Cationic Antimicrobials Research Articles

Campylobacter capsule and lipooligosaccharide confer resistance to serum and cationic antimicrobials

The innate immune system plays a critical role in host defense against mucosal bacteria. Campylobacter jejuni is a major cause of human gastroenteritis that usually resolves spontaneously within several days, suggesting that innate mechanisms are important to control the infection. However, the specific means by which this occurs is not well understood. While diarrheal isolates of C. jejuni usually are susceptible to human serum, we found that a systemic strain of C. jejuni, isolated from the cerebrospinal fluid of an infant with meningitis, is relatively more resistant to human serum, the Bactericidal/Permeability-Increasing Protein (BPI), an endogenous cationic antimicrobial protein, and the cationic peptide antibiotic polymyxin B. To test the hypothesis that the surface properties of this strain contributed to its ability to withstand these innate host defenses, we constructed isogenic mutants in capsule (kpsM) and lipooligosaccharide (waaF) and complemented these mutants by insertion of the complementation construct in trans into hipO, a chromosomal locus. We found that capsule expression was essential for serum resistance, whereas lipooligosaccharide played no substantial role. In contrast, the lipooligosaccharide mutant showed increased sensitivity to polymyxin B, α-defensins, cathelicidins, and BPI. These findings suggest that the polysaccharides of C. jejuni strains contribute differently to resistance against host innate immunity; whereby capsule is more important for resisting human complement and lipooligosaccharide is more important for protection against killing mediated by cationic antimicrobial peptides and proteins.

The Antimicrobial Activity, Hydrophobicity and Toxicity of Sulfonium Compounds, and Their Relationship

The sulfonium compound is a kind of cationic surfactant as well as a quaternary ammonium which has been used widely around the globe. This study investigated the antimicrobial activity, the hydrophobicity, the toxicity of several sulfoniums and their relationship with the aim of clarifying their antimicrobial activity and toxicity, and, furthermore, of predicting their usefulness availability as antimicrobials. As a result, the antimicrobial activity, expressed as the minimum inhibitory concentration (MIC) of the sulfoniums examined in this study, tended to decrease with the increase of their hydrophobicity, estimated by ClogP, and their antimicrobial activity against the gram-positive bacteria was higher than that against the gram-negative bacteria used in this study. The antimicrobial activities of several sulfoniums against the gram-positive bacteria were higher than those of some common cationic antimicrobials including quaternary ammoniums such as cetylpyridinium chloride (CPC) and benzalkonium chloride (BKC). In contrast, the antimicrobial activities of the sulfoniums against the gram-negative bacteria were lower than those of some common cationic antimicrobials. Meanwhile the toxicity, in particular, the acute dermal irritation/corrosion of the sulfoniums, tended to be lower than that of common cationic antimicrobials which were toxic in many cases indices. These results suggest that the sulfoniums might become useful antimicrobials which are less hazardous to human health than common cationic antimicrobials.

Corrections to Unnatural Amino Acid Side Chains as S1, S1′, and S2′ Probes Yield Cationic Antimicrobial Peptides with Stability toward Chymotryptic Degradation

Corrections to Unnatural Amino Acid Side Chains as S1, S1′, and S2′ Probes Yield Cationic Antimicrobial Peptides with Stability toward Chymotryptic Degradation

Unnatural Amino Acid Side Chains as S1, S1′, and S2′ Probes Yield Cationic Antimicrobial Peptides with Stability toward Chymotryptic Degradation

This work describes how the systematic incorporation of a range of unnatural amino acid derivatives in the P1, P1', and P2' positions allows for the generation of short lactoferricin based cationic antimicrobial peptides with a stability toward chymotryptic degradation. The necessary pharmacophore sets the peptides up for degradation by chymotrypsin, and a heavily truncated native tripeptide was rapidly digested despite its short sequence. Degradation studies indicated that increased half-lives could be obtained by altering the binding to each subsite surrounding the active site without sacrificing the antimicrobial activity. Important structural and mechanistic features were revealed in a fashion not feasible through the use of native peptide substrates. The results, which are generally applicable to a range of relevant peptides, further show that not only the S1 pocket, but also to the notably less studied S1' site can be used to control the proteolytic stability by incorporating different analogues of tryptophan and arginine.

Adaptive Resistance to the “Last Hope” Antibiotics Polymyxin B and Colistin in Pseudomonas aeruginosa Is Mediated by the Novel Two-Component Regulatory System ParR-ParS

As multidrug resistance increases alarmingly, polymyxin B and colistin are increasingly being used in the clinic to treat serious Pseudomonas aeruginosa infections. In this opportunistic pathogen, subinhibitory levels of polymyxins and certain antimicrobial peptides induce resistance toward higher, otherwise lethal, levels of these antimicrobial agents. It is known that the modification of lipid A of lipopolysaccharide (LPS) is a key component of this adaptive peptide resistance, but to date, the regulatory mechanism underlying peptide regulation in P. aeruginosa has remained elusive. The PhoP-PhoQ and PmrA-PmrB two-component systems, which control this modification under low-Mg2+ conditions, do not appear to play a major role in peptide-mediated adaptive resistance, unlike in Salmonella where PhoQ is a peptide sensor. Here we describe the identification and characterization of a novel P. aeruginosa two-component regulator affecting polymyxin-adaptive resistance, ParR-ParS (PA1799-PA1798). This system was required for activation of the arnBCADTEF LPS modification operon in the presence of subinhibitory concentrations of polymyxin, colistin, or the bovine peptide indolicidin, leading to increased resistance to various polycationic antibiotics, including aminoglycosides. This study highlights the complexity of the regulatory network controlling resistance to cationic antibiotics and host peptides in P. aeruginosa, which has major relevance in the development and deployment of cationic antimicrobials.

Biophysical studies of the interaction of squalamine and other cationic amphiphilic molecules with bacterial and eukaryotic membranes: importance of the distribution coefficient in membrane selectivity

The interaction of squalamine (SQ) with eukaryotic and prokaryotic membranes was studied and compared with the interaction of two other cationic amphipathic antimicrobials (CAAs), i.e. the antibiotic polymyxin B (PMB) and the detergent hexadecyltrimethylammonium bromide (CTAB). Whole cell experiments showed that the three CAA have in common the ability to interact with lipopolysaccharide-containing membranes through a divalent cation sensitive process. Differences were found regarding their kinetics of membrane permeabilisation and their selectivity for bacteria, with a preferential permeabilisation of bacteria by PMB>SQ and no selectivity for CTAB. Experiments with lipid monolayers and bilayers showed that this selectivity did not correlate with a preferential interaction of the CAAs with lipids but rather relies on differences in their ability to penetrate lipid bilayers and to cause electrically active lesions. Incidentally, our results also suggest that the distribution coefficient of CAAs could be used to predict their selectivity for bacteria.

Identification of the Asparagine Synthase Responsible for d -Asp Amidation in the Lactococcus lactis Peptidoglycan Interpeptide Crossbridge

We show that in Lactococcus lactis, the gene asnH encodes the asparagine synthase involved in amidation of D-Asp present in peptidoglycan side chains and crossbridges. The level of D-Asp amidation in peptidoglycan has a strong effect on the sensitivity of bacteria to endogenous autolysins and to the cationic antimicrobials nisin and lysozyme.

Cationic Amphiphiles Increase Activity of Aminoglycoside Antibiotic Tobramycin in the Presence of Airway Polyelectrolytes

It is empirically known that anionic polyelectrolytes present in cystic fibrosis (CF) airways due to bacterial infection significantly decrease the activity of cationic antimicrobials via electrostatic binding. In this work, we use synchrotron small-angle X-ray scattering to investigate the interaction between tobramycin, an aminoglycoside antibiotic commonly administered to CF patients via inhalation, with DNA, which is found in high concentrations in the CF airway. We find that interactions between DNA and tobramycin are significantly modified by the presence of mixtures of amphiphilic molecules. We measure a hierarchy of self-assembled structures formed between tobramycin, DNA, and the amphiphile mixtures and show how interactions between these components can be controlled. Results indicate that mixtures of cationic and negative curvature amphiphiles optimized for DNA binding via charge matching and curvature matching can competitively displace bound tobramycin from DNA and thereby drastically suppress tobramycin-DNA binding and resultant antimicrobial inactivation. Growth inhibition assays confirm the increased activity of tobramycin in the presence of DNA with the addition of the amphiphiles. These results suggest that optimized cationic amphiphile solutions have the potential to enhance antimicrobial function in highly infected environments that contain increased concentrations of anionic inflammatory polymers.

139 Antimicrobial Activity of Synthetic Cationic Steroids on Burn Wound Pathogens

Sepsis remains the single most important cause for multiple organ failure and death following burn injury for which no effective treatment is currently available. Burn injury is associated with immunosuppression which promotes sepsis. Studies from our laboratory have demonstrated that levels of the antimicrobial peptide (AMP), human β-defensin-2, was significantly decreased in burned skin epithelia, absent in burn blister fluid and poorly expressed in lung during inhalation injury.We hypothesize that 1) deficient antimicrobial peptides in burn wound promote progressive colonization of pathogens in burn wounds and 2) replacing active peptide components will restore the antimicrobial activity in burn wounds. We have synthesized several cationic steroid antimicrobials (CSAs) which mimic naturally occurring AMPs functionally. In vitro experiments using one of these synthetic drugs with locally isolated common burn pathogen P. aeruginosa and S aureus demonstrate MIC values of 2.0 ± 0.3 μg/ml and 0.4 ± 0.1 μg/ml, respectively, indicating effectiveness of these compounds against burn pathogens. These drugs were tested for cytotoxicity on cultured human keratinocytes using MTT cell viability assay. Results indicate that these drugs are nontoxic to cultured keratinocytes at the level required for bacterial killing. Since theses drugs are membrane active compounds it will be hard for bacteria to develop resistance and there is a great potential to be developed for clinical treatment.

Variations in the Degree of d -Alanylation of Teichoic Acids in Lactococcus lactis Alter Resistance to Cationic Antimicrobials but Have No Effect on Bacterial Surface Hydrophobicity and Charge

An increase of the degree of d-alanylation of teichoic acids in Lactococcus lactis resulted in a significant increase of bacterial resistance toward the cationic antimicrobials nisin and lysozyme, whereas the absence of D-alanylation led to a decreased resistance toward the same compounds. In contrast, the same variations of the D-alanylation degree did not modify bacterial cell surface charge and hydrophobicity. Bacterial adhesion to polystyrene and glass surfaces was not modified either.

P1971 Induction of resistance and cross-resistance offiuconazole, voriconazole, caspofungin and cationic antimicrobials against Candida species

P1971 Induction of resistance and cross-resistance offiuconazole, voriconazole, caspofungin and cationic antimicrobials against Candida species

In vitro activity and synergism of amphotericin B, azoles and cationic antimicrobials against the emerging pathogen Trichoderma spp.

The uncommon fungal pathogen Trichoderma shows increasing medical importance particularly in immunocompromised patients. Despite systemic antifungal therapy, prognosis of Trichoderma infection is poor regardless of the type of infection and the therapy used. The aim of the present study was to evaluate the in vitro activity and synergism of double antifungal combinations including amphotericin B, voriconazole, fluconazole, chlorhexidine digluconate and Akacid plus against 15 isolates of Trichoderma longibrachiatum and 1 isolate of Trichoderma harzianum. Individual MICs were determined by using broth microdilution method following the NCCLS M38-A guidelines with standard RPMI 1640 broth. Synergy tests were performed using the chequerboard method. All clinical Trichoderma strains showed reduced susceptibility to fluconazole (MICs>or=64 mg/L) and amphotericin B (MICs=2 mg/L), whereas lower MICs of 0.5-1 mg/L were detected for voriconazole. Akacid plus reached the lowest MIC values in a range of 0.06-0.5 mg/L, 4- to 32-fold higher MICs were found for chlorhexidine. No antagonism was observed for any of the antifungal combinations tested. Interaction of amphotericin B and azoles was indifferent (fractional inhibitory concentration index, FICI 2-4). The combination of one azole and one cationic biocide showed different degree of synergism (FICI 0.07-2.03). Interaction of Akacid plus and chlorhexidine resulted in synergism for each Trichoderma isolate (FICI-range 0.05-0.5). These results demonstrate no interaction between antifungals and some degree of synergism between azoles and cationic antimicrobials against Trichoderma spp.

Collectins and Cationic Antimicrobial Peptides of the Respiratory Epithelia

The respiratory epithelium is a primary site for the deposition of microorganisms that are acquired during inspiration. The innate immune system of the respiratory tract eliminates many of these potentially harmful agents preventing their colonization. Collectins and cationic antimicrobial peptides are antimicrobial components of the pulmonary innate immune system produced by respiratory epithelia, which have integral roles in host defense and inflammation in the lung. Synthesis and secretion of these molecules are regulated by the developmental stage, hormones, as well as many growth and immunoregulatory factors. The purpose of this review is to discuss antimicrobial innate immune elements within the respiratory tract of healthy and pneumonic lung with emphasis on hydrophilic surfactant proteins and beta-defensins.

Synergy in a medicinal plant: antimicrobial action of berberine potentiated by 5'-methoxyhydnocarpin, a multidrug pump inhibitor.

Multidrug resistance pumps (MDRs) protect microbial cells from both synthetic and natural antimicrobials. Amphipathic cations are preferred substrates of MDRs. Berberine alkaloids, which are cationic antimicrobials produced by a variety of plants, are readily extruded by MDRs. Several Berberis medicinal plants producing berberine were found also to synthesize an inhibitor of the NorA MDR pump of a human pathogen Staphylococcus aureus. The inhibitor was identified as 5'-methoxyhydnocarpin (5'-MHC), previously reported as a minor component of chaulmoogra oil, a traditional therapy for leprosy. 5'-MHC is an amphipathic weak acid and is distinctly different from the cationic substrates of NorA. 5'-MHC had no antimicrobial activity alone but strongly potentiated the action of berberine and other NorA substrates against S. aureus. MDR-dependent efflux of ethidium bromide and berberine from S. aureus cells was completely inhibited by 5'-MHC. The level of accumulation of berberine in the cells was increased strongly in the presence of 5'-MHC, indicating that this plant compound effectively disabled the bacterial resistance mechanism against the berberine antimicrobial.

Cationic peptide antimicrobials induce selective transcription of micF and osmY in Escherichia coli

Cationic antimicrobial peptides, such as polymyxin and cecropin, activated transcription of osmY and micF in growing Escherichia coli independently of each other. The micF response required the presence of a functional rob gene. It is intriguing that in this and other assays an identical response profile was also seen with hyperosmotic salt or sucrose gradient, two of the most commonly used traditional food preservatives. The osmY and micF transcription was not induced by hypoosmotic gradient, ionophoric peptides, uncouplers, or with other classes of membrane perturbing agents. The antibacterial peptides did not promote transcription of genes that respond to macromolecular or oxidative damage, fatty acid biosynthesis, heat shock, or depletion of proton or ion gradients. These and other results show that the antibacterial cationic peptides induce stasis in the early growth phase, and the transcriptional efficacy of antibacterial peptides correlates with their minimum inhibitory concentration, and also with their ability to mediate direct exchange of phospholipids between vesicles. The significance of these results is developed as the hypothesis that the cationic peptide antimicrobials stress growth of Gram-negative organisms by making contacts between the two phospholipid interfaces in the periplasmic space and prevent the hyperosmotic wrinkling of the cytoplasmic membrane. Broader significance of these results, and of the hypothesis that the peptide mediated contacts between the periplasmic phospholipid interfaces are the primary triggers, is discussed in relation to antibacterial resistance.

The role of fundamental research and biotechnology in finding solutions to the global problem of antibiotic resistance.

Resistance to antibiotics is becoming a major problem worldwide. Exacerbating this situation is the fact that few new antibiotics are in the development pipeline, and, indeed, no novel class of antibiotics has been introduced into medical practice in more than 20 years. It is proposed that the solutions to the problem of antibiotic resistance will be found only through fundamental research that will probably use biotechnology as a tool. A variety of novel approaches being utilized in university laboratories and biotechnology companies are outlined. Two approaches in particular, namely, Synsorbs and recombinant cationic peptide antimicrobials, that have been developed through the Canadian Bacterial Diseases Network, a national research consortium, are discussed.

カチオン性殺菌剤の活性を保持する歯磨製剤

カチオン性殺菌剤の活性を保持する歯磨製剤

Moenomycin, an inhibitor of cell wall synthesis

The bacterial cell wall is composed of a thick layer of peptidoglycan and cell wall polymers, which are either embedded in the membrane or linked to the peptidoglycan backbone and referred to as lipoteichoic acid (LTA) and wall teichoic acid (WTA), respectively. Modifications of the peptidoglycan or WTA backbone can alter the susceptibility of the bacterial cell towards cationic antimicrobials and lysozyme. The human pathogen Listeria monocytogenes is intrinsically resistant towards lysozyme, mainly due to deacetylation and O-acetylation of the peptidoglycan backbone via PgdA and OatA. Recent studies identified additional factors, which contribute to the lysozyme resistance of this pathogen. One of these is the predicted ABC transporter, EslABC. An eslB mutant is hyper-sensitive towards lysozyme, likely due to the production of thinner and less O-acetylated peptidoglycan. Using a suppressor screen, we show here that suppression of eslB phenotypes could be achieved by enhancing peptidoglycan biosynthesis, reducing peptidoglycan hydrolysis or alterations in WTA biosynthesis and modification. The lack of EslB also leads to a higher negative surface charge, which likely stimulates the activity of peptidoglycan hydrolases and lysozyme. Based on our results, we hypothesize that the portion of cell surface exposed WTA is increased in the eslB mutant due to the thinner peptidoglycan layer and that latter one could be caused by an impairment in UDP-N-acetylglucosamine (UDP-GlcNAc) production or distribution.