Activities of LL-37, a Cathelin-Associated Antimicrobial Peptide of Human Neutrophils

Human neutrophils contain two structurally distinct types of antimicrobial peptides, β-sheet defensins (HNP-1 to HNP-4) and the α-helical peptide LL-37. We used radial diffusion assays and an improved National Committee for Clinical Laboratory Standards-type broth microdilution assay to compare the antimicrobial properties of LL-37, HNP-1, and protegrin (PG-1). Although generally less potent than PG-1, LL-37 showed considerable activity (MIC, <10 μg/ml) against Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli, Listeria monocytogenes, Staphylococcus epidermidis, Staphylococcus aureus, and vancomycin-resistant enterococci, even in media that contained 100 mM NaCl. Certain organisms (methicillin-resistant S. aureus, Proteus mirabilis, and Candida albicans) were resistant to LL-37 in media that contained 100 mM NaCl but were susceptible in low-salt media. Burkholderia cepacia was resistant to LL-37, PG-1, and HNP-1 in low- or high-salt media. LL-37 caused outer and inner membrane permeabilization of E. coli ML-35p. Chromogenic Limulus assays revealed that LL-37 bound to E. coli O111:B4 lipopolysaccharide (LPS) with a high affinity and that this binding showed positive cooperativity (Hill coefficient = 2.02). Circular dichroism spectrometry disclosed that LL-37 underwent conformational change in the presence of lipid A, transitioning from a random coil to an α-helical structure. The broad-spectrum antimicrobial properties of LL-37, its presence in neutrophils, and its inducibility in keratinocytes all suggest that this peptide and its precursor (hCAP-18) may protect skin and other tissues from bacterial intrusions and LPS-induced toxicity. The potent activity of LL-37 against P. aeruginosa, including mucoid and antibiotic-resistant strains, suggests that it or related molecules might have utility as topical bronchopulmonary microbicides in cystic fibrosis.     

Lipid Segregation Explains Selective Toxicity of a Series of Fragments Derived from the Human Cathelicidin LL-37

The only human cathelicidin, the 37-residue peptide LL-37, exhibits antimicrobial activity against both gram-positive and gram-negative bacteria. We studied the ability of several fragments of LL-37, exhibiting different antimicrobial activities, to interact with membranes whose compositions mimic the cytoplasmic membranes of gram-positive or of gram-negative bacteria. These fragments are as follows: KR-12, the smallest active segment of LL-37, with the sequence KRIVQRIKDFLR, which exhibits antimicrobial activity only against gram-negative bacteria; a slightly smaller peptide, RI-10, missing the two cationic residues at the N and C termini of KR-12, which has been shown not to have any antimicrobial activity; a longer peptide, GF-17, which shows antimicrobial activity against gram-positive as well as gram-negative bacteria; and GF-17D3, with 3 d-amino-acid residues, which is also selective only for gram-negative bacteria. Those fragments with the capacity to cluster anionic lipids away from zwitterionic lipids in a membrane exhibit selective toxicity toward bacteria containing zwitterionic as well as anionic lipids in their cytoplasmic membranes but not toward bacteria with only anionic lipids. This finding allows for the prediction of the bacterial-species selectivity of certain agents and paves the way for designing new antimicrobials targeted specifically toward gram-negative bacteria.The increasing emergence of bacterial strains that are resistant to traditional antibiotics makes the development of a new generation of antimicrobial agents an urgent matter. Naturally occurring antimicrobial peptides (there are more than 1,450 entries in the Antimicrobial Peptide Database) (33) are promising candidates because of their potency and low toxicity. These compounds are found in all life forms, indicating their important role as key defense molecules of the innate immune systems of nearly all life forms. Defensins and cathelicidins are the two major families of host defense peptides found in mammals. In humans, the peptide LL-37 represents the only member of the cathelicidin family that has been identified. LL-37 has an important role in protecting humans from infections. Patients lacking LL-37 are found to be more prone to infections (23), as are LL-37 knockout mice (19). LL-37 is found at lower concentrations in the airways of individuals with cystic fibrosis who are more prone to infection (5). In vitro, this human cathelicidin peptide shows antimicrobial activity against gram-positive bacteria, gram-negative bacteria, viruses, and fungi (30, 34). A variety of approaches were applied in identifying fragments of LL-37 that retain antimicrobial activity (4, 13, 18, 25, 26). In a recent study, a smaller fragment of LL-37, corresponding to residues 18 to 29 (designated KR-12), was found to be the smallest LL-37 fragment that retained activity against gram-negative bacteria (32), despite having only 12 of the 37 residues of the native peptide. KR-12 is similar to the core peptide identified by structural analysis of LL-37, using total correlated spectroscopy by trimming nonessential regions (12). The identification of short, active peptides from LL-37 facilitates peptide synthesis and structure-activity studies. It may also provide insights into novel mechanisms of action of antimicrobial agents. In this work, we evaluate the antimicrobial activities of a panel of LL-37-derived peptides against the gram-negative bacterium Escherichia coli (K-12) as well as two strains of the gram-positive bacteria Staphylococcus aureus UAMS-1 (methicillin [meticillin]-sensitive S. aureus [MSSA]) and USA300 (methicillin-resistant S. aureus [MRSA]). These two species of bacteria have very different lipid compositions in their membranes (Table ​(Table1),1), and these lipid compositions have a profound influence on the relative toxicities of different peptides. We measured the interactions of these peptides with membranes having lipid compositions corresponding to those of these two bacterial species. Our work not only provides evidence for a novel mechanism of action but also explains the specificities of certain agents toward different species of bacteria.

TABLE 1.

Lipid compositions of the bacterial strains used

Antimicrobial Peptide GL13K Is Effective in Reducing Biofilms of Pseudomonas aeruginosa

Human parotid secretory protein (PSP; BPIF2A) is predicted to be structurally similar to bactericidal/permeability-increasing protein and lipopolysaccharide (LPS)-binding protein. Based on the locations of known antimicrobial peptides in the latter two proteins, potential active peptides in the PSP sequence were identified. One such peptide, GL13NH₂ (PSP residues 141 to 153) was shown previously to interfere with LPS binding and agglutinate bacteria without bactericidal activity. By introducing three additional positively charged lysine residues, the peptide was converted to the novel bactericidal cationic peptide GL13K (MIC for Pseudomonas aeruginosa, 8 μg/ml [5.6 μM]). We investigated the antibiofilm activity of GL13K against static, monospecies biofilms of P. aeruginosa PAO1. Two-hour exposure of a 24-h biofilm to 64 μg/ml (44.8 μM) GL13K reduced biofilm bacteria by 10², and 100 μg/ml (70 μM) GL13K reduced bacteria by 10³. Similar results could be achieved on 48-h-old biofilms. Lower concentrations of GL13K (32 μg/ml [22.4 μM]) were successful in reducing biofilm cell numbers in combination with tobramycin. This combination treatment also achieved total eradication of the biofilm in a majority (67.5%) of tested samples. An alanine scan of GL13K revealed the importance of the leucine residue in position six of the peptide sequence, where replacement led to a loss of antibiofilm activity, whereas the impact of replacing charged residues was less pronounced. Bacterial metalloproteases were found to partially inactivate GL13K but not a d amino acid version of the peptide.     

Bactericidal Activities of Cathelicidin LL-37 and Select Cationic Lipids against the Hypervirulent Pseudomonas aeruginosa Strain LESB58

Pseudomonas aeruginosa Liverpool epidemic strain (LES) infections in cystic fibrosis (CF) patients are associated with transmissibility and increased patient morbidity. This study was designed to assess the in vitro activities of cathelicidin LL-37 peptide (LL-37) and select cationic lipids against Pseudomonas aeruginosa LESB58 in CF sputum and in a setting mimicking the CF airway. We found that LL-37 naturally present in airway surface fluid and some nonpeptide cationic lipid molecules such as CSA-13, CSA-90, CSA-131, and D2S have significant, but broadly differing, bactericidal activities against P. aeruginosa LESB58. We observed strong inhibition of LL-37 bactericidal activity in the presence of purified bacteriophage Pf1, which is highly expressed by P. aeruginosa LES, but the activities of the cationic lipids CSA-13 and CSA-131 were not affected by this polyanionic virus. Additionally, CSA-13 and CSA-131 effectively prevent LESB58 biofilm formation, which is stimulated by Pf1 bacteriophage, DNA, or F-actin. CSA-13 and CSA-131 display strong antibacterial activities against different clinical strains of P. aeruginosa, and their activities against P. aeruginosa LESB58 and Xen5 strains were maintained in CF sputum. These data indicate that synthetic cationic lipids (mimics of natural antimicrobial peptides) are suitable for developing an effective treatment against CF lung P. aeruginosa infections, including those caused by LES strains.     

Sodium Nitrite Blocks the Activity of Aminoglycosides against Pseudomonas aeruginosa Biofilms

Sodium nitrite has broad antimicrobial activity at pH 6.5, including the ability to prevent biofilm growth by Pseudomonas aeruginosa on the surfaces of airway epithelial cells. Because of its antimicrobial activity, nitrite is being investigated as an inhaled agent for chronic P. aeruginosa airway infections in cystic fibrosis patients. However, the interaction between nitrite and commonly used aminoglycosides is unknown. This paper investigates the interaction between nitrite and tobramycin in liquid culture, abiotic biofilms, and a biotic biofilm model simulating the conditions in the cystic fibrosis airway. The addition of nitrite prevented killing by aminoglycosides in liquid culture, with dose dependence between 1.5 and 15 mM. The effect was not blocked by the nitric oxide scavenger CPTIO or dependent on efflux pump activity. Nitrite shifted the biofilm minimal bactericidal concentration (MBC-biofilm) from 256 μg/ml to >1,024 μg/ml in an abiotic biofilm model. In a biotic biofilm model, the addition of 50 mM nitrite decreased the antibiofilm activity of tobramycin by up to 1.2 log. Respiratory chain inhibition recapitulated the inhibition of aminoglycoside activity by nitrite, suggesting a potential mechanism of inhibition of energy-dependent aminoglycoside uptake. In summary, sodium nitrite induces resistance to both gentamicin and tobramycin in P. aeruginosa grown in liquid culture, as an abiotic biofilm, or as a biotic biofilm.     

Dermcidin-Derived Peptides Show a Different Mode of Action than the Cathelicidin LL-37 against Staphylococcus aureus

Dermcidin (DCD) is an antimicrobial peptide which is constitutively expressed in eccrine sweat glands. By postsecretory proteolytic processing in sweat, the DCD protein gives rise to anionic and cationic DCD peptides with a broad spectrum of antimicrobial activity. Many antimicrobial peptides induce membrane permeabilization as part of their killing mechanism, which is accompanied by a loss of the bacterial membrane potential. In this study we show that there is a time-dependent bactericidal activity of anionic and cationic DCD-derived peptides which is followed by bacterial membrane depolarization. However, DCD-derived peptides do not induce pore formation in the membranes of gram-negative and gram-positive bacteria. This is in contrast to the mode of action of the cathelicidin LL-37. Interestingly, LL-37 as well as DCD-derived peptides inhibit bacterial macromolecular synthesis, especially RNA and protein synthesis, without binding to microbial DNA or RNA. Binding studies with components of the cell envelope of gram-positive and gram-negative bacteria and with model membranes indicated that DCD-derived peptides bind to the bacterial envelope but show only a weak binding to lipopolysaccharide (LPS) from gram-negative bacteria or to peptidoglycan, lipoteichoic acid, and wall teichoic acid, isolated from Staphylococcus aureus. In contrast, LL-37 binds strongly in a dose-dependent fashion to these components. Altogether, these data indicate that the mode of action of DCD-derived peptides is different from that of the cathelicidin LL-37 and that components of the bacterial cell envelope play a role in the antimicrobial activity of DCD.Antimicrobial peptides (AMPs) serve as a first line of innate host defense in many species such as plants, amphibians, insects, and mammals. AMPs show a broad-spectrum antimicrobial activity against a wide range of pathogens including bacteria, fungi, and enveloped viruses (51). Most gene-encoded AMPs are synthesized as proforms, which are subsequently processed into mature peptides of various lengths. A common feature of most of these peptides is that they are cationic and form amphipathic structures (3). The mode of action of most AMPs is incompletely understood. It is believed that most AMPs kill microorganisms by membrane permeation either via pore formation or via membrane disintegration like that induced by the human cathelicidin LL-37. However, membrane disruption may not reflect the complex processes involved in the killing of microorganisms (5, 48). In addition, several AMPs clearly act differently and intracellular target sites have been identified (15). The mode of action has been unraveled for only a few AMPs which act via defined targets, such as the lantibiotic nisin, which specifically binds to bacterial lipid II, a membrane-bound component involved in peptidoglycan (PG) synthesis (7, 46). Similarly, the lantibiotic mersacidin interferes with transglycosylation and PG synthesis in gram-positive bacteria by direct targeting of lipid II (6). Furthermore, buforin II kills microorganisms by disruption of critical intracellular processes such as the inhibition of macromolecular biosynthesis or by interacting with nucleic acids inside the microorganisms (33). For several AMPs it has been demonstrated that the charge and the composition of the bacterial cell envelope determine sensitivity to AMPs (37). Staphylococcus aureus mutants lacking specific modifications in the bacterial envelope are highly susceptible to a variety of cationic AMPs. For example, incorporation of d-alanine into S. aureus teichoic acids by the dltA enzymes or the lysinylation of phosphatidylglycerol by mprF confers resistance to defensins, protegrins, and other AMPs by repulsion of the cationic peptides (36).Dermcidin (DCD) was identified by our group as a human AMP which is constitutively expressed in eccrine sweat glands and secreted into sweat (40). By postsecretory proteolytic processing in human sweat, the precursor protein gives rise to several truncated DCD peptides varying in length from 25 to 48 amino acids and with net charges between −2 and +2 (2, 10, 38). Several DCD peptides show antimicrobial activity against pathogenic microorganisms such as S. aureus, Escherichia coli, Enterococcus faecalis, Candida albicans, Staphylococcus epidermidis, Pseudomonas putida, and methicillin-resistant S. aureus as well as rifampin- and isoniazid-resistant Mycobacterium tuberculosis (9, 25, 40, 41, 45). We were able to show that DCD-derived peptides are also active under high-salt conditions and in a buffer resembling human sweat (40). Antimicrobially active DCD peptides, namely, the anionic peptides DCD-1L (48-mer) and DCD-1 (47-mer) and the cationic peptides SSL-25 (25-mer) and SSL-23 (23-mer), are derived from the C-terminal region of the precursor protein. Interestingly, these peptides have diverse and overlapping spectra of activity which are independent of the net peptide charge (41). In previous studies we showed that DCD peptides interact with the bacterial cell envelope and kill gram-negative bacteria without forming pores in membranes (41). In this study we investigated the mode of antimicrobial activity of DCD-derived peptides in more detail and studied bacterial factors that govern sensitivity or tolerance to DCD in the model microorganism S. aureus. In our first approach, we tried to identify the bacterial surface molecules to which DCD peptides bind. Second, we analyzed the bacterial response to DCD peptide challenge. Finally, we analyzed bacterial mutants to elucidate the mechanisms determining DCD sensitivity.     

In Vitro Efficacy of a Novel Active-Release Antimicrobial Coating To Eradicate Biofilms of Pseudomonas aeruginosa

Implant-related infections are becoming increasingly difficult to treat due to the formation of biofilms on implant surfaces. This study analyzed the in vitro efficacy of a novel antimicrobial coating against biofilms of Pseudomonas aeruginosa, using a flow cell system. Results indicated that P. aeruginosa biofilms were reduced by greater than 8 log₁₀ units in less than 24 h. Data indicated that this active-release coating may be promising for preventing biofilm implant-related infections.     

Evaluation of Strategies for Improving Proteolytic Resistance of Antimicrobial Peptides by Using Variants of EFK17, an Internal Segment of LL-37

Methods for increasing the proteolytic stability of EFK17 (EFKRIVQRIKDFLRNLV), a new peptide sequence with antimicrobial properties derived from LL-37, were evaluated. EFK17 was modified by four d-enantiomer or tryptophan (W) substitutions at known protease cleavage sites as well as by terminal amidation and acetylation. The peptide variants were studied in terms of proteolytic resistance, antibacterial potency, and cytotoxicity but also in terms their adsorption at model lipid membranes, liposomal leakage generation, and secondary-structure behavior. The W substitutions resulted in a marked reduction in the proteolytic degradation caused by human neutrophil elastase, Staphylococcus aureus aureolysin, and V8 protease but not in the degradation caused by Pseudomonas aeruginosa elastase. For the former two endoproteases, amidation and acetylation of the terminals also reduced proteolytic degradation but only when used in combination with W substitutions. The d-enantiomer substitutions rendered the peptides indigestible by all four proteases; however, those peptides displayed little antimicrobial potency. The W- and end-modified peptides, on the other hand, showed an increased bactericidal potency compared to that of the native peptide sequence, coupled with a moderate cytotoxicity that was largely absent in serum. The bactericidal, cytotoxic, and liposome lytic properties correlated with each other as well as with the amount of peptide adsorbed at the lipid membrane and the extent of helix formation associated with the adsorption. The lytic properties of the W-substituted peptides were less impaired by increased ionic strength, presumably by a combination of W-mediated stabilization of the largely amphiphilic helix conformation and a nonelectrostatic W affinity for the bilayer interface. Overall, W substitutions constitute an interesting means to reduce the proteolytic susceptibility of EFK17 while also improving antimicrobial performance.     

Identification of Novel Human Immunodeficiency Virus Type 1-Inhibitory Peptides Based on the Antimicrobial Peptide Database

To identify novel anti-HIV-1 peptides based on the antimicrobial peptide database (APD; http://aps.unmc.edu/AP/main.php), we have screened 30 candidates and found 11 peptides with 50% effective concentrations (EC₅₀) of <10 μM and therapeutic indices (TI) of up to 17. Furthermore, among the eight peptides (with identical amino acid compositions but different sequences) generated by shuffling the sequence of an aurein 1.2 analog, two had a TI twice that of the original sequence. Because antiviral peptides in the database have an arginine/lysine (R/K) ratio of >1, increases in the Arg contents of amphibian maximin H5 and dermaseptin S9 peptides and the database-derived GLK-19 peptide improved the TIs. These examples demonstrate that the APD is a rich resource and a useful tool for developing novel HIV-1-inhibitory peptides.AIDS has become the fourth leading cause of death worldwide, and the majority of human immunodeficiency virus (HIV) infections are acquired through heterosexual intercourse. The United Nations estimates that there are now 40 million people living with HIV infection/AIDS. Thus, it is urgent to develop novel therapeutic and preventative agents. Effective HIV vaccines are not yet available, making the development of topical microbicides that prevent the sexual transmission of HIV desirable (2, 16). Naturally occurring antimicrobial peptides (AMPs) are potent host defense molecules in all life forms (8, 9, 23). More than 1,500 such peptides have been registered in the antimicrobial peptide database (APD; http://aps.unmc.edu/AP/main.html) (19, 20). Prior to this study, a search of the database found entries for 68 peptides with known HIV-inhibitory activity. Some typical examples are melittin and cecropin (17), polyphemusin-derived T22 (21), LL-37 (1, 18), α-, β-, and θ-defensins (5, 9), cyclotides (7), and indolicidin (15). To date, less than 5% of the AMPs in the database have been evaluated, and thus, we hypothesize that the APD constitutes a useful resource for identifying novel HIV-inhibitory peptides. To test this hypothesis, we illustrate database-aided identification of new anti-HIV peptides using three different approaches: database screening, peptide sequence shuffling, and database-based peptide design.All the peptides (>95% pure) were purchased from Genemed Synthesis, Inc., TX. The well-accepted and highly standardized cytopathic effect (CPE) inhibition assay with CEM-SS cells was utilized as the primary screening assay to evaluate the efficacies and toxicities of compounds with unknown anti-HIV activities (3). The titer of the laboratory strain HIV-1_IIIB used is equivalent to a 50% tissue culture infective dose of approximately 1,000 infectious units per ml and results in an in-well multiplicity of infection of 0.0005 to 0.001 (14). CEM-SS cells are human T4 lymphoblastoid cells that have been cloned for virus-induced syncytial and fusogenic sensitivity following infection with HIV-1_IIIB (6, 11, 12). Both HIV-1_IIIB and the CEM-SS cells were obtained from the NIH AIDS Research and Reference Reagent Program (Rockville, MD). The cells were grown in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 5% penicillin-streptomycin, and 5% l-glutamine. CEM-SS cells at a concentration of 2.5 × 10³ cells per well and HIV-1_IIIB at the above-mentioned predetermined titer were sequentially added to 96-well round-bottom microtiter plates. Serially diluted peptides were added to the plates in triplicate. Each peptide was evaluated at six concentrations ranging from 0.32 to 100 μg/ml. The cultures were incubated at 5% CO₂ and 37°C for 6 days. Following incubation, the microtiter plates were stained with XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] dye (Sigma-Aldrich, St. Louis, MO) to evaluate the efficacies and toxicities of the test compounds (22). By using Microsoft Excel, the 50% effective concentration (EC₅₀; the concentration causing 50% inhibition of virus replication), the 50% toxic concentration (TC₅₀; the concentration causing 50% reduction in cell viability), and the therapeutic index (TI; the TC₅₀/EC₅₀ ratio) were calculated for each peptide. For Tables ​Tables11 to ​to3,3, the EC₅₀s and TC₅₀s were converted into micromolar values based on peptide molecular weights.

TABLE 1.

Findings from database-based anti-HIV-1_IIIB peptide screening

Bactericidal Efficiency and Modes of Action of the Novel Antimicrobial Peptide T9W against Pseudomonas aeruginosa

The antipseudomonal efficiency and mechanism of action of a novel engineered antimicrobial peptide, T9W, were evaluated in this study. T9W displayed high activity, with a lethal concentration (LC) of 1 to 4 μM against Pseudomonas aeruginosa, including against ciprofloxacin-, gentamicin-, and ceftazidime-resistant strains, even in the presence of 50 to 300 mM NaCl, 1 to 5 mM Ca²⁺, or 0.5 to 2 mM Mg²⁺. The time-kill curve (TKC) analysis demonstrated concentration-dependent activity, with T9W achieving complete killing in less than 30 min at 1× LC and in less than 5 min at 4× LC. Combination TKC analyses additionally demonstrated a synergistic effect with ciprofloxacin and gentamicin. The selectivity of T9W was further supported by its ability to specifically eliminate P. aeruginosa in a coculture with macrophages without toxicity to the mammalian cells. The results from fluorescent measurement indicated that T9W bound to lipopolysaccharide (LPS) and induced P. aeruginosa membrane depolarization, and microscopic observations and flow cytometry further indicated that T9W targeted the P. aeruginosa cell membrane and disrupted cytoplasmic membrane integrity, thereby causing cellular content release leading to cell death. This study revealed the potential usefulness of T9W as a novel antimicrobial agent against P. aeruginosa.     

Antipseudomonal Agents Exhibit Differential Pharmacodynamic Interactions with Human Polymorphonuclear Leukocytes against Established Biofilms of Pseudomonas aeruginosa

Pseudomonas aeruginosa is the most common pathogen infecting the lower respiratory tract of cystic fibrosis (CF) patients, where it forms tracheobronchial biofilms. Pseudomonas biofilms are refractory to antibacterials and to phagocytic cells with innate immunity, leading to refractory infection. Little is known about the interaction between antipseudomonal agents and phagocytic cells in eradication of P. aeruginosa biofilms. Herein, we investigated the capacity of three antipseudomonal agents, amikacin (AMK), ceftazidime (CAZ), and ciprofloxacin (CIP), to interact with human polymorphonuclear leukocytes (PMNs) against biofilms and planktonic cells of P. aeruginosa isolates recovered from sputa of CF patients. Three of the isolates were resistant and three were susceptible to each of these antibiotics. The concentrations studied (2, 8, and 32 mg/liter) were subinhibitory for biofilms of resistant isolates, whereas for biofilms of susceptible isolates, they ranged between sub-MIC and 2 × MIC values. The activity of each antibiotic alone or in combination with human PMNs against 48-h mature biofilms or planktonic cells was determined by XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] assay. All combinations of AMK with PMNs resulted in synergistic or additive effects against planktonic cells and biofilms of P. aeruginosa isolates compared to each component alone. More than 75% of CAZ combinations exhibited additive interactions against biofilms of P. aeruginosa isolates, whereas CIP had mostly antagonistic interaction or no interaction with PMNs against biofilms of P. aeruginosa. Our findings demonstrate a greater positive interaction between AMK with PMNs than that observed for CAZ and especially CIP against isolates of P. aeruginosa from the respiratory tract of CF patients.     

Efficacy of LL-37 and granulocyte colony-stimulating factor in a neutropenic murine sepsis due to Pseudomonas aeruginosa

A promising therapeutic strategy for the management of severe Pseudomonas infection in neutropenic patients may result from the coadministration of colony-stimulating factors (CSFs) that help maintain immune competence and antimicrobial peptides, a novel generation of adjunctive therapeutic agents with antimicrobial and anti-inflammatory properties. A promising peptide with these properties is LL-37, the only member of the cathelicidin family of antimicrobial peptides found in humans. BALB/c male mice were rendered neutropenic by intraperitoneal administration of cyclophosphamide on days -4 and -2 preinfection. Septic shock was induced at time 0 by intraperitoneal injection of 2x10 colony-forming units of P. aeruginosa American Type Culture Collection (ATCC) 27853. All animals were randomized to receive intravenously isotonic sodium chloride solution, 1 mg/kg of LL-37, 20 mg/kg of imipenem, 0.1 mg/kg of granulocyte CSF (G-CSF), 1 mg/kg of LL-37+0.1 mg/kg of G-CSF, or 20 mg/kg of imipenem+0.1 mg/kg of G-CSF. Lethality and bacterial growth in blood, peritoneum, spleen, liver, and kidney were evaluated. All regimens were significantly superior to controls at reducing the mouse lethality rate and bacterial burden in organs. Particularly, the combination between LL-37 and G-CSF was the most effective in protecting neutropenic mice from the onset of sepsis and in vitro significantly reduced the apoptosis of neutrophils. Combination therapy between LL-37 and G-CSF is a promising therapeutic strategy for the management of severe Pseudomonas infection complicated by neutropenia.     

Therapeutic Potential of the Antimicrobial Peptide OH-CATH30 for Antibiotic-Resistant Pseudomonas aeruginosa Keratitis

The therapeutic potential of antimicrobial peptides (AMPs) has been evaluated in many infectious diseases. However, the topical application of AMPs for ocular bacterial infection has not been well investigated. The AMP OH-CATH30, which was identified in the king cobra, exhibits potent antimicrobial activity. In this study, we investigated the therapeutic potential of OH-CATH30 for Pseudomonas aeruginosa keratitis. Ten isolates of P. aeruginosa from individuals with keratitis were susceptible to OH-CATH30 but not to cefoperazone, ciprofloxacin, gentamicin, and levofloxacin. The microdilution checkerboard assay showed that OH-CATH30 exhibited synergistic activity with ciprofloxacin and levofloxacin against antibiotic-resistant P. aeruginosa. Meanwhile, P. aeruginosa did not develop resistance to OH-CATH30, even after exposure at 0.5× the MIC for up to 25 subcultures. Furthermore, treatment with OH-CATH30, alone or in combination with levofloxacin, significantly improved the clinical outcomes of rabbit keratitis induced by antibiotic-resistant P. aeruginosa. Taken together, our data indicate that the topical application of OH-CATH30 is efficacious against drug-resistant P. aeruginosa keratitis. In addition, our study highlights the potential application of AMPs in treating ocular bacterial infections.     

In Vitro Activity of the Novel Antimicrobial Peptide Dendrimer G3KL against Multidrug-Resistant Acinetobacter baumannii and Pseudomonas aeruginosa

The in vitro activity of the novel antimicrobial peptide dendrimer G3KL was evaluated against 32 Acinetobacter baumannii (including 10 OXA-23, 7 OXA-24, and 11 OXA-58 carbapenemase producers) and 35 Pseudomonas aeruginosa (including 18 VIM and 3 IMP carbapenemase producers) strains and compared to the activities of standard antibiotics. Overall, both species collections showed MIC_50/90 values of 8/8 μg/ml and minimum bactericidal concentrations at which 50% or 90% of strains tested are killed (MBC_50/90) of 8/8 μg/ml. G3KL is a promising molecule with antibacterial activity against multidrug-resistant and extensively drug-resistant A. baumannii and P. aeruginosa isolates.     

Antimicrobial Activity of Fosfomycin-Tobramycin Combination against Pseudomonas aeruginosa Isolates Assessed by Time-Kill Assays and Mutant Prevention Concentrations

The antibacterial activity of fosfomycin-tobramycin combination was studied by time-kill assay in eight Pseudomonas aeruginosa clinical isolates belonging to the fosfomycin wild-type population (MIC = 64 μg/ml) but with different tobramycin susceptibilities (MIC range, 1 to 64 μg/ml). The mutant prevention concentration (MPC) and mutant selection window (MSW) were determined in five of these strains (tobramycin MIC range, 1 to 64 μg/ml) in aerobic and anaerobic conditions simulating environments that are present in biofilm-mediated infections. Fosfomycin-tobramycin was synergistic and bactericidal for the isolates with mutations in the mexZ repressor gene, with a tobramycin MIC of 4 μg/ml. This effect was not observed in strains displaying tobramycin MICs of 1 to 2 μg/ml due to the strong bactericidal effect of tobramycin alone. Fosfomycin presented higher MPC values (range, 2,048 to >2,048 μg/ml) in aerobic and anaerobic conditions than did tobramycin (range, 16 to 256 μg/ml). Interestingly, the association rendered narrow or even null MSWs in the two conditions. However, for isolates with high-level tobramycin resistance that harbored aminoglycoside nucleotidyltransferases, time-kill assays showed no synergy, with wide MSWs in the two environments. glpT gene mutations responsible for fosfomycin resistance in P. aeruginosa were determined in fosfomycin-susceptible wild-type strains and mutant derivatives recovered from MPC studies. All mutant derivatives had changes in the GlpT amino acid sequence, which resulted in a truncated permease responsible for fosfomycin resistance. These results suggest that fosfomycin-tobramycin can be an alternative for infections due to P. aeruginosa since it has demonstrated synergistic and bactericidal activity in susceptible isolates and those with low-level tobramycin resistance. It also prevents the emergence of resistant mutants in either aerobic or anaerobic environments.     

d-Amino Acids Enhance the Activity of Antimicrobials against Biofilms of Clinical Wound Isolates of Staphylococcus aureus and Pseudomonas aeruginosa

Within wounds, microorganisms predominantly exist as biofilms. Biofilms are associated with chronic infections and represent a tremendous clinical challenge. As antibiotics are often ineffective against biofilms, use of dispersal agents as adjunctive, topical therapies for the treatment of wound infections involving biofilms has gained interest. We evaluated in vitro the dispersive activity of d-amino acids (d-AAs) on biofilms from clinical wound isolates of Staphylococcus aureus and Pseudomonas aeruginosa; moreover, we determined whether combinations of d-AAs and antibiotics (clindamycin, cefazolin, oxacillin, rifampin, and vancomycin for S. aureus and amikacin, colistin, ciprofloxacin, imipenem, and ceftazidime for P. aeruginosa) enhance activity against biofilms. d-Met, d-Phe, and d-Trp at concentrations of ≥5 mM effectively dispersed preformed biofilms of S. aureus and P. aeruginosa clinical isolates, an effect that was enhanced when they were combined as an equimolar mixture (d-Met/d-Phe/d-Trp). When combined with d-AAs, the activity of rifampin was significantly enhanced against biofilms of clinical isolates of S. aureus, as indicated by a reduction in the minimum biofilm inhibitory concentration (MBIC) (from 32 to 8 μg/ml) and a >2-log reduction of viable biofilm bacteria compared to treatment with antibiotic alone. The addition of d-AAs was also observed to enhance the activity of colistin and ciprofloxacin against biofilms of P. aeruginosa, reducing the observed MBIC and the number of viable bacteria by >2 logs and 1 log at 64 and 32 μg/ml in contrast to antibiotics alone. These findings indicate that the biofilm dispersal activity of d-AAs may represent an effective strategy, in combination with antimicrobials, to release bacteria from biofilms, subsequently enhancing antimicrobial activity.     

Antimicrobial activity of subinhibitory concentrations of aminoglycosides against Pseudomonas aeruginosa as determined by the killing-curve method and the postantibiotic effect

This investigation used the postantibiotic effect (PAE) and killing curves to provide data on the antimicrobial activity of subinhibitory (1/8x, 1/4x and 1/2x minimum inhibitory concentration; MIC) and inhibitory (1x MIC) concentrations of amikacin, gentamicin and tobramycin against Pseudomonas aeruginosa. Subinhibitory concentrations (1/4x and 1/2x MIC) of aminoglycosides demonstrated a reproducible PAE. At 1/4x MIC, the order of duration of the PAE was approximately 15 min for all aminoglycosides, while at 1/2x MIC all three aminoglycosides displayed a similar PAE of approximately 40 min. Killing-curve studies demonstrated that subinhibitory concentrations of aminoglycosides either decrease bacterial growth for several hours (1/4x and 1/2x MIC) or produce stasis of growth for several hours (1/8x MIC). Only inhibitory aminoglycoside concentrations (1x MIC) proved to be bactericidal. Subinhibitory concentrations of aminoglycosides decrease bacterial growth and produce a PAE against P. aeruginosa.     

隐球菌保护性肽表位GMFDGLSGV多聚体重组表达及鉴定

目的重组表达隐球菌保护性肽表位GMFDGLSGV二聚体、四聚体、八聚体，以进一步探讨其免疫效应。方法选取隐球菌抗原表位GMFDGLSGV，引入Th表位PADRE及木马肽序列（RKKRRQRRR），并以RVKR序列作为接头，分别合成隐球菌保护性肽表位GMFDGLSGV二聚体、四聚体、八聚体全基因序列，经PCR扩增后分别插入融合表达载体pGEX-4T-1，将重组质粒转入大肠埃希氏菌BL21并以IPTG诱导表达，进一步经GST亲舍层析柱纯化，经SDS-PAGE和Western—blot鉴定重组表达蛋白。结果成功构建了隐球菌保护性表位GMFDGLSGV二聚体、四聚体、八聚体的表达质粒，并经IPTG诱导表达了大小约为32000，36000，42000的融合蛋白。结论隐球菌保护性肽表位GMFDGLSGV多聚体的重组表达为进一步开发隐球菌疫苗提供有价值的依据。     

Evaluation of Antimicrobial and Lipopolysaccharide-Neutralizing Effects of a Synthetic CAP18 Fragment against Pseudomonas aeruginosa in a Mouse Model

CAP18 (cationic antimicrobial protein; 18 kDa) is a neutrophil-derived protein that can bind to and inhibit various activities of lipopolysaccharide (LPS). The 37 C-terminal amino acids of CAP18 make up the LPS-binding domain. A truncated 32-amino-acid C-terminal fragment of CAP18 had potent activity against Pseudomonas aeruginosa in vitro. We studied the antimicrobial and LPS-neutralizing effects of this synthetic truncated CAP18 peptide (CAP18_106–137) on lung injury in mice infected with cytotoxic P. aeruginosa. To determine its maximal effect, the CAP18_106–137 peptide was mixed with bacteria just prior to tracheal instillation, and lung injury was evaluated by determining the amount of leakage of an alveolar protein tracer (¹²⁵I-albumin) into the circulation and by the quantification of lung edema. The lung injury caused by the instillation of 5 × 10⁵ CFU of P. aeruginosa was significantly reduced by the concomitant instillation of CAP18_106–137. However, the administration of CAP18_106–137 alone, without bacteria, induced lung edema, suggesting that it has some toxicity. Also, the peptide did not significantly reduce the number of bacteria that had been simultaneously instilled, nor did it significantly improve the survival of the infected mice. The addition of CAP18_106–137 to aztreonam along with the bacteria did decrease the level of antibiotic-induced release of inflammatory mediators including tumor necrosis factor alpha, interleukin-6, and nitric oxide and also improved the survival of the mice. Therefore, more investigations are needed to confirm the toxicities and the therapeutic benefits of CAP18_106–137 as an adjunctive therapy to antibiotics in the treatment of infections caused by gram-negative bacteria.