Metabolite compositions on skins of eastern hellbenders Cryptobranchus alleganiensis alleganiensis differ with location and captivity.

Eastern hellbenders Cryptobranchus alleganiensis alleganiensis, large aquatic salamanders, are declining over most of their range. The amphibian-killing fungus Batrachochytrium dendrobatidis (Bd) has contributed to global amphibian declines and has been detected on eastern hellbenders, but infection intensities were lower than those of species that are more susceptible to Bd. The factors limiting Bd on hellbenders may include antifungal metabolites produced by their skin microbiota. We used a metabolite fingerprinting technique to noninvasively identify the presence, but not identity, of metabolites associated with eastern hellbenders. We surveyed the skin of wild eastern hellbenders to test whether the composition and richness (i.e. number of metabolites) of their metabolites are explained by Bd status or location. Furthermore, we surveyed for metabolites on captive eastern hellbenders to test whether metabolite compositions were different between captive and wild eastern hellbenders. Bd detection was not associated with either metabolite richness or composition. Both metabolite composition and richness differed significantly on hellbenders from different locations (i.e. states). For metabolite composition, there was a statistical interaction between location and Bd status. Metabolite richness was greater on captive eastern hellbenders compared to wild hellbenders, and metabolite compositions differed between wild and captive eastern hellbenders. The methods we employed to detect metabolite profiles effectively grouped individuals by location even though metabolite composition and richness have high levels of intraspecific variation. Understanding the drivers and functional consequences of assemblages of skin metabolites on amphibian health will be an important step toward understanding the mechanisms that result in disease vulnerability.

Lymphocyte Inhibition by the Salamander-Killing Chytrid Fungus, Batrachochytrium salamandrivorans.

Amphibian populations have been declining around the world for more than five decades, and the losses continue. Although causes are complex, major contributors to these declines are two chytrid fungi, Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans, which both cause the disease termed chytridiomycosis. Previously, we showed that B. dendrobatidis impedes amphibian defenses by directly inhibiting lymphocytes in vitro and in vivo by release of soluble metabolites, including kynurenine (KYN), methylthioadenosine (MTA), and spermidine (SPD). Here, we show that B. salamandrivorans cells and cell-free supernatants also inhibit amphibian lymphocytes as well as a human T cell line. As we have shown for B. dendrobatidis, high-performance liquid chromatography (HPLC) and mass spectrometry revealed that KYN, MTA, and SPD are key metabolites found in the B. salamandrivorans supernatants. Production of inhibitory factors by B. salamandrivorans is limited to mature zoosporangia and can occur over a range of temperatures between 16°C and 26°C. Taken together, these results suggest that both pathogenic Batrachochytrium fungi have evolved similar mechanisms to inhibit lymphocytes in order to evade clearance by the amphibian immune system.

Diastereoselective Copper-Mediated Conjugate Addition of Functionalized Magnesiates for the Preparation of Bisaryl Nrf2 Activators.

A two-step metal-halogen exchange and diastereoselective copper-mediated Michael addition onto a complex α,ß-unsaturated system has been developed and applied toward the synthesis of bisaryl Nrf2 activators. Optimization of metal-halogen exchange using (n-Bu)3MgLi allowed for the preparation of custom aryl-functionalized magnesiate reagents at noncryogenic temperatures. Following transmetalation, these reagents were used in highly diastereoselective Michael addition reactions.

Trivalent sulfonium compounds (TSCs): Tetrahydrothiophene-based amphiphiles exhibit similar antimicrobial activity to analogous ammonium-based amphiphiles.

Recent advances in the development of quaternary ammonium compounds (QACs) have focused on new structural motifs to increase bioactivity, but significantly less studied has been the change from ammonium- to sulfonium-based disinfectants. Herein, we report the synthesis of structurally analogous series of quaternary ammonium and trivalent sulfonium compounds (TSCs). The bioactivity profiles of these compounds generally mirror each other, and the antibacterial activity of sulfonium-based THT-18 was found to be comparable to the commercial disinfectant, BAC. The development of these compounds presents a new avenue for further study of disinfectants to combat the growing threat of bacterial resistance.

Analysis of the Destabilization of Bacterial Membranes by Quaternary Ammonium Compounds: A Combined Experimental and Computational Study.

The mechanism of action of quaternary ammonium compound (QAC) antiseptics has long been assumed to be straightforward membrane disruption, although the process of approaching and entering the membrane has little modeling precedent. Furthermore, questions have more recently arisen regarding bacterial resistance mechanisms, and why select classes of QACs (specifically, multicationic QACs) are less prone to resistance. In order to better understand such subtleties, a series of molecular dynamics simulations were utilized to help identify these molecular determinants, directly comparing mono-, bis-, and triscationic QACs in simulated membrane intercalation models. Three distinct membranes were simulated, mimicking the surfaces of Escherichia coli and Staphylococcus aureus, as well as a neutral phospholipid control. By analyzing the resulting trajectories in the form of a timeseries analysis, insight was gleaned regarding the significant steps and interactions involved in the destabilization of phospholipid bilayers within the bacterial membranes. Finally, to more specifically probe the effect of the hydrophobic section of the amphiphile that presumably penetrates the membrane, a series of alkyl- and ester-based biscationic quaternary ammonium compounds were prepared, tested for antimicrobial activity against both Gram-positive and Gram-negative bacteria, and modeled.

Metabolites Involved in Immune Evasion by Batrachochytrium dendrobatidis Include the Polyamine Spermidine.

Amphibians have been declining around the world for more than four decades. One recognized driver of these declines is the chytrid fungus Batrachochytrium dendrobatidis, which causes the disease chytridiomycosis. Amphibians have complex and varied immune defenses against B. dendrobatidis, but the fungus also has a number of counterdefenses. Previously, we identified two small molecules produced by the fungus that inhibit frog lymphocyte proliferation, methylthioadenosine (MTA) and kynurenine (KYN). Here, we report on the isolation and identification of the polyamine spermidine (SPD) as another significant immunomodulatory molecule produced by B. dendrobatidis SPD and its precursor, putrescine (PUT), are the major polyamines detected, and SPD is required for growth. The major pathway of biosynthesis is from ornithine through putrescine to spermidine. An alternative pathway from arginine to agmatine to putrescine appears to be absent. SPD is inhibitory at concentrations of ≥10 µM and is found at concentrations between 1 and 10 µM in active fungal supernatants. Although PUT is detected in the fungal supernatants, it is not inhibitory to lymphocytes even at concentrations as high as 100 µM. Two other related polyamines, norspermidine (NSP) and spermine (SPM), also inhibit amphibian lymphocyte proliferation, but a third polyamine, cadaverine (CAD), does not. A suboptimal (noninhibitory) concentration of MTA (10 µM), a by-product of spermidine synthesis, enhances the inhibition of SPD at 1 and 10 µM. We interpret these results to suggest that B. dendrobatidis produces an "armamentarium" of small molecules that, alone or in concert, may help it to evade clearance by the amphibian immune system.

More QACs, more questions: Recent advances in structure activity relationships and hurdles in understanding resistance mechanisms.

Quaternary ammonium compounds (QACs) are a class of antimicrobials that have been around for over a century; nevertheless, they have found continued renewal in the structures to which they can be appended. Ranging from antimicrobial polymers to adding novel modes of action to existing antibiotics, QACs have found ongoing use due to their potent properties. However, resistance against QACs has begun to emerge, and the mechanism of resistance is still only partially understood. In this review, we aim to summarize the current state of the field and what is known about the mechanisms of resistance so that the QACs of the future can be designed to be evermore efficacious and utilized to unearth the remaining mysteries that surround bacteria's resistance to them.

Prodigiosin, Violacein, and Volatile Organic Compounds Produced by Widespread Cutaneous Bacteria of Amphibians Can Inhibit Two Batrachochytrium Fungal Pathogens.

Symbiotic bacteria can produce secondary metabolites and volatile compounds that contribute to amphibian skin defense. Some of these symbionts have been used as probiotics to treat or prevent the emerging disease chytridiomycosis. We examined 20 amphibian cutaneous bacteria for the production of prodigiosin or violacein, brightly colored defense compounds that pigment the bacteria and have characteristic spectroscopic properties making them readily detectable, and evaluated the antifungal activity of these compounds. We detected violacein from all six isolates of Janthinobacterium lividum on frogs from the USA, Switzerland, and on captive frogs originally from Panama. We detected prodigiosin from five isolates of Serratia plymuthica or S. marcescens, but not from four isolates of S. fonticola or S. liquefaciens. All J. lividum isolates produced violacein when visibly purple, while prodigiosin was only detected on visibly red Serratia isolates. When applied to cultures of chytrid fungi Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), prodigiosin caused significant growth inhibition, with minimal inhibitory concentrations (MIC) of 10 and 50 µM, respectively. Violacein showed a MIC of 15 µM against both fungi and was slightly more active against Bsal than Bd at lower concentrations. Although neither violacein nor prodigiosin showed aerosol activity and is not considered a volatile organic compound (VOC), J. lividum and several Serratia isolates did produce antifungal VOCs. White Serratia isolates with undetectable prodigiosin levels could still inhibit Bd growth indicating additional antifungal compounds in their chemical arsenals. Similarly, J. lividum can produce antifungal compounds such as indole-3-carboxaldehyde in addition to violacein, and isolates are not always purple, or turn purple under certain growth conditions. When Serratia isolates were grown in the presence of cell-free supernatant (CFS) from the fungi, CFS from Bd inhibited growth of the prodigiosin-producing isolates, perhaps indicative of an evolutionary arms race; Bsal CFS did not inhibit bacterial growth. In contrast, growth of one J. lividum isolate was facilitated by CFS from both fungi. Isolates that grow and continue to produce antifungal compounds in the presence of pathogens may represent promising probiotics for amphibians infected or at risk of chytridiomycosis. In a global analysis, 89% of tested Serratia isolates and 82% of J. lividum isolates were capable of inhibiting Bd and these have been reported from anurans and caudates from five continents, indicating their widespread distribution and potential for host benefit.

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.

Variation in Metabolite Profiles of Amphibian Skin Bacterial Communities Across Elevations in the Neotropics.

Both the structure and function of host-associated microbial communities are potentially impacted by environmental conditions, just as the outcomes of many free-living species interactions are context-dependent. Many amphibian populations have declined around the globe due to the fungal skin pathogen, Batrachochytrium dendrobatidis (Bd), but enivronmental conditions may influence disease dynamics. For instance, in Panamá, the most severe Bd outbreaks have occurred at high elevation sites. Some amphibian species harbor bacterial skin communities that can inhibit the growth of Bd, and therefore, there is interest in understanding whether environmental context could also alter these host-associated microbial communities in a way that might ultimately impact Bd dynamics. In a field survey in Panamá, we assessed skin bacterial communities (16S rRNA amplicon sequencing) and metabolite profiles (HPLC-UV/Vis) of Silverstoneia flotator from three high- and three low-elevation populations representing a range of environmental conditions. Across elevations, frogs had similar skin bacterial communities, although one lowland site appeared to differ. Interestingly, we found that bacterial richness decreased from west to east, coinciding with the direction of Bd spread through Panamá. Moreover, metabolite profiles suggested potential functional variation among frog populations and between elevations. While the frogs have similar bacterial community structure, the local environment might shape the metabolite profiles. Ultimately, host-associated community structure and function could be dependent on environmental conditions, which could ultimately influence host disease susceptibility across sites.

Ester- and amide-containing multiQACs: Exploring multicationic soft antimicrobial agents.

Quaternary ammonium compounds (QACs) are ubiquitous antiseptics whose chemical stability is both an aid to prolonged antibacterial activity and a liability to the environment. Soft antimicrobials, such as QACs designed to decompose in relatively short times, show the promise to kill bacteria effectively but not leave a lasting footprint. We have designed and prepared 40 soft QAC compounds based on both ester and amide linkages, in a systematic study of mono-, bis-, and tris-cationic QAC species. Antimicrobial activity, red blood cell lysis, and chemical stability were assessed. Antiseptic activity was strong against a panel of six bacteria including two MRSA strains, with low micromolar activity seen in many compounds; amide analogs showed superior activity over ester analogs, with one bisQAC displaying average MIC activity of ∼1µM. For a small subset of highly bioactive compounds, hydrolysis rates in pure water as well as buffers of pH =4, 7, and 10 were tracked by LCMS, and indicated good stability for amides while rapid hydrolysis was observed for all compounds in acidic conditions.

Identification of Bufadienolides from the Boreal Toad, Anaxyrus boreas, Active Against a Fungal Pathogen.

Amphibian granular glands provide a wide range of compounds on the skin that defend against pathogens and predators. We identified three bufadienolides-the steroid-like compounds arenobufagin, gamabufotalin, and telocinobufagin-from the boreal toad, Anaxyrus boreas, through liquid chromatography mass spectrometry (LC/MS). Compounds were detected both after inducing skin gland secretions and in constitutive mucosal rinses from toads. We described the antimicrobial properties of each bufadienolide against Batrachochytrium dendrobatidis (Bd), an amphibian fungal pathogen linked with boreal toad population declines. All three bufadienolides were found to inhibit Bd growth at similar levels. The maximum Bd inhibition produced by arenobufagin, gamabufotalin, and telocinobufagin were approximately 50%, in contrast to the complete Bd inhibition shown by antimicrobial skin peptides produced by some amphibian species. In addition, skin mucus samples significantly reduced Bd viability, and bufadienolides were detected in 15 of 62 samples. Bufadienolides also appeared to enhance growth of the anti-Bd bacterium Janthinobacterium lividum, and thus may be involved in regulation of the skin microbiome. Here, we localized skin bacteria within the mucus layer and granular glands of toads with fluorescent in situ hybridization. Overall, our results suggest that bufadienolides can function in antifungal defense on amphibian skin and their production is a potentially convergent trait similar to antimicrobial peptide defenses found on the skin of other species. Further studies investigating bufadienolide expression across toad populations, their regulation, and interactions with other components of the skin mucosome will contribute to understanding the complexities of amphibian immune defense.

Short-Term Exposure to Coal Combustion Waste Has Little Impact on the Skin Microbiome of Adult Spring Peepers (Pseudacris crucifer).

UNLABELLED: Disruptions to the microbiome can impact host health as can exposure to environmental contaminants. However, few studies have addressed how environmental contaminants impact the microbiome. We explored this question for frogs that breed in wetlands contaminated with fly ash, a by-product of coal combustion that is enriched in trace elements. We found differences in the bacterial communities among a fly ash-contaminated site and several reference wetlands. We then experimentally assessed the impacts of fly ash on the skin microbiome of adult spring peepers (Pseudacris crucifer). Frogs were exposed to fly ash in the laboratory for 12 h, the duration of a typical breeding event, and the skin microbiome was assessed after 5 days (experiment 1) or after 5 and 15 days (experiment 2). We examined bacterial community structure using 16S rRNA gene amplicon sequencing and metabolite profiles using high-pressure liquid chromatography-mass spectrometry (HPLC-MS). We found little impact as the result of acute exposure to fly ash on the bacterial communities or metabolite profiles in either experiment, suggesting that the bacterial symbiont communities of adults may be relatively resistant to brief contaminant exposure. However, housing frogs in the laboratory altered bacterial community structure in the two experiments, which supports prior research suggesting that environmental source pools are important for maintaining the amphibian skin microbiome. Therefore, for contaminants like fly ash that may alter the potential source pool of symbionts, we think it may be important to explore how contaminants affect the initial assembly of the amphibian skin microbiome in larval amphibians that develop within contaminated sites. IMPORTANCE: Animals are hosts to many symbiotic microorganisms, collectively called the microbiome, that play critical roles in host health. Therefore, environmental contaminants that alter the microbiome may impact hosts. Some of the most widespread contaminants, produced worldwide, are derived from the mining, storage, and combustion of coal for energy. Fly ash, for example, is a by-product of coal combustion. It contains compounds such as arsenic, selenium, cadmium, and strontium and is a recognized source of ground and surface water contamination. Here, we experimentally investigated the impacts of short-term fly ash exposure on the skin microbiome of spring peepers, one of many species of amphibian that sometimes breed in open fly ash disposal ponds. This research provides a look into the potential impacts of fly ash on an animal's microbiome and suggests important future directions for research on the effects of environmental contaminants on the microbiome.

Immunomodulatory metabolites released by the frog-killing fungus Batrachochytrium dendrobatidis.

Batrachochytrium dendrobatidis is a fungal pathogen in the phylum Chytridiomycota that causes the skin disease chytridiomycosis. Chytridiomycosis is considered an emerging infectious disease linked to worldwide amphibian declines and extinctions. Although amphibians have well-developed immune defenses, clearance of this pathogen from the skin is often impaired. Previously, we showed that the adaptive immune system is involved in the control of the pathogen, but B. dendrobatidis releases factors that inhibit in vitro and in vivo lymphocyte responses and induce lymphocyte apoptosis. Little is known about the nature of the inhibitory factors released by this fungus. Here, we describe the isolation and characterization of three fungal metabolites produced by B. dendrobatidis but not by the closely related nonpathogenic chytrid Homolaphlyctis polyrhiza. These metabolites are methylthioadenosine (MTA), tryptophan, and an oxidized product of tryptophan, kynurenine (Kyn). Independently, both MTA and Kyn inhibit the survival and proliferation of amphibian lymphocytes and the Jurkat human T cell leukemia cell line. However, working together, they become effective at much lower concentrations. We hypothesize that B. dendrobatidis can adapt its metabolism to release products that alter the local environment in the skin to inhibit immunity and enhance the survival of the pathogen.

Composition of symbiotic bacteria predicts survival in Panamanian golden frogs infected with a lethal fungus.

Symbiotic microbes can dramatically impact host health and fitness, and recent research in a diversity of systems suggests that different symbiont community structures may result in distinct outcomes for the host. In amphibians, some symbiotic skin bacteria produce metabolites that inhibit the growth of Batrachochytrium dendrobatidis (Bd), a cutaneous fungal pathogen that has caused many amphibian population declines and extinctions. Treatment with beneficial bacteria (probiotics) prevents Bd infection in some amphibian species and creates optimism for conservation of species that are highly susceptible to chytridiomycosis, the disease caused by Bd. In a laboratory experiment, we used Bd-inhibitory bacteria from Bd-tolerant Panamanian amphibians in a probiotic development trial with Panamanian golden frogs, Atelopus zeteki, a species currently surviving only in captive assurance colonies. Approximately 30% of infected golden frogs survived Bd exposure by either clearing infection or maintaining low Bd loads, but this was not associated with probiotic treatment. Survival was instead related to initial composition of the skin bacterial community and metabolites present on the skin. These results suggest a strong link between the structure of these symbiotic microbial communities and amphibian host health in the face of Bd exposure and also suggest a new approach for developing amphibian probiotics.

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.

Phylogenetic distribution of symbiotic bacteria from Panamanian amphibians that inhibit growth of the lethal fungal pathogen Batrachochytrium dendrobatidis.

The introduction of next-generation sequencing has allowed for greater understanding of community composition of symbiotic microbial communities. However, determining the function of individual members of these microbial communities still largely relies on culture-based methods. Here, we present results on the phylogenetic distribution of a defensive functional trait of cultured symbiotic bacteria associated with amphibians. Amphibians are host to a diverse community of cutaneous bacteria and some of these bacteria protect their host from the lethal fungal pathogen Batrachochytrium dendrobatidis (Bd) by secreting antifungal metabolites. We cultured over 450 bacterial isolates from the skins of Panamanian amphibian species and tested their interactions with Bd using an in vitro challenge assay. For a subset of isolates, we also completed coculture experiments and found that culturing isolates with Bd had no effect on inhibitory properties of the bacteria, but it significantly decreased metabolite secretion. In challenge assays, approximately 75% of the bacterial isolates inhibited Bd to some extent and these inhibitory isolates were widely distributed among all bacterial phyla. Although there was no clear phylogenetic signal of inhibition, three genera, Stenotrophomonas, Aeromonas and Pseudomonas, had a high proportion of inhibitory isolates (100%, 77% and 73%, respectively). Overall, our results demonstrate that antifungal properties are phylogenetically widespread in symbiotic microbial communities of Panamanian amphibians and that some functional redundancy for fungal inhibition occurs in these communities. We hope that these findings contribute to the discovery and development of probiotics for amphibians that can mitigate the threat of chytridiomycosis.

Biofilm-eradicating properties of quaternary ammonium amphiphiles: simple mimics of antimicrobial peptides.

Bacterial biofilms are difficult to eradicate because of reduced antibiotic sensitivity and altered metabolic processes; thus, the development of new approaches to biofilm eradication is urgently needed. Antimicrobial peptides (AMPs) and quaternary ammonium cations (QACs) are distinct, yet well-known, classes of antibacterial compounds. By mapping the general regions of charge and hydrophobicity of QACs onto AMP structures, we designed a small library of QACs to serve as simple AMP mimics. In order to explore the role that cationic charge plays in biofilm eradication, structures were varied with respect to cationic character, distribution of charge, and alkyl side chain. The reported compounds possess minimum biofilm eradication concentrations (MBEC) as low as 25 µM against Gram-positive biofilms, making them the most active anti-biofilm structures reported to date. These potent AMP mimics were synthesized in 1-2 steps and hint at the minimal structural requirements for biofilm destruction.

Beyond paraquats: dialkyl 3,3'- and 3,4'-bipyridinium amphiphiles as antibacterial agents.

Dialkyl 4,4'-bipyridinium compounds, known as 'paraquats' (PQs), have a long history of use as herbicides, as redox indicators, and more recently as potent antibacterial agents. However, due to their ability to form reactive oxygen species (ROS) in vivo, PQs are also known to be toxic. We proposed that altering the electrochemical properties of PQ, specifically by preparing isomeric bipyridinium structures with 3,3'- and 3,4'-substitution of the nitrogen heteroatoms on the biaryl core, would maintain antibacterial activity, yet decrease toxicity. We have thus prepared a series of 17 amphiphiles, dubbed 'metaquat' (MQ) and 'parametaquat' (PMQ), respectively, and investigated their antibacterial and electrochemical properties. Optimal inhibition of bacterial growth was observed in symmetric, biscationic structures; minimum inhibitory concentration (MIC) values measured as low as 0.5 µM against both Gram-positive and Gram-negative bacteria for the compound PMQ-11,11. Electrochemical analysis demonstrated the redox properties of the dialkyl 3,3'- and 3,4'-bipyridinium amphiphiles to be distinct from those of the 4,4'-bipyridinium isomer. Thus MQ and PMQ amphiphiles maintain the strong antibacterial activity of the PQ isomers, but show promise for reduced ROS toxicity.

The antimicrobial activity of mono-, bis-, tris-, and tetracationic amphiphiles derived from simple polyamine platforms.

A series of 34 amphiphilic compounds varying in both number of quaternary ammonium groups and length of alkyl chains has been assembled. The synthetic preparations for these structures are simple and generally high-yielding, proceeding in 1-2 steps without the need for chromatography. Antibacterial MIC data for these compounds were determined, and over half boast single digit MIC values against a series of gram-positive and gram-negative bacteria. MIC variation mostly hinged on the length of the alkyl chain, where a dodecyl group led to optimal activity; surprisingly, the number of cations and/or basic nitrogens was less important in dictating bioactivity. Additional structural variation was prepared in a trisamine series dubbed 12,3,X,3,12, providing a series of potent amphiphiles functionalized with varied allyl, alkyl, and benzyl groups. Tetraamines were also investigated, culminating in a two-step preparation of a tetracationic structure that showed only modestly improved bioactivity versus amphiphiles with two or three cations.