Antimicrobials: An update on new strategies to diversify treatment for bacterial infections.

Ninety-five years after Fleming's discovery of penicillin, a bounty of antibiotic compounds have been discovered, modified, or synthesised. Diversification of target sites, improved stability and altered activity spectra have enabled continued antibiotic efficacy, but overwhelming reliance and misuse has fuelled the global spread of antimicrobial resistance (AMR). An estimated 1.27 million deaths were attributable to antibiotic resistant bacteria in 2019, representing a major threat to modern medicine. Although antibiotics remain at the heart of strategies for treatment and control of bacterial diseases, the threat of AMR has reached catastrophic proportions urgently calling for fresh innovation. The last decade has been peppered with ground-breaking developments in genome sequencing, high throughput screening technologies and machine learning. These advances have opened new doors for bioprospecting for novel antimicrobials. They have also enabled more thorough exploration of complex and polymicrobial infections and interactions with the healthy microbiome. Using models of infection that more closely resemble the infection state in vivo, we are now beginning to measure the impacts of antimicrobial therapy on host/microbiota/pathogen interactions. However new approaches are needed for developing and standardising appropriate methods to measure efficacy of novel antimicrobial combinations in these contexts. A battery of promising new antimicrobials is now in various stages of development including co-administered inhibitors, phages, nanoparticles, immunotherapy, anti-biofilm and anti-virulence agents. These novel therapeutics need multidisciplinary collaboration and new ways of thinking to bring them into large scale clinical use.

The Efficacy and Adverse Events of Delafloxacin for Treating Acute Bacterial Skin and Skin Structure Infections: A Systematic Review and Meta-Analysis.

BACKGROUND: Acute bacterial skin and skin structure infections (ABSSSI) cause significant morbidity and mortality in hospitalized patients and outpatients as well. Newer fluoroquinolones such as delafloxacin might be a useful medication for treating infections of skin caused by gram-positive bacterial species that are resistant. AIMS AND OBJECTIVES: The aim of this study was to evaluate all the literature on delafloxacin in databases and make comparisons of its efficacy with antimicrobial drugs routinely used to treat skin infections. MATERIALS AND METHODS: A detailed search on different databases was conducted using, Cochrane Central Register of Controlled Trials, PubMed, and Embase. Primary outcome was microbiological cure at the end of the follow-up period. Absence of the signs and symptoms at the termination of the follow-up period and clinical response to medications was regarded as the secondary outcome. RESULTS: The pooled efficacy of delafloxacin was at 80% (95% confidence interval 1.01 [0.97, 1.06]; P = 0.51). No statistically significant difference was found between intravenous delafloxacin and comparator drugs. CONCLUSION: The effectiveness of delafloxacin was found to be non-inferior to tigecycline and linezolid. Efficacy and pooled cure rate of delafloxacin was also found to be superior to vancomycin.

A Myristoyl Amide Derivative of Doxycycline Potently Targets Cancer Stem Cells (CSCs) and Prevents Spontaneous Metastasis, Without Retaining Antibiotic Activity.

Here, we describe the chemical synthesis and biological activity of a new Doxycycline derivative, designed specifically to more effectively target cancer stem cells (CSCs). In this analog, a myristic acid (14 carbon) moiety is covalently attached to the free amino group of 9-amino-Doxycycline. First, we determined the IC50 of Doxy-Myr using the 3D-mammosphere assay, to assess its ability to inhibit the anchorage-independent growth of breast CSCs, using MCF7 cells as a model system. Our results indicate that Doxy-Myr is >5-fold more potent than Doxycycline, as it appears to be better retained in cells, within a peri-nuclear membranous compartment. Moreover, Doxy-Myr did not affect the viability of the total MCF7 cancer cell population or normal fibroblasts grown as 2D-monolayers, showing remarkable selectivity for CSCs. Using both gram-negative and gram-positive bacterial strains, we also demonstrated that Doxy-Myr did not show antibiotic activity, against Escherichia coli and Staphylococcus aureus. Interestingly, other complementary Doxycycline amide derivatives, with longer (16 carbon; palmitic acid) or shorter (12 carbon; lauric acid) fatty acid chain lengths, were both less potent than Doxy-Myr for the targeting of CSCs. Finally, using MDA-MB-231 cells, we also demonstrate that Doxy-Myr has no appreciable effect on tumor growth, but potently inhibits tumor cell metastasis in vivo, with little or no toxicity. In summary, by using 9-amino-Doxycycline as a scaffold, here we have designed new chemical entities for their further development as anti-cancer agents. These compounds selectively target CSCs, e.g., Doxy-Myr, while effectively minimizing the risk of driving antibiotic resistance. Taken together, our current studies provide proof-of-principle, that existing FDA-approved drugs can be further modified and optimized, to successfully target the anchorage-independent growth of CSCs and to prevent the process of spontaneous tumor cell metastasis.

Visualization and Quantification of the Oral Hygiene Effects of Brushing, Dentifrice Use, and Brush Wear Using a Tooth Brushing Simulator.

Approaches that reproduce dental hygiene regimens under controlled conditions have applications in preclinical research. We have applied standardized, reproducible brushing regimes to typodonts coated in simulated or biological plaques to assess the effects on tooth cleaning of toothbrush/dentifrice regimens. Replicated typodonts were coated with OccludeTM or GlogermTM indicators to simulate plaque, and brushed reproducibly using a mechanical brushing simulator to compare the cleaning of occlusal surfaces before and after brushing with water or a dentifrice. An in vitro model using salivary inocula to cultivate oral biofilms on typodont surfaces was then developed to evaluate removal of disclosed plaque by new toothbrushes in comparison to toothbrushes with wear equivalent to 3 months of use. Analyses of typodonts brushed under controlled conditions significantly (p < 0.01) distinguished between brushed and unbrushed surfaces and between the use of water vs. dentifrice for the removal of simulated interproximal plaque (p < 0.05). New toothbrushes removed significantly (p < 0.05) more biological plaque from typodont surfaces than brushes that had been worn by repeated brushing. Through controlled and defined brushing of typodonts with simulated and biological plaques, the effectiveness of dental hygiene regimens was compared under reproducible conditions. Data indicate that the cleaning effectiveness of brushing was augmented by the addition of dentifrice and that new brushes were significantly more effective than brushes with substantial wear from previous use. Whilst we have focussed on the occlusal surfaces of molars and worn brushes, the method could be applied to a range of other tooth surfaces and oral hygiene regimens.

Simultaneous Assessment of Acidogenesis-Mitigation and Specific Bacterial Growth-Inhibition by Dentifrices.

Dentifrices can augment oral hygiene by inactivating bacteria and at sub-lethal concentrations may affect bacterial metabolism, potentially inhibiting acidogenesis, the main cause of caries. Reported herein is the development of a rapid method to simultaneously measure group-specific bactericidal and acidogenesis-mitigation effects of dentifrices on oral bacteria. Saliva was incubated aerobically and anaerobically in Tryptone Soya Broth, Wilkins-Chalgren Broth with mucin, or artificial saliva and was exposed to dentifrices containing triclosan/copolymer (TD); sodium fluoride (FD); stannous fluoride and zinc lactate (SFD1); or stannous fluoride, zinc lactate and stannous chloride (SFD2). Minimum inhibitory concentrations (MIC) were determined turbidometrically whilst group-specific minimum bactericidal concentrations (MBC) were assessed using growth media and conditions selective for total aerobes, total anaerobes, streptococci and Gram-negative anaerobes. Minimum acid neutralization concentration (MNC) was defined as the lowest concentration of dentifrice at which acidification was inhibited. Differences between MIC and MNC were calculated and normalized with respect to MIC to derive the combined inhibitory and neutralizing capacity (CINC), a cumulative measure of acidogenesis-mitigation and growth inhibition. The overall rank order for growth inhibition potency (MIC) under aerobic and anaerobic conditions was: TD> SFD2> SFD1> FD. Acidogenesis-mitigation (MNC) was ordered; TD> FD> SFD2> SFD1. CINC was ordered TD> FD> SFD2> SFD1 aerobically and TD> FD> SFD1> SFD2 anaerobically. With respect to group-specific bactericidal activity, TD generally exhibited the greatest potency, particularly against total aerobes, total anaerobes and streptococci. This approach enables the rapid simultaneous evaluation of acidity mitigation, growth inhibition and specific antimicrobial activity by dentifrices.

Antibacterial efficacy of a cetylpyridinium chloride-based mouthrinse against Fusobacterium nucleatum and in vitro plaques.

PURPOSE: To assess the antimicrobial effects of a fluoride-free and alcohol-free mouthrinse containing 0.075% CPC (test rinse, TR) compared with an otherwise-identical CPC-free control rinse (CR). METHODS: Activity against laboratory cultures of Fusobacterium nucleatum, a bacterium associated with gingival disease, was determined using viable counting following 30-second exposures to TR and CR. Effects against intact saliva-derived plaque biofilms were quantified using confocal microscopy coupled with three-dimensional image analyses (viability profiling). RESULTS: Short exposures to TR caused significant inactivation of F. nucleatum, as determined by viable counting (c. 3 log reduction compared to the control rinse, P < 0.05). Confocal microscopy revealed extensive inactivation of complex oral biofilms following treatment with TR; biofilms were significantly less viable than those exposed to CR and three-dimensional images revealed extensive zones of dead bacteria even within plaque depths. In conclusion, this investigation demonstrates that the CPC-containing mouthrinse has significant antibacterial efficacy against oral bacteria associated with gingival disease and significantly inactivated plaque biofilm in comparison to a relevant control.

Antibacterial and anti-biofilm activity of mouthrinses containing cetylpyridinium chloride and sodium fluoride.

BACKGROUND: Cetylpyridinium chloride (CPC) and sodium fluoride augment oral hygiene by inactivating bacteria and inhibiting enamel demineralisation, respectively. However, there are few reports in the literature documenting the antibacterial efficacy of their combined use in mouthrinses. We have used six experimental systems to compare the antibacterial effects of mouthrinses containing 0.075% CPC (test rinse, TR) or 0.075% CPC with sodium fluoride (test fluoride rinse, TFR). RESULTS: Effects against planktonic bacteria were determined using viable counting (for Streptococcus mutans and salivary bacteria), a redox dye (for Actinomyces viscosus and salivary bacteria) and viable counting (for ex vivo oral rinses). Effects against saliva-derived biofilms were quantified using confocal microscopy and differential viable counting. Inhibition of biofilm formation was evaluated by pre-treating hydroxyapatite coupons with mouthrinses prior to inoculation. Otherwise-identical controls without CPC (control rinse and control fluoride rinse, CR and CFR, respectively), were included throughout. Compared to the controls, TFR and TR demonstrated significant antimicrobial effects in the redox assays, by viable counts (>3 log reductions) and in oral rinse samples (>1.25 log reductions, p < 0.05). TFR and TR also significantly reduced the viability of oral biofilms. Pre-treatment of hydroxyapatite with TFR and TR significantly inhibited biofilm formation (>3 log difference, p < 0.05). Overall, there were no consistent differences in the activities of TR and TFR. CONCLUSIONS: Sodium fluoride did not influence the antibacterial and anti-biofilm potency of CPC-containing formulations, supporting the combined use of CPC and sodium fluoride in mouthrinses to control oral bacteria and protect tooth enamel.

Altered Competitive Fitness, Antimicrobial Susceptibility, and Cellular Morphology in a Triclosan-Induced Small-Colony Variant of Staphylococcus aureus.

Staphylococcus aureus can produce small-colony variants (SCVs) that express various phenotypes. While their significance is unclear, SCV propagation may be influenced by relative fitness, antimicrobial susceptibility, and the underlying mechanism. We have investigated triclosan-induced generation of SCVs in six S. aureus strains, including methicillin-resistant S. aureus (MRSA). Parent strains (P0) were repeatedly passaged on concentration gradients of triclosan using a solid-state exposure system to generate P10. P10 was subsequently passaged without triclosan to generate X10. Susceptibility to triclosan and 7 antibiotics was assessed at all stages. For S. aureus ATCC 6538, SCVs were further characterized by determining microbicide susceptibility and competitive fitness. Cellular morphology was examined using electron microscopy, and protein expression was evaluated through proteomics. Triclosan susceptibility in all SCVs (which could be generated from 4/6 strains) was markedly decreased, while antibiotic susceptibility was significantly increased in the majority of cases. An SCV of S. aureus ATCC 6538 exhibited significantly increased susceptibility to all tested microbicides. Cross-wall formation was impaired in this bacterium, while expression of FabI, a target of triclosan, and IsaA, a lytic transglycosylase involved in cell division, was increased. The P10 SCV was 49% less fit than P0. In summary, triclosan exposure of S. aureus produced SCVs in 4/6 test bacteria, with decreased triclosan susceptibility but with generally increased antibiotic susceptibility. An SCV derived from S. aureus ATCC 6538 showed reduced competitive fitness, potentially due to impaired cell division. In this SCV, increased FabI expression could account for reduced triclosan susceptibility, while IsaA may be upregulated in response to cell division defects.

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.

Fluorescent probe for detection of bacteria: conformational trigger upon bacterial reduction of an azo bridge.

Using biomimetic chemical reduction or Clostridium perfringens cell extract containing azoreductase, the dimer-fluorescent probe 2,4-O-bisdansyl-6,7-diazabicyclooct-6-ene, which possesses a conformationally constrained cis-azo bridge, is reduced to the tetra-equatorial 2,4-O-bisdansyl-cyclohexyl-3,5-bisammonium salt which exhibits fluorescence indicative of a dansyl monomer.

Attenuated virulence and biofilm formation in Staphylococcus aureus following sublethal exposure to triclosan.

Subeffective exposure of Staphylococcus aureus to the biocide triclosan can reportedly induce a small-colony variant (SCV) phenotype. S. aureus SCVs are characterized by low growth rates, reduced pigmentation, and lowered antimicrobial susceptibility. While they may exhibit enhanced intracellular survival, there are conflicting reports regarding their pathogenicity. The current study reports the characteristics of an SCV-like strain of S. aureus created by repeated passage on sublethal triclosan concentrations. S. aureus ATCC 6538 (the passage 0 [P0] strain) was serially exposed 10 times to concentration gradients of triclosan to generate strain P10. This strain was then further passaged 10 times on triclosan-free medium (designated strain ×10). The MICs and minimum bactericidal concentrations of triclosan for P0, P10, and ×10 were determined, and growth rates in biofilm and planktonic cultures were measured. Hemolysin, DNase, and coagulase activities were measured, and virulence was determined using a Galleria mellonella pathogenicity model. Strain P10 exhibited decreased susceptibility to triclosan and characteristics of an SCV phenotype, including a considerably reduced growth rate and the formation of pinpoint colonies. However, this strain also had delayed coagulase production, had impaired hemolysis (P < 0.01), was defective in biofilm formation and DNase activity, and displayed significantly attenuated virulence. Colony size, hemolysis, coagulase activity, and virulence were only partially restored in strain ×10, whereas the planktonic growth rate was fully restored. However, ×10 was at least as defective in biofilm formation and DNase production as P10. These data suggest that although repeated exposure to triclosan may result in an SCV-like phenotype, this is not necessarily associated with increased virulence and adapted bacteria may exhibit other functional deficiencies.

A novel method for exploring elemental composition of microbial communities: laser ablation-inductively coupled plasma-mass spectrometry of intact bacterial colonies.

Bacterial colonies are spatially complex structures whose physiology is profoundly dependent on interactions between cells and with the underlying semi-solid substratum. Here, we use bacterial colonies as a model of a microbial community to evaluate the potential of laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) to delineate elemental distributions within colonies with minimal pre-treatment. To reduce water content of the colony and limit undesirable absorption of laser energy, we compared methods of preparing 24h-old colonies of Escherichia coli TG1 on agar for laser ablation. Colonies on excised agar segments dried on chromatography paper were superior to colonies dried in a dessicator or by prolonged incubation, with respect to signal magnitude, signal:noise ratio and background signal. Having optimised laser scan speed (10 microm s(-1)) and laser beam diameter (100 microm), further improvements were achieved by growing colonies on nylon membranes over agar, which were then transferred to the ablation chamber without further treatment. Repeated line rasters across individual membrane-supported colonies yielded three-dimensional elemental maps of colonies, revealing a convex morphology consistent with visual inspection. By normalising isotope counts for P, Mn, Zn, Fe and Ca against Mg, the most abundant cellular divalent cation, we sought elemental heterogeneity within the colony. The normalised concentration of Mn in the perimeter was higher than in the colony interior, whereas the converse was true for Ca. LA-ICP-MS is a novel and powerful method for probing elemental composition and organisation within microbial communities and should find numerous applications in, for example, biofilm studies.