Building a Better Quaternary Ammonium Compound (QAC): Branched Tetracationic Antiseptic Amphiphiles

Bacteria contaminate surfaces in a wide variety of environments, causing severe problems across a number of industries. In a continuation of our campaign to develop novel antibacterial quaternary ammonium compounds (QACs) as useful antiseptics, we have identified a starting material bearing four tertiary amines, enabling the rapid synthesis of several tris‐ and tetracationic QACs. Herein we report the synthesis and biological activity of a series of 24 multiQACs deemed the “superT” family, and an investigation of the role of cationic charge in antimicrobial and anti‐biofilm activity, as well as toxicity. This class represents the most potent series of QACs reported to date against methicillin‐resistant Staphylococcus aureus (MRSA), with minimum inhibitory concentrations (MICs) and minimum biofilm eradication concentrations (MBECs) as low as 0.25 and 25 μm , respectively. Based on the significant cell‐surface‐charge differences between bacterial and eukaryotic cells, in certain cases we observed excellent efficacy‐to‐toxicity profiles, exceeding a 100‐fold differential. This work further elucidates the chemical underpinnings of disinfectant efficacy versus toxicity based on cationic charge.     

Scaffold‐Hopping of Multicationic Amphiphiles Yields Three New Classes of Antimicrobials

Quaternary ammonium compounds (QACs) are a vital class of antiseptics. Recent investigations into their construction are uncovering novel and potent multicationic variants. Based on a trisQAC precedent, we have implemented a scaffold‐hopping approach to develop alternative QAC architectures that display 1–3 long alkyl chains in specific projections from cyclic and branched core structures bearing 3–4 nitrogen atoms. The preparation of 30 QAC structures allowed for correlation of scaffold structure with antimicrobial activity. We identified QACs with limited conformational flexibility that have improved bioactivity against planktonic bacteria as compared to their linear counterparts. We also confirmed that resistance, as evidenced by an increased minimum inhibitory concentration (MIC) for methicillin‐resistant Staphylococcus aureus (MRSA) compared to methicillin‐susceptible Staphylococcus aureus (MSSA), can reduce efficacy up to 64‐fold for monocationic QACs. Differentiation of antimicrobial and anti‐biofilm activity, however, was not observed, suggesting that these compounds utilize a non‐specific mode of eradication.     

Identification of N‐Arylated NH125 Analogues as Rapid Eradicating Agents against MRSA Persister Cells and Potent Biofilm Killers of Gram‐Positive Pathogens

Bacterial biofilms housing dormant persister cells are innately tolerant to antibiotics and disinfectants, yet several membrane‐active agents are known to eradicate tolerant bacterial cells. NH125, a membrane‐active persister killer and starting point for development, led to the identification of two N‐arylated analogues ( 1 and 2 ) that displayed improved biofilm eradication potencies compared to the parent compound and rapid persister‐cell‐killing activities in stationary cultures of methicillin‐resistant Staphylococcus aureus (MRSA). We found 1 and 2 to be superior to other membrane‐active agents in biofilm eradication assays, with 1 demonstrating minimum biofilm eradication concentrations (MBEC) of 23.5, 11.7, and 2.35 μm against MRSA, methicillin‐resistant Staphylococcus epidermidis (MRSE), and vancomycin‐resistant Enterococcus faecium (VRE) biofilms, respectively. We tested our panel of membrane‐active agents against MRSA stationary cultures and found 1 to rapidly eradicate MRSA stationary cells by 4 log units (99.99 %) in 30 min. The potent biofilm eradication and rapid persister‐cell‐killing activities exhibited by N‐arylated NH125 analogues could have significant impact in addressing biofilm‐associated problems.     

Treatment of ampicillin‐loaded wastewater by combined adsorption and biodegradation

BACKGROUND: This study investigated the treatment of ampicillin (AMP)‐loaded wastewater in airlift reactors where biofilms were developed on granular activated carbon (GAC). A series of batch experiments were thus carried out in order to differentiate potentials of adsorption and biodegradation which would jointly contribute to the AMP removal. RESULTS: Results showed that almost all influent AMP was removed in two reactors supplemented with 4 and 8 mg L^?1 AMP, respectively. Batch experiments revealed that the percentage of the AMP removed through biodegradation increased along with the development of biofilms on GAC. For the mature biofilm‐covered GAC, adsorption accounted for about 60% of the observed AMP removal, whereas the other 40% could be attributed to biodegradation. Possible degraders of AMP were also identified, such as Acinetobacter sp., Flavobacterium sp., Pseudoxanthomonas sp., Delftia sp. and Sphingobium sp. CONCLUSION: The airlift biofilm reactor with GAC as carrier would be a feasible technology for treating AMP‐loaded wastewater due to the joint action of adsorption and biodegradation of AMP by the biofilm‐covered GAC. Copyright © 2010 Society of Chemical Industry     

Lipidation of Temporin-1CEb Derivatives as a Tool for Activity Improvement,Pros and Cons of the Approach

The alarming raise of multi-drug resistance among human microbial pathogens makes the development of novel therapeutics a priority task. In contrast to conventional antibiotics, antimicrobial peptides (AMPs), besides evoking a broad spectrum of activity against microorganisms, could offer additional benefits, such as the ability to neutralize toxins, modulate inflammatory response, eradicate bacterial and fungal biofilms or prevent their development. The latter properties are of special interest, as most antibiotics available on the market have limited ability to diffuse through rigid structures of biofilms. Lipidation of AMPs is considered as an effective approach for enhancement of their antimicrobial potential and in vivo stability; however, it could also have undesired impact on selectivity, solubility or the aggregation state of the modified peptides. In the present work, we describe the results of structural modifications of compounds designed based on cationic antimicrobial peptides DK5 and CAR-PEG-DK5, derivatized at their N-terminal part with fatty acids with different lengths of carbon chain. The proposed modifications substantially improved antimicrobial properties of the final compounds and their effectiveness in inhibition of biofilm development as well as eradication of pre-formed 24 h old biofilms of Candida albicans and Staphylococcus aureus. The most active compounds (C5-DK5, C12-DK5 and C12-CAR-PEG-DK5) were also potent against multi-drug resistant Staphylococcus aureus USA300 strain and clinical isolates of Pseudomonas aeruginosa. Both experimental and in silico methods revealed strong correlation between the length of fatty acid attached to the peptides and their final membranolytic properties, tendency to self-assemble and cytotoxicity.     

Photodynamic Inactivation of Legionella pneumophila Biofilm Formation by Cationic Tetra- and Tripyridylporphyrins in Waters of Different Hardness

The bacterium Legionella pneumophila is still one of the probable causes of waterborne diseases, causing serious respiratory illnesses. In the aquatic systems, L. pneumophila exists inside free-living amoebae or can form biofilms. Currently developed disinfection methods are not sufficient for complete eradication of L. pneumophila biofilms in water systems of interest. Photodynamic inactivation (PDI) is a method that results in an antimicrobial effect by using a combination of light and a photosensitizer (PS). In this work, the effect of PDI in waters of natural origin and of different hardness, as a treatment against L. pneumophila biofilm, was investigated. Three cationic tripyridylporphyrins, which were previously described as efficient agents against L. pneumophila alone, were used as PSs. We studied how differences in water hardness affect the PSs’ stability, the production of singlet oxygen, and the PDI activity on L. pneumophila adhesion and biofilm formation and in biofilm destruction. Amphiphilic porphyrin showed a stronger tendency for aggregation in hard and soft water, but its production of singlet oxygen was higher in comparison to tri- and tetracationic hydrophilic porphyrins that were stable in all water samples. All three studied porphyrins were shown to be effective as PDI agents against the adhesion of the L. pneumophila to polystyrene, against biofilm formation, and in the destruction of the formed biofilm, in their micromolar concentrations. However, a higher number of dissolved ions, i.e., water hardness, generally reduced somewhat the PDI activity of all the porphyrins at all tested biofilm growth stages.     

Discovery and Biological Characterization of the Auromomycin Chromophore as an Inhibitor of Biofilm Formation in Vibrio cholerae

Bacterial biofilms pose a significant challenge in clinical environments due to their inherent lack of susceptibility to antibiotic treatment. It is widely recognized that most pathogenic bacterial strains in the clinical setting persist in the biofilm state, and are the root cause of many recrudescent infections. The discovery and development of compounds capable of either inhibiting biofilm formation or initiating biofilm dispersal might provide new therapeutic avenues for reducing the number of hospital‐acquired, biofilm‐mediated infections. We detail here the application of our recently reported image‐based, high‐throughput screen to the discovery of microbially derived natural products with inhibitory activity against Vibrio cholerae biofilm. Examination of a prefractionated library of microbially derived marine natural products has led to the identification of a new biofilm inhibitor that is structurally unrelated to previously reported inhibitors and is one of the most potent inhibitors of V. cholerae reported to date. Combination of this compound with sub‐MIC concentrations of a number of clinically relevant antibiotics was shown to improve the inhibitory efficacy of this new compound compared to monotherapy treatments, and provides evidence for the potential therapeutic benefit of biofilm inhibitors in treating persistent biofilm‐mediated infections.     

Peptoid Efficacy against Polymicrobial Biofilms Determined by Using Propidium Monoazide‐Modified Quantitative PCR

Biofilms containing Candida albicans are responsible for a wide variety of clinical infections. The protective effects of the biofilm matrix, the low metabolic activity of microorganisms within a biofilm and their high mutation rate, significantly enhance the resistance of biofilms to conventional antimicrobial treatments. Peptoids are peptide‐mimics that share many features of host defence antimicrobial peptides but have increased resistance to proteases and therefore have better stability in vivo. The activity of a library of peptoids was tested against monospecies and polymicrobial bacterial/fungal biofilms. Selected peptoids showed significant bactericidal and fungicidal activity against the polymicrobial biofilms. This coupled with low cytotoxicity suggests that peptoids could offer a new option for the treatment of clinically relevant polymicrobial infections.     

Efflux Pumps Might Not Be the Major Drivers of QAC Resistance in Methicillin‐Resistant Staphylococcus aureus

Quaternary ammonium compounds (QACs) are commonly used antiseptics that are now known to be subject to bacterial resistance. The prevalence and mechanisms of such resistance, however, remain underexplored. We investigated a variety of QACs, including those with multicationic structures (multiQACs), and the resistance displayed by a variety of Staphylococcus aureus strains with and without genes encoding efflux pumps, the purported main driver of bacterial resistance in MRSA. Through minimum inhibitory concentration (MIC)‐, kinetic‐, and efflux‐based assays, we found that neither the qacR/qacA system present in S. aureus nor another efflux pump system is the main reason for bacterial resistance to QACs. Our findings suggest that membrane composition could be the predominant driver that allows CA‐MRSA to withstand the assault of conventional QAC antiseptics.     

Combined application of 13C NMR spectroscopy and confocal laser scanning microscopy—Investigation on biofilm structure and physico-chemical properties

Long-term biofilm processes are influenced by the interplay of biofilm accumulation and detachment, which in turn depend partially on the biofilm structure and composition. In this study a combination of confocal laser scanning microscopy (CLSM) and nuclear magnetic resonance (NMR) spectroscopy was applied to analyze biofilm structure, composition and molecular mobility. Whereas CLSM delivers information about the structure of biofilms the NMR measurement provides detailed but not locally resolved information about the chemical composition of biofilm constituents. Heterotrophic mixed-species biofilms were cultivated in rotating annular reactors exposed to different flow conditions and glucose concentrations in order to obtain biofilms with diverse architectural structures. The growth state of the biofilms appeared to influence the composition of biofilm and detached biomass. The difference in the ¹³C NMR spectra between the differently structured biofilms or between biofilm and detached biomass was small, except for the still exponential growing biofilm supplied with the highest glucose concentration. More information was gained from the mobility of specific molecular groups within the biofilm biomass. Molecules within the biofilm biomass of the non-filamentous biofilms were more strongly bound than the molecules within the respective detached biomass. Glucose starvation resulted in a reduction in the biofilm molecular mobility. The opposite was observed in the filamentous biofilm. In this case, the molecular mobility in the biofilm increased after starvation and the molecules in the detached biomass were bound more strongly than in the respective biofilm biomass. It could be shown that the combination of CLSM and ¹³C NMR spectroscopy is a promising approach to analyze the interactions between biofilm architecture, composition or growth state and biofilm detachment.     

Metallocene QACs: The Incorporation of Ferrocene Moieties into monoQAC and bisQAC Structures

Inspired by the incorporation of metallocene functionalities into a variety of bioactive structures, particularly antimicrobial peptides, we endeavored to broaden the structural variety of quaternary ammonium compounds (QACs) by the incorporation of the ferrocene moiety. Accordingly, 23 ferrocene-containing mono- and bisQACs were prepared in high yields and tested for activity against a variety of bacteria, including Gram-negative strains and a panel of clinically isolated MRSA strains. Ferrocene QACs were shown to be effective antiseptics with some displaying single-digit micromolar activity against all bacteria tested, demonstrating yet another step in the expansion of structural variety of antiseptic QACs.     

Listeria monocytogenes repellence by enzymatically modified PES surfaces

The effect of enzyme‐catalyzed modification of poly(ethersulfone) (PES) on the adhesion and biofilm formation of two Listeria monocytogenes strains is evaluated under static and dynamic flow conditions. PES has been modified with gallic acid, ferulic acid and 4‐hydroxybenzoic acid. The surfaces modified with any of these compounds show up to 70% reduced adhesion of L. monocytogenes under static conditions and up to 95% under dynamic flow conditions compared with unmodified surfaces. Also, under static conditions the formation of biofilms is reduced by ～70%. These results indicate that the brush structures that are formed by the polymers on the PES surface directly influence the ability of microorganisms to interact with the surface, thereby reducing attachment and biofilm formation of L. monocytogenes. Based on these results, it is expected that enzyme‐catalyzed surface modification is a promising tool to reduce microbial adhesion and biofilm formation. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41576.     

Photoinactivation of Pseudomonas aeruginosa Biofilm by Dicationic Diaryl-Porphyrin

In recent years, antimicrobial photodynamic therapy (aPDT) has received increasing attention as a promising tool aimed at both treating microbial infections and sanitizing environments. Since biofilm formation on biological and inert surfaces makes difficult the eradication of bacterial communities, further studies are needed to investigate such tricky issue. In this work, a panel of 13 diaryl-porphyrins (neutral, mono- and di-cationic) was taken in consideration to photoinactivate Pseudomonas aeruginosa. Among cationic photosensitizers (PSs) able to efficiently bind cells, in this study two dicationic showed to be intrinsically toxic and were ruled out by further investigations. In particular, the dicationic porphyrin (P11) that was not toxic, showed a better photoinactivation rate than monocationic in suspended cells. Furthermore, it was very efficient in inhibiting the biofilms produced by the model microorganism Pseudomonas aeruginosa PAO1 and by clinical strains derived from urinary tract infection and cystic fibrosis patients. Since P. aeruginosa represents a target very difficult to inactivate, this study confirms the potential of dicationic diaryl-porphyrins as photo-activated antimicrobials in different applicative fields, from clinical to environmental ones.     

(+)-Dehydroabietic Acid,an Abietane-Type Diterpene,Inhibits Staphylococcus aureus Biofilms in Vitro

Potent drugs are desperately needed to counteract bacterial biofilm infections, especially those caused by gram-positive organisms, such as Staphylococcus aureus. Moreover, anti-biofilm compounds/agents that can be used as chemical tools are also needed for basic in vitro or in vivo studies aimed at exploring biofilms behavior and functionability. In this contribution, a collection of naturally-occurring abietane-type diterpenes and their derivatives was tested against S. aureus biofilms using a platform consisting of two phenotypic assays that have been previously published by our group. Three active compounds were identified: nordehydroabietylamine (1), (+)-dehydroabietic acid (2) and (+)-dehydroabietylamine (3) that prevented biofilm formation in the low micromolar range, and unlike typical antibiotics, only 2 to 4-fold higher concentrations were needed to significantly reduce viability and biomass of existing biofilms. Compound 2, (+)-dehydroabietic acid, was the most selective towards biofilm bacteria, achieving high killing efficacy (based on log Reduction values) and it was best tolerated by three different mammalian cell lines. Since (+)-dehydroabietic acid is an easily available compound, it holds great potential to be used as a molecular probe in biofilms-related studies as well as to serve as inspirational chemical model for the development of potent drug candidates.     

Mitochondria‐Penetrating Peptides: Sequence Effects and Model Cargo Transport

A class of mitochondria‐penetrating peptides (MPPs) was studied in an effort to optimize their applications in the delivery of bioactive cargo to this therapeutically important organelle. The sequence requirements for mitochondrial entry were monitored, and it was discovered that while an alternating cationic/hydrophobic residue motif is not required, the inclusion of a stretch of adjacent cationic amino acids can impede access to the organelle. In addition, a variety of N‐ and C‐terminal cargo were tested to determine if there are limitations to the lipophilicity, charge, or polarity of compounds that can be transported to mitochondria by MPPs. The results reported demonstrate that these peptide sequences are versatile transporters that will have a range of biological applications.     

Synthesis and Antibacterial Activity of Novel Amido-Amine-Based Cationic Gemini Surfactants

A series of novel cationic gemini surfactants were synthesized from corresponding amido-amines in a single step reaction. The amido-amines were obtained from long chain carboxylic acids and 3-N,N-dimethylamino-1-propyl-amine with excellent isolated yield (up to 95 %). All the synthesized quaternary ammonium compounds (QACs) were further investigated for surface active properties. The critical micelle concentration (CMC) and the effectiveness of surface tension reduction were determined. The surface tension measurements of newly synthesized gemini surfactants showed good water solubility, and low CMC values, had great efficiency in lowering the surface tension and a strong adsorption at the air/water interface than the corresponding monomeric surfactants. Further, the antibacterial activity of the synthesized QACs against both Gram-positive and Gram-negative bacteria was also investigated.     

Electrochemical insight into the mechanism of electron transport in biofilms of Geobacter sulfurreducens

Electroactive bacterial biofilms can be produced on a polarized electrode by forcing its use as the final electron acceptor for bacterial respiration. This strategy offers the researcher the unique possibility to control the respiration process with extreme precision. The production of current, the accumulation of charge and the conducting properties of electroactive biofilms has been interrogated in this work through very basic electrochemical techniques including chronopotentiometry, chronoamperometry and cyclic voltammetry. Presented results indicate that charge can be accumulated in the biofilm conductive network, that network conductivity does not represent a limit for current production and that both the steady state current and the amount of accumulated charge depend on the redox state of cytochromes wiring the cells to the electrode. A model of biofilm conduction is presented as well.     

Dental Chatter: Bacterial Cross-Talk in the Biofilm of the Oral Cavity

Dental biofilms are composed of hundreds of bacterial species. These biofilms are diverse biological structures due to the heterogeneity of the many different types of supports in the oral cavity. The bacteria immobilized in these biofilms are exposed to rapid environmental changes such as pH, temperature, nutrition and anti-plaque agents. One mode in which these bacteria adapt in the dental biofilm is by quorum sensing. This cell-cell communication regulates diverse sets of adhesion modes, physiological changes, virulence properties, allowing the bacteria to persist in the dental biofilm under rapid environmental changes. In this review, we will concentrate mostly on the cariogenic bacterium Streptococcus mutans as one of the pivotal microorganisms in the supra-gingival biofilm that plays a major role in dental caries.     

Cationic Antimicrobial Polymers and Their Assemblies

Cationic compounds are promising candidates for development of antimicrobial agents. Positive charges attached to surfaces, particles, polymers, peptides or bilayers have been used as antimicrobial agents by themselves or in sophisticated formulations. The main positively charged moieties in these natural or synthetic structures are quaternary ammonium groups, resulting in quaternary ammonium compounds (QACs). The advantage of amphiphilic cationic polymers when compared to small amphiphilic molecules is their enhanced microbicidal activity. Besides, many of these polymeric structures also show low toxicity to human cells; a major requirement for biomedical applications. Determination of the specific elements in polymers, which affect their antimicrobial activity, has been previously difficult due to broad molecular weight distributions and random sequences characteristic of radical polymerization. With the advances in polymerization control, selection of well defined polymers and structures are allowing greater insight into their structure-antimicrobial activity relationship. On the other hand, antimicrobial polymers grafted or self-assembled to inert or non inert vehicles can yield hybrid antimicrobial nanostructures or films, which can act as antimicrobials by themselves or deliver bioactive molecules for a variety of applications, such as wound dressing, photodynamic antimicrobial therapy, food packing and preservation and antifouling applications.     

Advancements in the Development of Non-Nitrogen-Based Amphiphilic Antiseptics to Overcome Pathogenic Bacterial Resistance

The prevalence of quaternary ammonium compounds (QACs) as common disinfecting agents for the past century has led bacteria to develop resistance to such compounds. Given the alarming increase in resistant strains, new strategies are required to combat this rise in resistance. Recent efforts to probe and combat bacterial resistance have focused on studies of multiQACs. Relatively unexplored, however, have been changes to the primary atom bearing positive charge in these antiseptics. Here we review the current state of the field of both phosphonium and sulfonium amphiphilic antiseptics, both of which hold promise as novel means to address bacterial resistance.