Bactericidal effect of bovine lactoferrin, LFcin, LFampin and LFchimera on antibiotic-resistant Staphylococcus aureus and Escherichia coli

Increased prevalence of antibiotic-resistant bacteria has become a major threat to the health sector worldwide due to their virulence, limited therapeutic options and distribution in both hospital and community settings. Discovery and development of new agents to combat antibiotic-resistant bacteria is thus needed. This study therefore aimed to evaluate the ability of bovine lactoferrin (LF), peptides from two antimicrobial domains lactoferricin B (LFcin17-30) and lactoferrampin (LFampin265-284) and a chimeric construct (LFchimera) containing both peptides, as potential bactericidal agents against clinical isolates of antibiotic-resistant Staphylococcus aureus and Escherichia coli. Results in kinetics of growth show that LF chimera and peptides inhibited the growth of both bacterial species. By confocal microscopy and flow cytometry it was observed that LF and FITC-labeled peptides are able to interact with these bacteria and cause membrane permeabilization, as monitored by propidium iodide staining, these effects were decreased by preincubation with lipopolysaccharide in E. coli. By electron microscopy, a clear cellular damage was observed in bacteria after treatments with LFchimera and peptides, suggesting that interaction and membrane disruption are probably involved as a mechanism of action. In conclusion, results show that LFchimera, LF and peptides have potential as bactericidal agents in the antibiotic-resistant strains of S. aureus and E. coli and also the work strongly suggest that LFcin17-30 and LFampin265-284 acts synergistically with antibiotics against multidrug resistant EPEC and MRSA in vitro.     

Isolation and identification of lipopeptide antibiotics from <Emphasis Type="Italic">Paenibacillus elgii</Emphasis> B69 with inhibitory activity against methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Two lipopeptide antibiotics, pelgipeptins C and D, were isolated from Paenibacillus elgii B69 strain. The molecular masses of the two compounds were both determined to be 1,086 Da. Mass-spectrometry, amino acid analysis and NMR spectroscopy indicated that pelgipeptin C was the same compound as BMY-28160, while pelgipeptin D was identified as a new antibiotic of the polypeptin family. These two peptides were active against all the tested microorganisms, including antibiotic-resistant pathogenic bacterial strains such as methicillin-resistant Staphylococcus aureus (MRSA). Time-kill assays demonstrated that pelgipeptin D exhibited rapid and effective bactericidal action against MRSA at 4×MIC. Based on acute toxicity test, the intraperitoneal LD50 value of pelgipeptin D was slightly higher than that of the structurally related antimicrobial agent polymyxin B. Pelgipeptins are highly potent antibacterial and antifungal agents, particularly against MRSA, and warrant further investigation as possible therapeutic agents for bacteria infections resistant to currently available antibiotics.     

StCT2, a new antibacterial peptide characterized from the venom of the scorpion Scorpiops tibetanus

Bacterial infection poses an increasing threat to global public health and new types of antibacterial agents are urgently needed to respond to the threat. Scorpion venom contains series of bioactive peptides, among which antibacterial peptide is an important part. Herein, a new antimicrobial peptide StCT2 was characterized from the venomous gland cDNA library of the Scorpiops tibetanus. The full-length cDNA of StCT2 is 369 nucleotides encoding the precursor that contains a putative 24 residues signal peptide, a presumed 14 residues mature peptide, and a putative 37 residues acidic propeptide at the C-terminus. The minimal inhibition concentrations (MICs) of StCT2 for Staphylococcus aureus were 6.25-25μg/ml, including antibiotic-resistant strains such as methicillin resistant S. aureus (MRSA). StCT2 was further found to show high in vivo antimicrobial activity by an S. aureus infection mouse model. StCT2 exerted its antimicrobial activity via a rapid bactericidal mechanism. Taken together, these results demonstrate the efficacy and general mechanism of StCT2 antimicrobial action and the therapeutic potential of StCT2 as a new antimicrobial peptide.     

Characterization of cell membrane parameters of clinical isolates of Staphylococcus aureus with varied susceptibility to alpha-melanocyte stimulating hormone

Staphylococcus aureus (S. aureus), a major human pathogen of hospital and community acquired infections, is becoming resistant to almost all commercially available antibiotics. This has prompted development of antimicrobial peptides as therapeutic options. Alpha melanocyte stimulating hormone (α-MSH) is one such peptide known to possess antimicrobial properties. In the present study, we analyzed the antimicrobial activity of α-MSH against 75 clinical strains of S. aureus including both methicillin susceptible S. aureus (MSSA) and methicillin resistant S. aureus (MRSA) strains. Results of our previous study showed that membrane damage is the major mechanism of staphylocidal activity of α-MSH. In this context, we compared the various bacterial membrane parameters, viz., membrane fluidity, lipid composition, and surface charge of a few selected MSSA and MRSA strains that showed variable susceptibility to the melanocortin peptide. Our results showed that α-MSH killed both type of strains efficiently (≥70% killing in 84% clinical strains after exposure with 6μM of α-MSH for 1h). It was observed that compared to the α-MSH-susceptible strains, the α-MSH-non-susceptible strains had a different membrane order and phospholipid pattern. There was no consistent pattern of cell surface charge to distinguish α-MSH-susceptible strain from a non-susceptible strain. In conclusion, α-MSH possessed potential staphylocidal activity for both against MSSA and MRSA strains. S. aureus strains not susceptible to the peptide exhibited a rigid membrane and a higher amount of the cationic phospholipid as compared to the α-MSH-susceptible strains.     

Synergistic interactions between cefalexin and kanamycin in Mueller–Hinton broth medium and in milk

Aims:  This study investigated the in vitro bactericidal activity of an intramammary drug product by comparing the kill kinetics of cefalexin and kanamycin, alone and in fixed ratio combination, against Streptococcus uberis , Staphylococcus aureus and Escherichia coli strains isolated from field cases of bovine mastitis. The effect of milk as a diluent on the rate of bacterial killing was also assessed.
Methods and Results:  Antibacterial kill kinetics was determined against each bacterial strain in Mueller–Hinton broth (MHB) and in milk. In MHB, the fixed cefalexin : kanamycin combination (1·5 : 1 w/w) exhibited a clear synergistic bactericidal activity against the strains tested. The combination also showed an enhanced killing activity in milk, as compared to either agent alone.
Conclusions:  The data show the occurrence of synergistic interactions between cefalexin and kanamycin, resulting in a faster and enhanced bactericidal activity against major mastitis pathogens.
Significance and Impact of the Study:  The study demonstrated that the combination exhibited a larger and faster rate of kill of S. aureus , S. uberis and E. coli compared to either cefalexin or kanamycin alone, while using a lower total amount of antibiotic. Synergistic and additive effects were also observed when milk was used as a medium. The results support the use of this combination of narrow spectrum antibiotics to treat clinical mastitis via the intramammary route and provide data on its killing kinetics.     

Beyond conventional antibiotics for the future treatment of methicillin-resistant Staphylococcus aureus infections: two novel alternatives

The majority of antibiotics currently used to treat methicillin-resistant Staphylococus aureus (MRSA) infections target bacterial cell wall synthesis or protein synthesis. Only daptomycin has a novel mode of action. Reliance on limited targets for MRSA chemotherapy, has contributed to antimicrobial resistance. Two alternative approaches to the treatment of S.?aureus infection, particularly those caused by MRSA, that have alternative mechanisms of action and that address the challenge of antimicrobial resistance are cationic host defence peptides and agents that target S.?aureus virulence. Cationic host defence peptides have multiple mechanisms of action and are less likely than conventional agents to select resistant mutants. They are amenable to modifications that improve their stability, effectiveness and selectivity. Some cationic defence peptides such as bactenecin, mucroporin and imcroporin have potent in vitro bactericidal activity against MRSA. Antipathogenic agents also have potential to limit the pathogenesis of S?aureus. These are generally small molecules that inhibit virulence targets in S.?aureus without killing the bacterium and therefore have limited capacity to promote resistance development. Potential antipathogenic targets include the sortase enzyme system, the accessory gene regulator (agr) and the carotenoid biosynthetic pathway. Inhibitors of these targets have been identified and these may have potential for further development.     

Antibiotic and synergistic effect of Leu-Lys rich peptide against antibiotic resistant microorganisms isolated from patients with cholelithiasis

Pseudomonas aeruginosa has eventually developed resistance against flomoxef sodium, isepamicin and cefpiramide. Therefore, in this study, the antibacterial activity and synergistic effects of the amphipathic-derived P5-18mer antimicrobial peptide were tested against pathogens associated with cholelithiasis that have developed resistance against commonly used antibiotics. The results were then compared with the activities of the amphipathic-derived peptide, P5-18mer, melittin and common antibiotics. Growth inhibition of planktonic bacteria was tested using the National Committee for Clinical Laboratory Standards (NCCLS). The bactericidal activity of the antimicrobial peptides was measured using time-kill curves. Synergistic effects were evaluated by testing the effects of P5-18mer alone and in combination with flomoxef sodium, isepamicin or cefpiramide at 0.5 × MIC. P5-18mer peptide displayed strong activity against pathogens and flomoxef sodium, isepamicin and cefpiramide-resistant bacteria cell lines obtained from a patient with gallstones; however, it did not exert cytotoxicity against the human keratinocyte HaCat cell line. In addition, the results of time-kill curves indicated that P5-18mer peptide exerted bactericidal activity against four strains of P. aeruginosa. Finally, the use of P5-18mer and antibiotics exerted synergistic effects against cell lines that were resistant to commonly used antibiotics. These results indicate that this class of peptides has a rapid microbicidal effect on flomoxef sodium, isepamicin and cefpiramide-resistant strains of P. aeruginosa. Therefore, these peptides may be used as a lead drug for the treatment of acquired pathogens from patients with cholelithiasis who are affected with antibiotic-resistant bacteria.     

Lactoferrin: an iron-binding antimicrobial protein against <Emphasis Type="Italic">Escherichia coli</Emphasis> infection

Escherichia coli (E. coli) are the most common aerobic gram-negative bacilli in a normal intestinal tract. They cause most of the intra-abdominal infections, wound infections associated with abdominal surgery, and septicemia. Most of these infections are of endogenous intestinal origin. Lactoferrin (LF) is an iron-binding glycoprotein found in milk and various external secretions. This protein has been found to have a number of biological functions, including antimicrobial, anti-cancer, antioxidant, and immunomodulatory effects. Partial degradation of LF by pepsin can give rise to peptides termed lactoferricin (LFcin) with more potent antimicrobial activity. LF and LFcin have been shown to inhibit the growth of a number of pathogenic bacteria (including E. coli and antibiotic-resistant strains), fungi, and even viruses in both in vitro and in vivo studies. We previously demonstrated that both recombinant porcine LF (pLF) produced from yeast and a synthetic 20-residue porcine LFcin peptide exhibit antimicrobial activity in vitro. In one of our recent studies, we performed pathogen challenges, including pathogenic E. coli, Staphylococcus aureus and Candida albicans, of the digestive tract of a transgenic milk-fed animal model. The results showed that LF has broad spectrum antimicrobial activity in the digestive tract and protects the mucosa of the small intestine from injury. Our following study also revealed that pLF as a feedstuff additive enhances avian immunity, including antibody formation and cell-mediated immunity. All of these results suggest that LF could be a novel natural protein in the treatment and prevention of infections with E. coli or antibiotic-resistant bacteria strains.     

A heterodimer comprised of two bovine lactoferrin antimicrobial peptides exhibits powerful bactericidal activity against Burkholderia pseudomallei

Melioidosis is a severe infectious disease that is endemic in Southeast Asia and Northern Australia. Burkholderia pseudomallei, the causative agent of this disease, has developed resistance to an increasing list of antibiotics, demanding a search for novel agents. Lactoferricin and lactoferrampin are two antimicrobial domains of lactoferrin with a broad spectrum of antimicrobial activity. A hybrid peptide (LFchimera) containing lactoferrampin (LFampin265–284) and a part of lactoferricin (LFcin17–30) has strikingly higher antimicrobial activities compared to the individual peptides. In this study, the antimicrobial activities of this chimeric construct (LFchimera1), as well as of another one containing LFcin17–30 and LFampin268–284, a shorter fragment of LFampin265–284 (LFchimera2), and the constituent peptides were tested against 7 isolates of B. pseudomallei and compared to the preferential antibiotic ceftazidime (CAZ). All isolates including B. pseudomallei 979b shown to be resistant to CAZ, at a density of 10⁵ CFU/ml, could be killed by 5–10 μM of LFchimera1 within 2 h, while the other peptides as well as the antibiotic CAZ only inhibited the B. pseudomallei strains resulting in an overgrowth in 24 h. These data indicate that LFchimera1 could be considered for development of therapeutic agents against B. pseudomallei.     

Antimicrobial activity of endemic Crataegus tanacetifolia (Lam.) Pers and observation of the inhibition effect on bacterial cells

Up to now an increasing number of antibiotic-resistant bacteria have been reported and thus new natural therapeutic agents are needed in order to eradicate these pathogens. Through the discovery of plants such as Crataegus tanacetifolia (Lam.) Pers that have antimicrobial activity, it will be possible to discover new natural drugs serving as chemotherapeutic agents for the treatment of nosocomial pathogens and take these antibiotic-resistant bacteria under control. The objective of the present study was to determine antimicrobial activity and the activity mechanism of C. tanacetifolia plant extract. The leaves of C. tanacetifolia, which is an endemic plant, were extracted using methanol and tested against 10 bacterial and 4 yeast strains by using a drop method. It was observed that the plant extract had antibacterial effects on Bacillus subtilis, Shigella, Staphylococcus aureus, and Listeria monocytogenes among the microorganisms that were tested. Minimum inhibitory concentration (MIC) results obtained at the end of an incubation of 24 h were found to be > or =6.16 mg ml(-1) for B. subtilis, < 394 mg ml(-1) for Shigella, and > or =3.08 mg ml(-1) for L. monocytogenes and S. aureus and minimum bactericidal concentration (MBC) were found as > or =24.63 mg ml(-1) for B. subtilis, > or =394 mg ml(-1) for Shigella, > or =6.16 mg ml(-1) for L. monocytogenes, and > or =98.5 mg ml(-1) for S. aureus. According to the MBC results, it was found that the plant extract had bactericidal effects and in order to explain the activity mechanism and cell deformation of bacterial strains treated with plant extract, the scanning electron microscopy (SEM) was used. The results of SEM showed that the treated cells appeared shrunken and there was degradation of the cell walls. This study, in which the antibacterial effect of C. tanacetifolia was demonstrated, will be a base for further investigations on advanced purification and effect mechanism of action of its active compounds.     

Pyrrolidine bis-cyclic guanidines with antimicrobial activity against drug-resistant Gram-positive pathogens identified from a mixture-based combinatorial library

The rapid rise in antibiotic-resistant Gram-positive bacterial infections prompted us to explore the development of novel strategies for synthesis of large chemical libraries amenable to high-throughput screening for antimicrobial activities. Here we report the solid-phase synthesis of a 738,192 member pyrrolidine bis-cyclic guanidine chemical library with 26 different amino acids at three positions of diversity and 42 carboxylic acids at the fourth position. This synthetic combinatorial library was developed for positional scanning and screened for bacteriostatic and bactericidal activities against the important human pathogen methicillin-resistant Staphylococcus aureus (MRSA). The eight compound mixtures exhibiting bactericidal activity (10 microg/mL) against MRSA were used to direct the synthesis of 36 individual compounds that were then screened for activity against MRSA, vancomycin-resistant Enterococcus faecalis (VRE), and two Gram-negative bacterial species. At least 20 individual compounds were bactericidal for MRSA at 2.5 microg/mL, with a subset of these compounds showing bactericidal activities (10 microg/mL) against the other species tested. This approach demonstrates the capability to synthesize and screen a complex library to yield promising antimicrobials that address a critical need for novel infectious disease therapeutics.     

Antibacterial activity and mechanism of a scorpion venom peptide derivative in vitro and in vivo

BmKn2 is an antimicrobial peptide (AMP) characterized from the venom of scorpion Mesobuthus martensii Karsch by our group. In this study, Kn2-7 was derived from BmKn2 to improve the antibacterial activity and decrease hemolytic activity. Kn2-7 showed increased inhibitory activity against both gram-positive bacteria and gram-negative bacteria. Moreover, Kn2-7 exhibited higher antibacterial activity against clinical antibiotic-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA). In addition, the topical use of Kn2-7 effectively protected the skin of mice from infection in an S. aureus mouse skin infection model. Kn2-7 exerted its antibacterial activity via a bactericidal mechanism. Kn2-7 killed S. aureus and E. coli rapidly by binding to the lipoteichoic acid (LTA) in the S. aureus cell wall and the lipopolysaccharides (LPS) in the E. coli cell wall, respectively. Finally, the hemolytic activity of Kn2-7 was significantly decreased, compared to the wild-type peptide BmKn2. Taken together, the Kn2-7 peptide can be developed as a topical therapeutic agent for treating bacterial infections.     

Comparison of bacteriostatic and bactericidal activity of 13 essential oils against strains with varying sensitivity to antibiotics

Aims:  To compare the bacteriostatic and bactericidal activity of 13 chemotyped essential oils (EO) on 65 bacteria with varying sensitivity to antibiotics.
Methods and Results:  Fifty-five bacterial strains were tested with two methods used for evaluation of antimicrobial activity (CLSI recommendations): the agar dilution method and the time-killing curve method. EO containing aldehydes ( Cinnamomum verum bark and Cymbopogon citratus ), phenols ( Origanum compactum , Trachyspermum ammi , Thymus satureioides , Eugenia caryophyllus and Cinnamomum verum leaf) showed the highest antimicrobial activity with minimum inhibitory concentration (MIC) <2% (v/v) against all strains except Pseudomonas aeruginosa . Alcohol-based EO ( Melaleuca alternifolia , Cymbopogon martinii and Lavandula angustifolia ) exhibited varying degrees of activity depending on Gram status. EO containing 1·8-cineole and hydrocarbons ( Eucalyptus globulus , Melaleuca cajeputii and Citrus sinensis ) had MIC_90% ≥ 10% (v/v). Against P. aeruginosa , only C. verum bark and O. compactum presented MIC ≤2% (v/v). Cinnamomum verum bark, O. compactum , T. satureioides , C. verum leaf and M. alternifolia were bactericidal against Staphylococcus aureus and Escherichia coli at concentrations ranging from to 0·31% to 10% (v/v) after 1 h of contact. Cinnamomum verum bark and O. compactum were bactericidal against P. aeruginosa within 5 min at concentrations <2% (v/v).
Conclusions:  Cinnamomum verum bark had the highest antimicrobial activity, particularly against resistant strains.
Significance and Impact of the Study:  Bacteriostatic and bactericidal activity of EO on nosocomial antibiotic-resistant strains.     

The Staphylococcus aureus surface protein IsdA mediates resistance to innate defenses of human skin

Resistance to human skin innate defenses is crucial for survival and carriage of Staphylococcus aureus, a common cutaneous pathogen and nasal colonizer. Free fatty acids extracted from human skin sebum possess potent antimicrobial activity against S. aureus. The mechanisms by which S. aureus overcomes this host defense during colonization remain unknown. Here, we show that S. aureus IsdA, a surface protein produced in response to the host, decreases bacterial cellular hydrophobicity rendering them resistant to bactericidal human skin fatty acids and peptides. IsdA is required for survival of S. aureus on live human skin. Reciprocally, skin fatty acids prevent the production of virulence determinants and the induction of antibiotic resistance in S. aureus and other Gram-positive pathogens. A purified human skin fatty acid was effective in treating systemic and topical infections of S. aureus suggesting that our natural defense mechanisms can be exploited to combat drug-resistant pathogens.     

Lactoferrin‐derived peptides and Lactoferricin chimera inhibit virulence factor production and biofilm formation in Pseudomonas aeruginosa

Aims: To investigate the bactericidal activity of lactoferrin‐derived peptides and a new LF‐derived peptides chimera (LFchimera) against P. aeruginosa and the influence on virulence factors of P. aeruginosa. Methods and Results: Lactoferricin (LFcin) and lactoferrampin (LFampin) are highly bioactive peptides isolated from the N‐terminal region of lactoferrin (LF) by pepsin digestion. In this study, we designed LFchimera containing LFcin amino acids 17‐30 and LFampin amino acids 268‐284. Pseudomonas aeruginosa cells were incubated in medium with peptides at different concentrations, and then the assays of viability, pyocyanin, elastase activity and biofilm formation of P. aeruginosa were performed. We found that the concentration‐dependent antibactericidal activity and down‐regulating pyocyanin, elastase and biofilm formation of LFchimera were significantly stronger than those of LF, LFcin, LFampin or LFcin plus LFampin. Conclusions: Our results indicated that LF, LFcin, LFampin and LFchimera were potential candidates to combat P. aeruginosa, and LFchimera was the most effective in them. Significance and Impact of the Study: The new LFchimera has better activity against P. aeruginosa than LF, LFcin and LFampin and may be a promising new compound for treatment of P. aeruginosa infection.     

A new class of nifuroxazide analogues: synthesis of 5-nitrothiophene derivatives with antimicrobial activity against multidrug-resistant Staphylococcus aureus

Hospital-acquired methicillin-resistant Staphylococcus aureus (MRSA) has been an increasing problem worldwide since the initial reports over 40 years ago. To examine new drug leads with potential antibacterial activities, 14 p-substituted benzoic acid [(5-nitro-thiophen-2-yl)-methylene]-hydrazides were designed, synthesized, and tested against standard and multidrug-resistant S. aureus strains by serial dilution tests. All compounds exhibited significant bacteriostatic activity and some of them also showed bactericidal activity. The results confirmed the potential of this class of compounds as an alternative for the development of selective antimicrobial agents.     

The antimicrobial potential of a new derivative of cathelicidin from <Emphasis Type="Italic">Bungarus fasciatus</Emphasis> against methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Cathelicidins are a family of antimicrobial peptides which exhibit broad antimicrobial activities against antibiotic-resistant bacteria. Considering the progressive antibiotic resistance, cathelicidin is a candidate for use as an alternative approach to treat and overcome the challenge of antimicrobial resistance. Cathelicidin-BF (Cath-BF) is a short antimicrobial peptide, which was originally extracted from the venom of Bungarus fasciatus. Recent studies have reported that Cath-BF and some related derivatives exert strong antimicrobial and weak hemolytic properties. This study investigates the bactericidal and cytotoxic effects of Cath-BF and its analogs (Cath-A and Cath-B). Cath-A and Cath-B were designed to increase their net positive charge, to have more activity against methicillin resistant S. aureus (MRSA). The results of this study show that Cath-A, with a +17-net charge, has the most noteworthy antimicrobial activity against MRSA strains, with minimum inhibitory concentration (MIC) ranging between 32–128 μg/ml. The bacterial kinetic analysis by 1 × MIC concentration of each peptide shows that Cath-A neutralizes the clinical MRSA isolate for 60 min. The present data support the notion that increasing the positive net charge of antimicrobial peptides can increase their potential antimicrobial activity. Cath-A also displayed the weakest cytotoxicity effect against human umbilical vein endothelial and H9c2 rat cardiomyoblast cell lines. Analysis of the hemolytic activity reveals that all three peptides exhibit minor hemolytic activity against human erythrocytes at concentrations up to 250 μg/ml. Altogether, these results suggest that Cath-A and Cath-B are competent candidates as novel antimicrobial compounds against MRSA and possibly other multidrug resistant bacteria.     

Pharmacological synergism of bee venom and melittin with antibiotics and plant secondary metabolites against multi-drug resistant microbial pathogens

The goal of this study was to investigate the antimicrobial activity of bee venom and its main component, melittin, alone or in two-drug and three-drug combinations with antibiotics (vancomycin, oxacillin, and amikacin) or antimicrobial plant secondary metabolites (carvacrol, benzyl isothiocyanate, the alkaloids sanguinarine and berberine) against drug-sensitive and antibiotic-resistant microbial pathogens. The secondary metabolites were selected corresponding to the molecular targets to which they are directed, being different from those of melittin and the antibiotics.The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were evaluated by the standard broth microdilution method, while synergistic or additive interactions were assessed by checkerboard dilution and time-kill curve assays. Bee venom and melittin exhibited a broad spectrum of antibacterial activity against 51 strains of both Gram-positive and Gram-negative bacteria with strong anti-MRSA and anti-VRE activity (MIC values between 6 and 800 µg/ml). Moreover, bee venom and melittin showed significant antifungal activity (MIC values between 30 and 100 µg/ml). Carvacrol displayed bactericidal activity, while BITC exhibited bacteriostatic activity against all MRSA and VRE strains tested (reference strains and clinical isolates), both compounds showed a remarkable fungicidal activity with minimum fungicidal concentration (MFC) values between 30 and 200 µg/ml. The DNA intercalating alkaloid sanguinarine showed bactericidal activity against MRSA NCTC 10442 (MBC 20 µg/ml), while berberine exhibited bacteriostatic activity against MRSA NCTC 10442 (MIC 40 µg/ml).Checkerboard dilution tests mostly revealed synergism of two-drug combinations against all the tested microorganisms with FIC indexes between 0.24 and 0.50, except for rapidly growing mycobacteria in which combinations exerted an additive effect (FICI = 0.75–1). In time-kill assays all three-drug combinations exhibited a powerful bactericidal synergistic effect against MRSA NCTC 10442, VRE ATCC 51299, and E. coli ATCC 25922 with a reduction of more than 3log₁₀ in the colony count after 24 h. Our findings suggest that bee venom and melittin synergistically enhanced the bactericidal effect of several antimicrobial agents when applied in combination especially when the drugs affect several and differing molecular targets. These results could lead to the development of novel or complementary antibacterial drugs against MDR pathogens.     

Synthesis and antimicrobial activity of N¹-benzyl or N¹-benzyloxy-1,6-dihydro-1,3,5-triazine-2,4-diamines

The emergence and spread of multidrug-resistant strains of Staphylococcus aureus and Mycobacterium tuberculosis are generating a threat to public health worldwide. In the current study, a series of N(1)-benzyl and N(1)-benzyloxy-1,6-dihydro-1,3,5-triazine-2,4-diamine derivatives were synthesized and investigated for their antimicrobial activity against S. aureus, and Mycobacterium smegmatis which is taxonomically related to M. tuberculosis. Most of the compounds exhibited good activity against M. smegmatis as determined by comparison of diameters of the zone of inhibition of test compounds and standard antibiotics. Compound 7o showed potent antimycobacterial activity against M. smegmatis without mammalian DHFR inhibition liability. The results from this study indicate that 1-benzyl derivatives of 1,6-dihydro-1,3,5-triazine-2,4-diamines may be used as lead compounds for the discovery of antimycobacterial agents.     

Direct antimicrobial properties of substance P

The structural similarity between substance P (SP, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH(2)) and Arg/Pro rich bactericidal peptides suggests a possible direct effect of SP on invasive microbes. We now present evidence that substance P possesses direct antimicrobial activity, highest against S. aureus. A substance P antagonist also possesses such activity but while less potent than substance P agonist S. aureus, is more potent than substance P against C. albicans. Our data also show that the endogenous peptides bradykinin and neurotensin, that also play role in modulation of the host-defense system in situ, have antimicrobial properties but are less potent than substance P.