C-Locked Analogs of the Antimicrobial Peptide BP214.

BP214 is an all-D antimicrobial peptide amide, kklfkkilryl, which shows an excellent activity against colistin-resistant Acinetobacter baumannii and a low hemolytic activity. The aim of the present work was to investigate how C-terminus-to-side chain macrocyclization and fatty acid modification affect the antimicrobial and hemolytic activity of this peptide. In total, 18 analogs of BP214 were synthesized using a combination of Fmoc-based solid-phase peptide synthesis and the submonomer approach. Cyclization was achieved by reacting the ε-amino group of a C-terminal lysine residue with a bromoacetylgroup attached to the Nα amino group of the N-terminal amino acid, generating a secondary amine at which the exocyclic lipopeptide tail was assembled. Three different ring sizes (i.e., 3-5 amino acid residues) of C-locked analogs combined with fatty acids of different lengths (i.e., C10-C14) were investigated. The antimicrobial activity of the analogs was tested against Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. The most promising compound was analog 13 (MIC = 4 µg/mL (2.4 µM) against E. coli and 36% hemolysis of red blood cells at 150 µM). In a time-kill assay, this peptide showed a significant, concentration-dependent reduction in viable E. coli cells comparable to that seen for colistin.

Antisense inhibition of the Escherichia coli NrdAB aerobic ribonucleotide reductase is bactericidal due to induction of DNA strand breaks.

BACKGROUND: Antisense peptide nucleic acids (PNAs) constitute an alternative to traditional antibiotics, by their ability to silence essential genes. OBJECTIVES: To evaluate the antibacterial effects of antisense PNA-peptide conjugates that target the gene encoding the alpha subunit (NrdA) of the Escherichia coli ribonucleotide reductase (RNR). METHODS: Bacterial susceptibility of a series of NrdA-targeting PNAs was studied by MIC determination and time-kill analysis. Western-blot analysis, gene complementation and synergy with hydroxyurea were employed to determine the efficiency of NrdA-PNA antisense treatment. The effect on chromosome replication was addressed by determining the DNA synthesis rate, by flow cytometry analysis, by quantitative PCR and by fluorescence microscopy. The use of DNA repair mutants provided insight into the bactericidal action of NrdA-PNA. RESULTS: Treatment with NrdA-PNA specifically inhibited growth of E. coli, as well as NrdA protein translation at 4 µM. Also, the DNA synthesis rate was reduced, preventing completion of chromosome replication and resulting in formation of double-stranded DNA breaks and cell death. CONCLUSIONS: These data present subunits of the NrdAB RNR as a target for future antisense microbial agents and provide insight into the bacterial physiological response to RNR-targeting antimicrobials.

New Insights into the Antimicrobial Action of Cinnamaldehyde towards Escherichia coli and Its Effects on Intestinal Colonization of Mice.

Escherichia coli is responsible for cases of diarrhea around the world, and some studies have shown the benefits of cinnamaldehyde in the treatment of bacterial disease. Therefore, the objective of this study was to evaluate the effects of cinnamaldehyde in mice colonized by pathogenic E. coli, as well as to provide more insights into its antimicrobial action mechanism. After determination of minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations, the interference of cinnamaldehyde in macromolecular pathways (synthesis of DNA, RNA, protein, and cell wall) was measured by incorporation of radioisotopes. The anti-adhesive properties of cinnamaldehyde towards E. coli 042 were evaluated using human epithelial type 2 (HEp-2) cells. Intestinal colonization was tested on mice, and the effect of cinnamaldehyde on Tenebrio molitor larvae. Cinnamaldehyde showed MIC and MBC values of 780 µg/mL and 1560 µg/mL, respectively; reduced the adhesion of E. coli 042 on HEp-2 cells; and affected all the synthetic pathways evaluated, suggesting that compost impairs the membrane/cell wall structure leading bacteria to total collapse. No effect on the expression of genes related to the SOS pathway (sulA and dinB1) was observed. The compound did not interfere with cell viability and was not toxic against T. molitor larvae. In addition, cinnamaldehyde-treated mice exhibited lower levels of colonization by E. coli 042 than the untreated group. Therefore, the results show that cinnamaldehyde is effective in treating the pathogenic E. coli strain 042 and confirm it as a promising lead molecule for the development of antimicrobial agents.

Novel Cyclic Lipopeptide Antibiotics: Effects of Acyl Chain Length and Position.

Multidrug-resistant bacteria are a global health problem. One of the last-resort antibiotics against Gram-negative bacteria is the cyclic lipopeptide colistin, displaying a flexible linker with a fatty acid moiety. The aim of the present project was to investigate the effect on antimicrobial activity of introducing fatty acid moieties of different lengths and in different positions in a cyclic peptide, S3(B), containing a flexible linker. The lipidated analogues of S3(B) were synthesized by 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis. Following assembly of the linear peptide by Fmoc solid-phase peptide synthesis, on-resin head-to-tail cyclization and fatty acid acylation were performed. The antimicrobial activity was determined against the ESKAPE pathogens, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli. Furthermore, hemolytic activity was determined against human erythrocytes. A total of 18 cyclic lipopeptides were synthesized and characterized. It was found that introduction of fatty acids in positions next to the flexible linker was more strongly linked to antimicrobial activity. The fatty acid length altered the overall hydrophobicity, which was the driving force for both high antimicrobial and hemolytic activity. Peptides became highly hemolytic when carbon-chain length exceeded 10 (i.e., C10), overlapping with the optimum for antimicrobial activity (i.e., C8-C12). The most promising candidate (C8)5 showed antimicrobial activity corresponding to that of S3(B), but with an improved hemolytic profile. Finally, (C8)5 was further investigated in a time-kill experiment.

Analogues of a Cyclic Antimicrobial Peptide with a Flexible Linker Show Promising Activity against Pseudomonas aeruginosa and Staphylococcus aureus

The emergence of multi-drug resistant bacteria is becoming a major health concern. New strategies to combat especially Gram-negative pathogens are urgently needed. Antimicrobial peptides (AMPs) found in all multicellular organisms act as a first line of defense in immunity. In recent years, AMPs have attracted increasing attention as potential antibiotics. Naturally occurring antimicrobial cyclic lipopeptides include colistin and daptomycin, both of which contain a flexible linker. We previously reported a cyclic AMP BSI-9 cyclo(Lys-Nal-Lys-Lys-Bip-O2Oc-Nal-Lys-Asn) containing a flexible linker, with a broad spectrum of activity against bacterial strains and low hemolytic activity. In this study, improvement of the antimicrobial activity of BSI-9, against the European Committee on Antimicrobial Susceptibility Testing (EUCAST) strains of S. aureus, E. coli, A. baumannii, and P. aeruginosa was examined. This led to synthesis of eighteen peptide analogues of BSI-9, produced in four individual stages, with a different focus in each stage; cyclization point, hydrophobicity, cationic side-chain length, and combinations of the last two. Specifically the modified compound 11, exhibited improved activity against Staphylococcus aureus and Pseudomonas aeruginosa with MIC of 4 µg/mL and 8 µg/mL, respectively, compared to the original BSI-9, which had an MIC of 16-32 µg/mL.

Structure-Activity Study of an All-d Antimicrobial Octapeptide D2D.

The increasing emergence of multi-drug resistant bacteria is a serious threat to public health worldwide. Antimicrobial peptides have attracted attention as potential antibiotics since they are present in all multicellular organisms and act as a first line of defence against invading pathogens. We have previously identified a small all-d antimicrobial octapeptide amide kk(1-nal)fk(1-nal)k(nle)-NH2 (D2D) with promising antimicrobial activity. In this work, we have performed a structure-activity relationship study of D2D based on 36 analogues aimed at discovering which elements are important for antimicrobial activity and toxicity. These modifications include an alanine scan, probing variation of hydrophobicity at lys5 and lys7, manipulation of amphipathicity, N-and C-termini deletions and lys-arg substitutions. We found that the hydrophobic residues in position 3 (1-nal), 4 (phe), 6 (1-nal) and 8 (nle) are important for antimicrobial activity and to a lesser extent cationic lysine residues in position 1, 2, 5 and 7. Our best analogue 5, showed MICs of 4 µg/mL against A. baumannii, E. coli, P. aeruginosa and S. aureus with a hemolytic activity of 47% against red blood cells. Furthermore, compound 5 kills bacteria in a concentration-dependent manner as shown by time-kill kinetics. Circular dichroism (CD) spectra of D2D and compounds 1-8 showed that they likely fold into α-helical secondary structure. Small angle x-ray scattering (SAXS) experiments showed that a random unstructured polymer-like chains model could explain D2D and compounds 1, 3, 4, 6 and 8. Solution structure of compound 5 can be described with a nanotube structure model, compound 7 can be described with a filament-like structure model, while compound 2 can be described with both models. Lipid interaction probed by small angle X-ray scattering (SAXS) showed that a higher amount of compound 5 (~50-60%) inserts into the bilayer compared to D2D (~30-50%). D2D still remains the lead compound, however compound 5 is an interesting antimicrobial peptide for further investigations due to its nanotube structure and minor improvement to antimicrobial activity compared to D2D.

Expanding the potential of NAI-107 for treating serious ESKAPE pathogens: synergistic combinations against Gram-negatives and bactericidal activity against non-dividing cells.

Objectives: To characterize NAI-107 and related lantibiotics for their in vitro activity against Gram-negative pathogens, alone or in combination with polymyxin, and against non-dividing cells or biofilms of Staphylococcus aureus. NAI-107 was also evaluated for its propensity to select or induce self-resistance in Gram-positive bacteria. Methods: We used MIC determinations and chequerboard experiments to establish the antibacterial activity of the examined compounds against target microorganisms. Time-kill assays were used to evaluate killing of exponential and stationary-phase cells. The effects on biofilms (growth inhibition and biofilm eradication) were evaluated using biofilm-coated pegs. The frequency of spontaneous resistant mutants was evaluated by either direct plating or by continuous sub-culturing at 0.5 × MIC levels, followed by population analysis profiles. Results: The results showed that NAI-107 and its brominated variant are highly active against Neisseria gonorrhoeae and some other fastidious Gram-negative pathogens. Furthermore, all compounds strongly synergized with polymyxin against Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, and showed bactericidal activity. Surprisingly, NAI-107 alone was bactericidal against non-dividing A. baumannii cells. Against S. aureus, NAI-107 and related lantibiotics showed strong bactericidal activity against dividing and non-dividing cells. Activity was also observed against S. aureus biofilms. As expected for a lipid II binder, no significant resistance to NAI-107 was observed by direct plating or serial passages. Conclusions: Overall, the results of the current work, along with previously published results on the efficacy of NAI-107 in experimental models of infection, indicate that this lantibiotic represents a promising option in addressing the serious threat of antibiotic resistance.

Modulation of Backbone Flexibility for Effective Dissociation of Antibacterial and Hemolytic Activity in Cyclic Peptides.

Bacterial resistance to antibiotic therapy is on the rise and threatens to evolve into a worldwide emergency: alternative solutions to current therapies are urgently needed. Cationic amphipathic peptides are potent membrane-active agents that hold promise as the next-generation therapy for multidrug-resistant infections. The peptides' behavior upon encountering the bacterial cell wall is crucial, and much effort has been dedicated to the investigation and optimization of this amphipathicity-driven interaction. In this study we examined the interaction of a novel series of nine-membered flexible cyclic AMPs with liposomes mimicking the characteristics of bacterial membranes. Employed techniques included circular dichroism and marker release assays, as well as microbiological experiments. Our analysis was aimed at correlating ring flexibility with their antimicrobial, hemolytic, and membrane activity. By doing so, we obtained useful insights to guide the optimization of cyclic antimicrobial peptides via modulation of their backbone flexibility without loss of activity.

The Lantibiotic NAI-107 Efficiently Rescues Drosophila melanogaster from Infection with Methicillin-Resistant Staphylococcus aureus USA300.

We used the fruit fly Drosophila melanogaster as a cost-effective in vivo model to evaluate the efficacy of novel antibacterial peptides and peptoids for treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections. A panel of peptides with known antibacterial activity in vitro and/or in vivo was tested in Drosophila Although most peptides and peptoids that were effective in vitro failed to rescue lethal effects of S. aureus infections in vivo, we found that two lantibiotics, nisin and NAI-107, rescued adult flies from fatal infections. Furthermore, NAI-107 rescued mortality of infection with the MRSA strain USA300 with an efficacy equivalent to that of vancomycin, a widely applied antibiotic for the treatment of serious MRSA infections. These results establish Drosophila as a useful model for in vivo drug evaluation of antibacterial peptides.

An Amphipathic Undecapeptide with All d-Amino Acids Shows Promising Activity against Colistin-Resistant Strains of Acinetobacter baumannii and a Dual Mode of Action.

Multiple strains of Acinetobacter baumannii have developed multidrug resistance (MDR), leaving colistin as the only effective treatment. The cecropin-α-melittin hybrid BP100 (KKLFKKILKYL-NH2) and its analogs have previously shown activity against a wide array of plant and human pathogens. In this study, we investigated the in vitro antibacterial activities of 18 BP100 analogs (four known and 14 new) against the MDR A. baumannii strain ATCC BAA-1605, as well as against a number of other clinically relevant human pathogens. Selected peptides were further evaluated against strains of A. baumannii that acquired resistance to colistin due to mutations of the lpxC, lpxD, pmrA, and pmrB genes. The novel analogue BP214 showed antimicrobial activity at 1 to 2 µM and a hemolytic 50% effective concentration (EC50) of >150 µM. The lower activity of its enantiomer suggests a dual, specific and nonspecific mode of action. Interestingly, colistin behaved antagonistically to BP214 when pmrAB and lpxC mutants were challenged.

Collateral Resistance and Sensitivity Modulate Evolution of High-Level Resistance to Drug Combination Treatment in Staphylococcus aureus.

As drug-resistant pathogens continue to emerge, combination therapy will increasingly be relied upon to treat infections and to help combat further development of multidrug resistance. At present a dichotomy exists between clinical practice, which favors therapeutically synergistic combinations, and the scientific model emerging from in vitro experimental work, which maintains that this interaction provides greater selective pressure toward resistance development than other interaction types. We sought to extend the current paradigm, based on work below or near minimum inhibitory concentration levels, to reflect drug concentrations more likely to be encountered during treatment. We performed a series of adaptive evolution experiments using Staphylococcus aureus. Interestingly, no relationship between drug interaction type and resistance evolution was found as resistance increased significantly beyond wild-type levels. All drug combinations, irrespective of interaction types, effectively limited resistance evolution compared with monotreatment. Cross-resistance and collateral sensitivity were found to be important factors in the extent of resistance evolution toward a combination. Comparative genomic analyses revealed that resistance to drug combinations was mediated largely by mutations in the same genes as single-drug-evolved lineages highlighting the importance of the component drugs in determining the rate of resistance evolution. Results of this work suggest that the mechanisms of resistance to constituent drugs should be the focus of future resistance evolution work.

Rapid selection of Plasmodium falciparum chloroquine resistance transporter gene and multidrug resistance gene-1 haplotypes associated with past chloroquine and present artemether-lumefantrine use in Inhambane District, southern Mozambique.

Chloroquine (CQ) use in Mozambique was stopped in 2002 and artemether-lumefantrine (AL) was implemented in 2008. In light of no use of CQ and extensive use of AL, we determined the frequency of molecular markers of Plasmodium falciparum drug resistance/tolerance to CQ and AL in persons living in Linga-Linga, an isolated peninsula and in Furvela village, which is located 8 km inland. The P. falciparum chloroquine resistance transporter gene CVMNK wild type increased in frequency from 43.9% in 2009 to 66.4% in 2010 (P ≤ 0.001), and combined P. falciparum multidrug resistance gene 1 N86-184F-D1246 haplotype increased significantly between years (P = 0.039). The combination of P. falciparum chloroquine resistance transporter gene CVMNK and P. falciparum multidrug resistance gene NFD increased from 24.3% (2009) to 45.3% in (2010, P = 0.017). The rapid changes observed may largely be caused by decreased use of CQ and large-scale use of AL. In the absence of a clear AL-resistance marker and the (almost) continent-wide use of AL in sub-Saharan Africa, and when considering CQ reintroduction, continued monitoring of these markers is needed.

Prevalence of single nucleotide polymorphisms in the Plasmodium falciparum multidrug resistance gene (Pfmdr-1) in Korogwe District in Tanzania before and after introduction of artemisinin-based combination therapy.

Tanzania implemented artemether-lumefantrine (AL) as the first-line treatment for uncomplicated malaria in November of 2006 because of resistance to sulfadoxine-pyrimethamine. AL remains highly efficacious, but widespread use may soon facilitate emergence of artemisinin tolerance/resistance, which initially may be detected at the molecular level as temporal changes in the frequency of single nucleotide polymorphisms (SNPs) in the Pfmdr-1 gene associated with AL resistance. In Tanzania, 830 Plasmodium falciparum-positive samples collected between 2003 and 2010 were examined for SNPs of Pfmdr-1 at codons 86, 184, and 1246. Both the N86 and 184F increased from 2006 to 2010 (logistic regression; N86: odds ratio [95% confidence interval] = 1.35 [1.07-1.71], P = 0.01; 184F: odds ratio = 1.42 [1.07-1.88], P = 0.02), and no change was found for D1246 (odds ratio = 1.01 [0.80-1.28], P = 0.9). The observed changes may be because of introduction of AL, and if so, this finding gives cause for concern and argues for continued surveillance of these molecular markers.