An efflux-susceptible antibiotic-adjuvant with systemic efficacy against mouse infections.

Scarcity of effective treatments against sepsis is daunting, especially under the contemporary standpoints on antibiotics resistance, entailing the development of alternative treatment strategies. Here, we describe the design and antibiotic adjuvant properties of a new lipopeptide-like pentamer, decanoyl-bis.diaminobutyrate-aminododecanoyl-diaminobutyrate-amide (C10BBc12B), whose sub-maximal tolerated doses combinations with inefficient antibiotics demonstrated systemic efficacies in murine models of peritonitis-sepsis and urinary-tract infections. Attempts to shed light into the mechanism of action using membrane-active fluorescent probes, suggest outer-membrane interactions to dominate the pentamer's adjuvant properties, which were not associated with typical inner-membrane damages or with delayed bacterial growth. Yet, checkerboard titrations with low micromolar concentrations of C10BBc12B exhibited unprecedented capacities in potentiation of hydrophobic antibiotics towards Gram-negative ESKAPE pathogens, with an apparent low propensity for prompting resistance to the antibiotics. Assessment of the pentamer's potentiating activities upon efflux inhibition incites submission of a hitherto unreported, probable action mechanism implicating the pentamer's de-facto capacity to hijack bacterial efflux pumps for boosting its adjuvant activity through repetitive steps including outer-membrane adhesion, translocation and subsequent expulsion.

Mechanistic Studies of Antibiotic Adjuvants Reducing Kidney's Bacterial Loads upon Systemic Monotherapy.

We describe the design and attributes of a linear pentapeptide-like derivative (C14(ω5)OOc10O) screened for its ability to elicit bactericidal competences of plasma constituents against Gram-negative bacteria (GNB). In simpler culture media, the lipopeptide revealed high aptitudes to sensitize resilient GNB to hydrophobic and/or efflux-substrate antibiotics, whereas in their absence, C14(ω5)OOc10O only briefly delayed bacterial proliferation. Instead, at low micromolar concentrations, the lipopeptide has rapidly lowered bacterial proton and ATP levels, although significantly less than upon treatment with its bactericidal analog. Mechanistic studies support a two-step scenario providing a plausible explanation for the lipopeptide's biological outcomes against GNB: initially, C14(ω5)OOc10O permeabilizes the outer membrane similarly to polymyxin B, albeit in a manner not necessitating as much LPS-binding affinity. Subsequently, C14(ω5)OOc10O would interact with the inner membrane gently yet intensively enough to restrain membrane-protein functions such as drug efflux and/or ATP generation, while averting the harsher inner membrane perturbations that mediate the fatal outcome associated with bactericidal peers. Preliminary in vivo studies where skin wound infections were introduced in mice, revealed a significant efficacy in affecting bacterial viability upon topical treatment with creams containing C14(ω5)OOc10O, whereas synergistic combination therapies were able to secure the pathogen's eradication. Further, capitalizing on the finding that C14(ω5)OOc10O plasma-potentiating concentrations were attainable in mice blood at sub-maximal tolerated doses, we used a urinary tract infection model to acquire evidence for the lipopeptide's systemic capacity to reduce the kidney's bacterial loads. Collectively, the data establish the role of C14(ω5)OOc10O as a compelling antibacterial potentiator and suggest its drug-like potential.

Sub-inhibitory membrane damage undermines Staphylococcus aureus virulence.

We investigated antibacterial properties of a recently described membrane-active lipopeptide, C10OOc12O (decanoyl-ornithyl-ornithyl-dodecanoyl-ornithyl-amide) against Gram-positive bacteria (GPB). Minimal inhibitory concentrations (MICs) and kinetics were compared in culture media and plasma. Chemo-sensitization to antibiotics was determined using the checkerboard assay. Membrane damages were estimated using diverse membrane potential sensitive dyes. ATP levels and relevant enzymes activities were measured using commercial bioassay kits. While relatively weakly active in simple culture media, sub-MIC levels (~ten-fold) of C10OOc12O have significantly improved the antibacterial function of Human plasma. Mechanistic studies indicated that C10OOc12O-treated bacteria have sustained mild membrane damage(s) in association with rapid (within 2 min) but low (<10%) dissipation of the trans-membrane potential; Intracellular ATP levels were transiently reduced (~20%) whereas extracellular ATP increased only at MIC values; Sub-inhibitory concentrations were sufficient for inhibiting major agr-regulated virulence factors (lipase and α-toxin) and for sensitizing MRSA USA300 to the antibiotic oxacillin to the point of reverting the bacteria status from oxacillin-resistant to oxacillin-sensitive (i.e., oxacillin MIC was reduced from 32 to 0.1 mg/l). These findings argue that by means of mild depolarization, C10OOc12O affects the quorum sensing regulator in a manner that transiently weakens bacterial defenses, thereby enforcing studies that support the potential usefulness of fighting S. aureus (and possibly other GPB) infections, by targeting its virulence.

A broad-spectrum bactericidal lipopeptide with anti-biofilm properties.

Previous studies of the oligoacyllysyl (OAK) series acyl-lysyl-lysyl-aminoacyl-lysine-amide, suggested their utility towards generating robust linear lipopeptide-like alternatives to antibiotics, although to date, none exhibited potent broad-spectrum bactericidal activity. To follow up on this premise, we produced a new analog (C14KKc12K) and investigated its properties in various media. Mechanistic studies suggest that C14KKc12K uses a non-specific membrane-disruptive mode of action for rapidly reducing viability of Gram-negative bacteria (GNB) similarly to polymyxin B (PMB), a cyclic lipopeptide used as last resort antibiotic. Indeed, C14KKc12K displayed similar affinity for lipopolysaccharides and induced cell permeabilization associated with rapid massive membrane depolarization. Unlike PMB however, C14KKc12K was also bactericidal to Gram-positive bacteria (GPB) at or near the minimal inhibitory concentration (MIC), as assessed against a multispecies panel of >50 strains, displaying MIC50 at 3 and 6 µM, respectively for GPB and GNB. C14KKc12K retained activity in human saliva, reducing the viability of cultivable oral microflora by >99% within two minutes of exposure, albeit at higher concentrations, which, nonetheless, were similar to the commercial gold standard, chlorhexidine. This equipotent bactericidal activity was also observed in pre-formed biofilms of Streptococcus mutans, a major periodontal pathogen. Such compounds therefore, may be useful for eradication of challenging poly-microbial infections.

Controlling bacterial infections by inhibiting proton-dependent processes.

Bacterial resistance to antibiotics is recognized as one of the greatest threats in modern healthcare, taking a staggering toll worldwide. New approaches for controlling bacterial infections must be designed, eventually combining multiple strategies for complimentary therapies. This review explores an old/new paradigm for multi-targeted antibacterial therapy, focused at disturbing bacterial cytoplasmic membrane functions at sub minimal inhibitory concentrations, namely through superficial physical alterations of the bilayer, thereby perturbing transmembrane signals transduction. Such a paradigm may have the advantage of fighting the infection while avoiding many of the known resistance mechanisms. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

Improved bacterial detection using immobilized acyl-lysyl oligomers.

The global need to improve bacterial detection in liquid media has motivated multidisciplinary research efforts toward developing new approaches that overcome the shortcomings of traditional techniques. We recently proposed the use of oligomers of acylated lysyls (OAKs) in their resin-linked form (ROAKs) for the efficient, robust, and inexpensive filtration of bacteria. Here, to investigate the potential for the use of ROAKs in downstream applications, we first examined the capacity of ROAKs to capture bacteria as a function of environmental conditions and structure-activity relationships (SARs). We next assessed their ability to release the captured bacteria and then combined both abilities to improve real-time PCR outcomes. ROAKs were able to deplete liquid samples of bacterial content after incubation or continuous flow, illustrating the efficient capture of different bacterial species under a wide range of ionic strength and pH conditions. We also show circumstances for the significant release of captured bacteria, live or dead, for further analysis. Finally, the SAR study revealed a shorter ROAK derivative exhibiting a capture capacity similar to that of the parent construct but the increased recovery of ROAK-bound bacteria, enabling improvement of the detection sensitivity by 20-fold. Collectively, the data support the potential usefulness of a simple, robust, and efficient approach for rapid capture/analysis of bacteria from tap water and, possibly, from more complex media.

Antiplasmodial properties of acyl-lysyl oligomers in culture and animal models of malaria.

Our previous analysis of antiplasmodial properties exhibited by dodecanoyl-based oligo-acyl-lysyls (OAKs) has outlined basic attributes implicated in potent inhibition of parasite growth and underlined the critical role of excess hydrophobicity in hemotoxicity. To dissociate hemolysis from antiplasmodial effect, we screened >50 OAKs for in vitro growth inhibition of Plasmodium falciparum strains, thus revealing the minimal requirements for antiplasmodial potency in terms of sequence and composition, as confirmed by efficacy studies in vivo. The most active sequence, dodecanoyllysyl-bis(aminooctanoyllysyl)-amide (C(12)K-2α(8)), inhibited parasite growth at submicromolar concentrations (50% inhibitory concentration [IC(50)], 0.3 ± 0.1 µM) and was devoid of hemolytic activity (<0.4% hemolysis at 150 µM). Unlike the case of dodecanoyl-based analogs, which equally affect ring and trophozoite stages of the parasite developmental cycle, the ability of various octanoyl-based OAKs to distinctively affect these stages (rings were 4- to 5-fold more sensitive) suggests a distinct antiplasmodial mechanism, nonmembranolytic to host red blood cells (RBCs). Upon intraperitoneal administration to mice, C(12)K-2α(8) demonstrated sustainable high concentrations in blood (e.g., 0.1 mM at 25 mg/kg of body weight). In Plasmodium vinckei-infected mice, C(12)K-2α(8) significantly affected parasite growth (50% effective dose [ED(50)], 22 mg/kg) but also caused mortality in 2/3 mice at high doses (50 mg/kg/day × 4).