotic resistance profiles. Minimum inhibitory, minimum bactericidal, and fractional inhibitory concentrations were performed to determine the efficacy of the metal ions and graphene composites individually and their synergy in combination. Crystal violet biofilm and XTT assays measured the biofilm inhibition and metabolic activity, respectively. Molybdenum, platinum, tin, gold, and palladium ions exhibited the greatest antimicrobial activity (MIC = 7.8-26.0 mg/L), whilst GO and Gr demonstrated moderate-to-no effect against the planktonic bacterial cells, irrespective of their antibiograms. Biofilms were inhibited by zinc, palladium, silver, and graphene. In combination, silver-graphene and molybdenum-graphene inhibited both the planktonic and biofilm forms of the bacteria making them potential candidates for development into topical antimicrobials for burns patients infected with antibiotic-resistant P. aeruginosa.

Fatty Acid Supplementation Reverses the Small Colony Variant Phenotype in Triclosan-Adapted Staphylococcus aureus: Genetic, Proteomic and Phenotypic Analyses.

Staphylococcus aureus can develop a small colony variant (SCV) phenotype in response to sub-lethal exposure to the biocide triclosan. In the current study, whole genome sequencing was performed and changes in virulence were investigated in five Staphylococcus aureus strains following repeated exposure to triclosan. Following exposure, 4/5 formed SCV and exhibited point mutations in the triclosan target gene fabI with 2/4 SCVs showing mutations in both fabI and fabD. The SCV phenotype was in all cases immediately reversed by nutritional supplementation with fatty acids or by repeated growth in the absence of triclosan, although fabI mutations persisted in 3/4 reverted SCVs. Virulence, determined using keratinocyte invasion and Galleria mellonella pathogenicity assays was significantly (p < 0.05) attenuated in 3/4 SCVs and in the non-SCV triclosan-adapted bacterium. Proteomic analysis revealed elevated FabI in 2/3 SCV and down-regulation in a protein associated with virulence in 1/3 SCV. In summary, attenuated keratinocyte invasion and larval virulence in triclosan-induced SCVs was associated with decreases in growth rate and virulence factor expression. Mutation occurred in fabI, which encodes the main triclosan target in all SCVs and the phenotype was reversed by fatty acid supplementation, demonstrating an association between fatty acid metabolism and triclosan-induced SCV.

Bacteriological effects of dentifrices with and without active ingredients of natural origin.

Compounds of natural origin are increasingly used as adjuncts to oral hygiene. We have adopted four distinct approaches to assess the antibacterial activity of dentifrices containing natural active ingredients against oral bacteria in several test systems. Corsodyl Daily (CD), Kingfisher Mint (KM), and Parodontax fluoride (PF) were compared to a dentifrice containing fluoride (Colgate Cavity Protection [CCP]) and one containing triclosan (Colgate Total [CT]). The growth inhibitory and bactericidal potency of the formulations were determined for 10 isolated oral bacteria. Effects of single exposures of simulated supragingival plaques were then determined by epifluorescence and confocal microscopy, while the effects of repeated exposures were quantified by viable counting. Additionally, dense plaques, maintained in continuous culture, were repeatedly dosed, and the outcome was assessed by viable counting and eubacterial DNA profiling. The test dentifrices exhibited variable specificity and potency against oral bacteria in axenic culture. Of the herbal formulations, KM caused the largest viability reductions in simulated supragingival plaques, with CT causing the greatest reductions overall. Following single exposures, CD caused moderate reductions, while PF had no effect. After multiple dosing, all formulations significantly reduced numbers of total, facultative, and Gram-negative anaerobes, but only KM and CT caused greater reductions than the fluoride control. KM also reduced counts of streptococci (rank order of effectiveness: CT > KM > CCP > PF > CD). Marked changes in eubacterial DNA profiles were not detected for any herbal formulation in dense plaques, although KM markedly reduced viable counts of streptococci, in agreement with supragingival data. While both nonherbal comparators displayed antibacterial activity, the triclosan-containing formulation caused greater viability reductions than the herbal and nonherbal formulations.

Plants as sources of new antimicrobials and resistance-modifying agents.

Infections caused by multidrug-resistant bacteria are an increasing problem due to the emergence and propagation of microbial drug resistance and the lack of development of new antimicrobials. Traditional methods of antibiotic discovery have failed to keep pace with the evolution of resistance. Therefore, new strategies to control bacterial infections are highly desirable. Plant secondary metabolites (phytochemicals) have already demonstrated their potential as antibacterials when used alone and as synergists or potentiators of other antibacterial agents. The use of phytochemical products and plant extracts as resistance-modifying agents (RMAs) represents an increasingly active research topic. Phytochemicals frequently act through different mechanisms than conventional antibiotics and could, therefore be of use in the treatment of resistant bacteria. The therapeutic utility of these products, however, remains to be clinically proven. The aim of this article is to review the advances in in vitro and in vivo studies on the potential chemotherapeutic value of phytochemical products and plant extracts as RMAs to restore the efficacy of antibiotics against resistant pathogenic bacteria. The mode of action of RMAs on the potentiation of antibiotics is also described.

An in vitro comparison of dentifrice formulations in three distinct oral microbiotas.

OBJECTIVES: In vitro biofilm models, representative of some aspects of nascent, supra-gingival plaques (Hydroxyapatite Disc Biofilm Models), developed supra-gingival plaques (Modified Drip-flow Biofilm Reactors) and sub-gingival plaques (Multiple Sorbarod Devices) were used to compare the antimicrobial effects of a triclosan-containing dentifrice with a stannous fluoride and zinc lactate combination. DESIGN: Triplicate salivary biofilm microcosms were maintained for 2d (hydroxyapatite discs), 5d (Sorbarods) or up to 6d (drip flow reactors). Dentifrice slurries (10%, w/v) were added once to the discs and repeatedly to the Drip Flow Reactors and Sorbarods. Plaques were analysed by differential culture and gravimetrically. RESULTS: Whilst both dentifrices were comparably effective at reducing viability and plaque accumulation in mature supragingival plaques, the triclosan dentifrice produced comparatively larger reductions in total streptococci and anaerobes in nascent plaques (p<0.05) and greater reductions in Gram-negative anaerobes and streptococci in subgingival plaques. CONCLUSIONS: We have used a multi-model approach to determine the effectiveness and specificity of dentifrices against compositionally distinct plaques. Whilst both formations reduced bacterial viability and plaque accumulation, their effects could be differentiated in nascent and deep plaques where the triclosan dentifrice caused larger viability reductions.