Inhibition of biofilm formation by the antisense peptide nucleic acids targeted at the <Emphasis Type="Italic">motA</Emphasis> gene in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PAO1 strain

Pseudomonas aeruginosa is a ubiquitous bacterium which is able to attach to many abiotic and biotic surfaces and form biofilms resulting in infections. The motA gene was an essential gene in the early phase of biofilm formation of P. aeruginosa PAO1. In this study, antisense peptide nucleic acids (PNAs) and PNAs conjugated with the peptide (KFF)₃K were used to investigate whether they could mediate gene-specific antisense effects in P. aeruginosa PAO1. We found that antisense (KFF)₃K-PNA targeted at motA gene could inhibit biofilm formation in P. aeruginosa PAO1 in a dose-dependent manner. The minimal effective concentration of this antisense agent was 1 μmol l⁻¹, and the inhibited effect could last for at least 8 h. When compared with the control group, the value of OD570 of P. aeruginosa PAO1 reduced apparently when treated with (KFF)₃K-PNA. The expression of motA was sharply reduced when treated with (KFF)₃K-PNA, but reduced slightly when treated with PNA, and had no reduction when treated with (KFF)₃K. Our results demonstrated that the cell-penetrating peptide of (KFF)₃K improved significantly the antisense inhibition effect of PNA. The (KFF)₃K-PNA conjugates might be used as antisense agent for inhibition of the biofilm formation. This provides exciting possibility for developing new tool for microbial genetic treatment.     

Antibacterial Activity of Novel Cationic Peptides against Clinical Isolates of Multi-Drug Resistant Staphylococcus pseudintermedius from Infected Dogs

Staphylococcus pseudintermedius is a major cause of skin and soft tissue infections in companion animals and has zoonotic potential. Additionally, methicillin-resistant S. pseudintermedius (MRSP) has emerged with resistance to virtually all classes of antimicrobials. Thus, novel treatment options with new modes of action are required. Here, we investigated the antimicrobial activity of six synthetic short peptides against clinical isolates of methicillin-susceptible and MRSP isolated from infected dogs. All six peptides demonstrated potent anti-staphylococcal activity regardless of existing resistance phenotype. The most effective peptides were RRIKA (with modified C terminus to increase amphipathicity and hydrophobicity) and WR-12 (α-helical peptide consisting exclusively of arginine and tryptophan) with minimum inhibitory concentration₅₀ (MIC₅₀) of 1 µM and MIC₉₀ of 2 µM. RR (short anti-inflammatory peptide) and IK8 “D isoform” demonstrated good antimicrobial activity with MIC₅₀ of 4 µM and MIC₉₀ of 8 µM. Penetratin and (KFF)₃K (two cell penetrating peptides) were the least effective with MIC₅₀ of 8 µM and MIC₉₀ of 16 µM. Killing kinetics revealed a major advantage of peptides over conventional antibiotics, demonstrating potent bactericidal activity within minutes. Studies with propidium iodide and transmission electron microscopy revealed that peptides damaged the bacterial membrane leading to leakage of cytoplasmic contents and consequently, cell death. A potent synergistic increase in the antibacterial effect of the cell penetrating peptide (KFF)₃K was noticed when combined with other peptides and with antibiotics. In addition, all peptides displayed synergistic interactions when combined together. Furthermore, peptides demonstrated good therapeutic indices with minimal toxicity toward mammalian cells. Resistance to peptides did not evolve after 10 passages of S. pseudintermedius at sub-inhibitory concentration. However, the MICs of amikacin and ciprofloxacin increased 32 and 8 fold, respectively; under similar conditions. Taken together, these results support designing of peptide-based therapeutics for combating MRSP infections, particularly for topical application.     

Value Addition in the Efficacy of Conventional Antibiotics by Nisin against Salmonella

Frequent and indiscriminate use of existing battery of antibiotics has led to the development of multi drug resistant (MDR) strains of pathogens. As decreasing the concentration of the antibiotic required to treat Salmonellosis might help in combating the development of resistant strains, the present study was designed to assess the synergistic effects, if any, of nisin, in combination with conventional anti-Salmonella antibiotics against Salmonella enterica serovar Typhimurium. Minimum inhibitory concentrations (MICs) of the selected antimicrobial agents were determined by micro and macro broth dilution assays. In-vitro synergy between the agents was evaluated by radial diffusion assay, fractional inhibitory concentration (FIC) index (checkerboard test) and time-kill assay. Scanning electron microscopy (SEM) was also performed to substantiate the effect of the combinations. In-vivo synergistic efficacy of the combinations selected on the basis of in-vitro results was also evaluated in the murine model, in terms of reduction in the number of Salmonellae in liver, spleen and intestine. Nisin-ampicillin and nisin-EDTA combinations were observed to have additive effects, whereas the combinations of nisin-ceftriaxone and nisin-cefotaxime were found to be highly synergistic against serovar Typhimurium as evident by checkerboard test and time-kill assay. SEM results revealed marked changes on the outer membrane of the bacterial cells treated with various combinations. In-vivo synergy was evident from the larger log unit decreases in all the target organs of mice treated with the combinations than in those treated with drugs alone. This study thus highlights that nisin has the potential to act in conjunction with conventional antibiotics at much lower MICs. These observations seem to be significant, as reducing the therapeutic concentrations of antibiotics may be a valuable strategy for avoiding/reducing the development of emerging antibiotic resistance. Value added potential of nisin in the efficacy of conventional antibiotics may thus be exploited not only against Salmonella but against other Gram-negative infections as well.     

Inhibition of gene expression and growth of multidrug-resistant Acinetobacter baumannii by antisense peptide nucleic acids

Acinetobacter baumannii causes common and severe community- and hospital-acquired infections. The increasing emergence of multidrug-resistant (MDR) and pan-drug resistant A. baumannii has limited the therapeutic options, highlighting the need for new therapeutic strategies. The goal of this study was to investigate whether antisense peptide nucleic acids (PNAs) could mediate gene-specific inhibition effects in MDR A. baumannii. We described a screening strategy based on computational prediction and dot hybridization for identifying potential inhibitory PNAs, and evaluated the in vitro growth inhibition potency of two PNAs conjugated to the (KFF)₃K peptide (pPNA1 and pPNA2), both of which targeted the growth essential gene gyrA of A. baumannii. Both pPNAs showed strong inhibition effects on bacterial growth and gyrA mRNA expression in a dose-dependent manner. The lowest inhibitory and bactericidal concentration were 5 and 10 μM, respectively. Combination of the two pPNAs showed superimposed effect other than synergistic effect. Control PNAs without (KFF)₃K peptide conjugation or with mismatched antisense sequence had no inhibition effects on bacterial growth or mRNA expression. Our study suggests that anti-gyrA pPNAs can efficiently inhibit gene expression and bacterial growth, and has the potential as a new therapeutic option for MDR A. baumannii.     

Antibacterial Efficacy of Dihydroxylated Chalcones in Binary and Ternary Combinations with Nalidixic Acid and Nalidix Acid–Rutin Against Escherichia coli ATCC 25 922

In order to determine the existence of synergism, the bacteriostatic action of flavonoids against Escherichia coli ATCC 25 922 between dihydroxylated chalcones and a clinically interesting conventional antibiotic, binary combinations of 2′,3-dihydroxychalcone, 2′,4-dihydroxychalcone and 2′,4′-dihydroxychalcone with nalidixic acid and its ternary combinations with rutin (inactive flavonoid) were assayed against this Gram negative bacterium. Using a kinetic-turbidimetric method, growth kinetics were monitored in broths containing variable amounts of dihydroxychalcone alone, combinations of dihydroxychalcone variable concentration–nalidixic acid constant concentration and dihydroxychalcone variable concentration–nalidixic acid constant concentration–rutin constant concentration, respectively. The minimum inhibitory concentrations of dihydroxychalcones alone and its binary and ternary combinations were evaluated. All chalcones, and their binary and ternary combinations showed antibacterial activity, being rutin an excellent synergizing for the dihydroxychalcone–nalidixic acid binary combination against E. coli ATCC 25 922. Thus, this synergistic effect is an important way that could lead to the development of new combination antibiotics against infections caused by E. coli.     

Expression of 4kD scorpion defensin and its in vitro synergistic activity with conventional antibiotics

The 4kD scorpion defensin (SD) is a potent disulfide-linked peptide. In this study, we expressed it in methylotrophic yeast Pichia pastoris and purified it using Ni–NTA His Bind Resin. We investigated its in vitro antibacterial activity and effect in combination with several conventional antibiotics. We first examined its antibacterial activity towards several Gram-positive and Gram-negative bacteria. Then we used the broth microdilution method to test drugs alone and in combination and used the fractional inhibitory concentration (FIC index) to classify the drug interactions. Our study showed the expressed SD peptide has antibacterial activity against Salmonella typhimurium, E. coli and S. aureus etc. Synergy or additive interaction was observed between SD and Norfloxacin, Polymyxin B and Ampicillin. Cell growth tests showed that combination of SD and Norfloxacin can improve their activity against bacteria. This result maybe permit lower using of the conventional antibiotic agents more effectively and safely.     

Membrane-Active Macromolecules Resensitize NDM-1 Gram-Negative Clinical Isolates to Tetracycline Antibiotics

Gram-negative ‘superbugs’ such as New Delhi metallo-beta-lactamase-1 (bla _NDM-1) producing pathogens have become world’s major public health threats. Development of molecular strategies that can rehabilitate the ‘old antibiotics’ and halt the antibiotic resistance is a promising approach to target them. We report membrane-active macromolecules (MAMs) that restore the antibacterial efficacy (enhancement by >80-1250 fold) of tetracycline antibiotics towards bla _NDM-1 Klebsiella pneumonia and bla _NDM-1 Escherichia coli clinical isolates. Organismic studies showed that bacteria had an increased and faster uptake of tetracycline in the presence of MAMs which is attributed to the mechanism of re-sensitization. Moreover, bacteria did not develop resistance to MAMs and MAMs stalled the development of bacterial resistance to tetracycline. MAMs displayed membrane-active properties such as dissipation of membrane potential and membrane-permeabilization that enabled higher uptake of tetracycline in bacteria. In-vivo toxicity studies displayed good safety profiles and preliminary in-vivo antibacterial efficacy studies showed that mice treated with MAMs in combination with antibiotics had significantly decreased bacterial burden compared to the untreated mice. This report of re-instating the efficacy of the antibiotics towards bla _NDM-1 pathogens using membrane-active molecules advocates their potential for synergistic co-delivery of antibiotics to combat Gram-negative superbugs.     

Synergistic Effect and Antibiofilm Activity Between the Antimicrobial Peptide Coprisin and Conventional Antibiotics Against Opportunistic Bacteria

Coprisin is a 43-mer defensin-like peptide from the dung beetle, Copris tripartitus. In this study, we tested its minimum inhibitory concentration and performed combination assays to confirm the antibacterial susceptibility of coprisin and synergistic effects with antibiotics. The synergistic effects were evaluated by testing the effects of coprisin in combination with ampicillin, vancomycin, and chloramphenicol. The results showed that coprisin possessed antibacterial properties and had synergistic activities with the antibiotics. To understand the synergistic mechanism(s), we conducted hydroxyl radical assays. Coprisin alone and in combination with antibiotics generated hydroxyl radicals, which are highly reactive oxygen forms and the major property of bactericidal agents. Furthermore, the antibiofilm effect of coprisin alone and in combination with antibiotics was investigated. Biofilm formation is the source of many relentless and chronic bacterial infections. The results indicated that coprisin alone and in combination with antibiotics also had antibiofilm activity. Therefore, we conclude that coprisin has the potential to be used as a combinatorial therapeutic agent for the treatment of infectious diseases caused by bacteria.     

Fusion expression of the PGLa-AM1 with native structure and evaluation of its anti-<Emphasis Type="Italic">Helicobacter pylori</Emphasis> activity

Helicobacter pylori (H. pylori) shows increasingly enhanced resistance to various antibiotics, and its eradication has become a major problem in medicine. The antimicrobial peptide PGLa-AM1 is a short peptide with 22 amino acids and exhibits strong antibacterial activity. In this study, we investigated whether it has anti-H. pylori activity for the further development of anti-H. pylori drugs to replace existing antibiotics. However, the natural antimicrobial peptide PGLa-AM1 shows a low yield and is difficult to separate, limiting its application. A good strategy to solve this problem is to express the antimicrobial peptide PGLa-AM1 using gene engineering at a high level and low cost. For getting PGLa-AM1 with native structure, in this study, a specific protease cleavage site of tobacco etch virus (TEV) was designed before the PGLa-AM1 peptide. For convenience to purify and identify high-efficiency expression PGLa-AM1, the PGLa-AM1 gene was fused with the polyhedrin gene of Bombyx mori (B. mori), and a 6 × His tag was designed to insert before the amino terminus of the fusion protein. The fusion antibacterial peptide PGLa-AM1 (FAMP) gene codon was optimized, and the gene was synthesized and cloned into the Escherichia coli (E. coli) pET-30a (+) expression vector. The results showed that the FAMP was successfully expressed in E. coli. Its molecular weight was approximately 34 kDa, and its expression level was approximately 30 mg/L. After the FAMP was purified, it was further digested with TEV protease. The acquired recombinant antimicrobial peptide PGLa-AM1 exerted strong anti-H. pylori activity and therapeutic effect in vitro and in vivo.     

Sensitization of Microcin J25-Resistant Strains by a Membrane-Permeabilizing Peptide

Microcin J25 (MccJ25) is a plasmid-encoded, 21-amino-acid, antibacterial peptide produced by Escherichia coli. MccJ25 inhibits RNA polymerase and the membrane respiratory chain. MccJ25 uptake into E. coli-sensitive strains is mediated by the outer membrane receptor FhuA and the inner membrane proteins TonB, ExbB, ExbD, and SbmA. This peptide is active on some E. coli, Salmonella, and Shigella species strains, while other Gram-negative bacteria, such as clinical isolates of Enterobacter cloacae, Citrobacter freundii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Moraxella catarrhalis, and Salmonella enterica serovar Typhimurium, are completely resistant. In the present work, we demonstrated that the membrane-permeabilizing peptide (KFF)₃K made some resistant strains sensitive to MccJ25, among them S. Typhimurium, where the antibiotic inhibits in vitro cell growth and bacterial replication within macrophages. The results demonstrate that the membrane permeabilization induced by (KFF)₃K allows MccJ25 penetration in an FhuA and SbmA-independent manner and suggest that the combination of both peptides could be considered as a therapeutic agent against pathogenic Salmonella strains.The antibiotic peptide microcin J25 (MccJ25), produced by an Escherichia coli strain, is ribosomally synthesized and consists of 21 amino acid residues (G¹-G-A-G-H⁵-V-P-E-Y-F¹⁰-V-G-I-G-T¹⁵-P-I-S-F-Y²⁰-G) (4, 12). MccJ25 is a lasso peptide (1, 10, 17), contains a lactam linkage between the α-amino group of Gly¹ and the γ-carboxyl of Glu⁸, forming an 8-residue ring (Gly¹ to Glu⁸), which is termed a lariat ring. The “tail” (Tyr⁹ to Gly²¹) passes through the ring, with Phe¹⁹ and Tyr²⁰ straddling each side of the tail, sterically trapping the tail within the ring. MccJ25 amino acids F¹⁰ to P¹⁶ form a β-hairpin structure comprising two β-strands (F¹⁰-V¹¹ and T¹⁵-P¹⁶) and a β-turn (V¹¹ to G¹⁴).The uptake of MccJ25 into the E. coli periplasmic space depends on the outer membrane receptor FhuA and the inner membrane proteins TonB, ExbD, and ExbB (11, 13). Additionally, the inner membrane protein SbmA transports MccJ25 from the periplasmic to the cytoplasmic space (13). Once inside the sensitive cell, the peptide is able to inhibit E. coli RNA polymerase (RNAP) and membrane respiratory chain, which represent the MccJ25 targets (2, 5, 7, 18). Several Salmonella enterica serovars showed high sensitivity against MccJ25, while others, like Salmonella enterica serovar Typhimurium, S. enterica serovar Derby, and some S. enterica serovar Enteritidis strains were completely resistant (16). Since introduction of the E. coli fhuA allele cloned in a multicopy plasmid into these bacteria rendered them hypersensitive to the antibiotic, we concluded that this intrinsic resistance is due to the inability of the FhuA receptor protein to mediate the penetration of MccJ25. In fact, MccJ25 was able to inhibit both intracellular targets in the transformed strains (16).The polianionic lipopolysaccharide (LPS) component of the outer membrane is stabilized by divalent cation bridges (15). It was suggested that many cationic peptides are able to bind to LPS and disrupt these bridges, resulting in an increased bacterial membrane permeabilization. Vaara and Porro (15) characterized a series of synthetic peptides that were able to sensitize Gram-negative bacteria to hydrophobic and amphipathic antibiotics. One of them, KFFKFFKFFK [(KFF)₃K], a peptide rich in cationic lysine and hydrophobic phenylalanine residues, showed a potent effect on outer membrane disorganization and weak damage to the cytoplasmic membrane (15).In this work, we have shown that the (KFF)₃K peptide allows MccJ25 uptake independently of the FhuA and SbmA receptors, turning in vitro microcin-resistant strains into susceptible ones. Moreover, we have demonstrated that (KFF)₃K was able to exert the same inhibitory effect in vivo on S. Typhimurium replicating within eukaryotic cells.     

Synthesis and in vitro evaluation of PNA-peptide-DETA conjugates as potential cell penetrating artificial ribonucleases

We report the synthesis of novel artificial ribonucleases with potentially improved cellular uptake. The design of trifunctional conjugates 1a and 1b is based on the specific RNA-recognizing properties of PNA, the RNA-cleaving abilities of diethylenetriamine (DETA), and the peptide (KFF)(3)K for potential uptake into E. coli. The conjugates were assembled in a convergent synthetic route involving native chemical ligation of a PNA, containing an N-terminal cysteine, with the C-terminal thioester of the cell-penetrating (KFF)(3)K peptide to give 12a and 12b. These hybrids contained a free cysteine side-chain, which was further functionalized with an RNA-hydrolyzing diethylenetriamine (DETA) moiety. The trifunctional conjugates (1a, 1b) were evaluated for RNA-cleaving properties in vitro and showed efficient degradation of the target RNA at two major cleavage sites. It was also established that the cleavage efficiency strongly depended on the type of spacer connecting the PNA and the peptide.     

Synergistic effects of short peptides and antibiotics against bacterial and fungal strains

There is a rising tide of concern about the antibiotic resistance issue. To reduce the possibility of antibiotic-resistant infections, a new generation of antimicrobials must be developed. Antimicrobial peptides are potential alternatives to antibiotics that can be used alone or together with conventional antibiotics to combat antimicrobial resistance. In this work, lead compounds LP-23, DP-23, SA4, and SPO from previously published studies were synthesized by solid-phase peptide synthesis and their antimicrobial evaluation was carried out against various bacterial and fungal strains. Peptide combinations with antibiotics were evaluated by using the checkerboard method and their minimal inhibitory concentration (MIC) in combination was calculated by using the fractional inhibitory concentration (FIC) index. Cytotoxicity evaluations of these peptides further confirmed their selectivity toward microbial cells. Based on the FIC values, LP-23, DP-23, and SPO demonstrated synergy in combination with gentamicin against a gentamicin-resistant clinical isolate of Escherichia coli. For Staphylococcus aureus, Escherichia coli, and Salmonella typhimurium, seven combinations exhibited synergistic effects between peptide/peptoids and the tested antibiotics. Additionally, almost all the combinations of peptides/peptoids with amphotericin B and fluconazole also showed effective synergy against Aspergillus niger and Aspergillus flavus. The synergy found between LP-23, DP-23, SA4, and SPO with the selected antibiotics may have the potential to be used as a combination therapy against various microbial infections.     

In Vitro Activity of Sodium New Houttuyfonate Alone and in Combination with Oxacillin or Netilmicin against Methicillin-Resistant Staphylococcus aureus

Background

Staphylococcus aureus can cause severe infections, including bacteremia and sepsis. The spread of methicillin-resistant Staphylococcus aureus (MRSA) highlights the need for novel treatment options. Sodium new houttuyfonate (SNH) is an analogue of houttuynin, the main antibacterial ingredient of Houttuynia cordata Thunb. The aim of this study was to evaluate in vitro activity of SNH and its potential for synergy with antibiotics against hospital-associated MRSA.

Methodology

A total of 103 MRSA clinical isolates recovered in two hospitals in Beijing were evaluated for susceptibility to SNH, oxacillin, cephalothin, meropenem, vancomycin, levofloxacin, minocycline, netilmicin, and trimethoprim/sulfamethoxazole by broth microdilution. Ten isolates were evaluated for potential for synergy between SNH and the antibiotics above by checkerboard assay. Time-kill analysis was performed in three isolates to characterize the kill kinetics of SNH alone and in combination with the antibiotics that engendered synergy in checkerboard assays. Besides, two reference strains were included in all assays.

Principal Findings

SNH inhibited all test strains with minimum inhibitory concentrations (MICs) ranging from 16 to 64 µg/mL in susceptibility tests, and displayed inhibition to bacterial growth in concentration-dependent manner in time-kill analysis. In synergy studies, the combinations of SNH-oxacillin, SNH-cephalothin, SNH-meropenem and SNH-netilmicin showed synergistic effects against 12 MRSA strains with median fractional inhibitory concentration (FIC) indices of 0.38, 0.38, 0.25 and 0.38 in checkerboard assays. In time-kill analysis, SNH at 1/2 MIC in combination with oxacillin at 1/128 to 1/64 MIC or netilmicin at 1/8 to 1/2 MIC decreased the viable colonies by ≥2log₁₀ CFU/mL.

Conclusions/Significance

SNH demonstrated in vitro antibacterial activity against 103 hospital-associated MRSA isolates. Combinations of sub-MIC levels of SNH and oxacillin or netilmicin significantly improved the in vitro antibacterial activity against MRSA compared with either drug alone. The SNH-based combinations showed promise in combating MRSA.     

Synthesis,antibacterial activities and molecular docking studies of peptide and Schiff bases as targeted antibiotics

A series of peptide and Schiff bases (PSB) were synthesized by reacting salicylic acid, primary diamines with salicylaldehyde or its derivatives, and 40 of which were newly reported. The inhibitory activities against Escherichia coli β-ketoacyl-acyl carrier protein synthase III (ecKAS III) were investigated in vitro and molecular docking simulation also surveyed. Top 10 PSB compounds which posses both good inhibitory activity and well binding affinities were picked out, and their antibacterial activities against Gram-negative and Gram-positive bacterial strains were tested, expecting to exploit potent antibacterial agent with broad-spectrum antibiotics activity. The results demonstrate compound N-(3-(5-bromo-2-hydroxybenzylideneamino)propyl)-2-hydroxybenzamide (2d) can be as a potential antibiotics agent, displaying minimal inhibitory concentration values in the range of 0.39–3.13 μg/mL against various bacteria.     

Peptide derived from the lipid binding domain of Group IB human pancreatic phospholipase A2 possesses antibacterial activity

Group 1B human pancreatic secretory phospholipase A₂ (hp-sPLA₂), a digestive enzyme synthesized by pancreatic acinar cells and present in pancreatic juice, do not have antibacterial activity towards Escherichia coli. Our earlier results suggest that the N-terminal first ten amino acid residues of hp-sPLA₂ constitute major portion of the membrane binding domain of full-length enzyme and is responsible for the precise orientation of enzyme on the membrane surface by inserting into the lipid bilayers (Pande et al. (2006) Biochemistry, 45,12436–12447). In this study we report the antibacterial properties of a peptide (AVWQFRKMIK-CONH₂; N10 peptide), which corresponds to the N-terminal first ten amino acid residues of hp-sPLA₂, against E. coli. Full-length hp-sPLA₂, which contains this peptide sequence as N-terminal α-helix, did not showed detectable antibacterial activity. Presence of physiological concentration of salt or preincubation of N10 peptide with soluble anionic polymer inhibits the antibacterial activity indicating the importance of electrostatic interaction in binding of peptide to bacterial membrane. Addition of peptide resulted in destabilization of outer as well as inner cytoplasmic membrane of E. coli suggesting bacterial membranes to be the main target of action. N10 peptide exhibits strong synergism with lysozyme and potentiates the antibacterial activity of lysozyme. The peptide was inactive against human erythrocyte. Our result shows for the first time that a peptide fragment of hp-sPLA₂ possesses antibacterial activity towards E. coli and at subinhibitory concentration and can potentiate the antibacterial activity of membrane active enzyme. These observations suggest that N10 peptide may play an important role in the antimicrobial activity of pancreatic juice.     

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.     

Novel strategies of essential oils,chitosan, and nano- chitosan for inhibition of multi-drug resistant: E. coli O157:H7 and Listeria monocytogenes

Despite the wide range of available antibiotics, food borne bacteria demonstrate a huge spectrum of resistance. The current study aims to use natural components such as essential oils (EOs), chitosan, and nano-chitosan that have very influential antibacterial properties with novel technologies like chitosan solution/film loaded with EOs against multi-drug resistant bacteria. Two strains of Escherichia coli O157:H7 and three strains of Listeria monocytogenes were used to estimate antibiotics resistance. Ten EOs and their mixture, chitosan, nano-chitosan, chitosan plus EO solutions, and biodegradable chitosan film enriched with EOs were tested as antibacterial agents against pathogenic bacterial strains. Results showed that E. coli O157:H7 51,659 and L. monocytogenes 19,116 relatively exhibited considerable resistance to more than one single antibiotic. Turmeric, cumin, pepper black, and marjoram did not show any inhibition zone against L. monocytogenes; Whereas, clove, thyme, cinnamon, and garlic EOs exhibited high antibacterial activity against L. monocytogenes with minimum inhibitory concentration (MIC) of 250–400 μl 100^?1 ml and against E. coli O157:H7 with an MIC of 350–500 μl 100^?1 ml, respectively. Among combinations, clove, and thyme EOs showed the highest antibacterial activity against E. coli O157:H7 with MIC of 170 μl 100^?1 ml, and the combination of cinnamon and clove EOs showed the strongest antibacterial activity against L. monocytogenes with an MIC of 120 μl 100^?1 ml. Both chitosan and nano-chitosan showed a promising potential as an antibacterial agent against pathogenic bacteria as their MICs were relatively lower against L. monocytogenes than for E. coli O157:H7. Chitosan combined with each of cinnamon, clove, and thyme oil have a more effective antibacterial activity against L. monocytogenes and E. coli O157:H7 than the mixture of oils alone. Furthermore, the use of either chitosan solution or biodegradable chitosan film loaded with a combination of clove and thyme EOs had the strongest antibacterial activity against L. monocytogenes and E. coli O157:H7. However, chitosan film without EOs did not exhibit an inhibition zone against the tested bacterial strains.