Zinc oxide nanoparticle inhibits the biofilm formation of <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis>

Biofilms are structured consortia of microbial cells that grow on living and non living surfaces and surround themselves with secreted polymers. Infections with bacterial biofilms have emerged as a foremost public health concern because biofilm growing cells can be highly resistant to both antibiotics and host immune defenses. Zinc oxide nanoparticles have been reported as a potential antimicrobial agent, thus, in the current study, we have evaluated the antimicrobial as well as antibiofilm activity of zinc oxide nanoparticles against the bacterium Streptococcus pneumoniae which is a significant cause of disease. Zinc oxide nanoparticles showed strong antimicrobial activity against S. pneumoniae, with an MIC value of 40 μg/ml. Biofilm inhibition of S. pneumoniae was also evaluated by performing a series of experiments such as crystal violet assay, microscopic observation, protein count, EPS secretion etc. using sub-MIC concentrations (3, 6 and 12 µg/ml) of zinc oxide nanoparticles. The results showed that the sub-MIC doses of zinc oxide nanoparticles exhibited significant anti-biofilm activity against S. pneumoniae, with maximum biofilm attenuation found at 12 μg/ml. Taken together, the results indicate that zinc oxide nanoparticles can be considered as a potential agent for the inhibition of microbial biofilms.     

Antimicrobial activity of camphor tree silver nano-particles against foulbrood diseases and finding out new strain of Serratia marcescens via DGGE-PCR,as a secondary infection on honeybee larvae

American foulbrood (AFB) and European foulbrood (EFB) are the two major bacterial diseases affecting honeybees, leading to a decrease in viability of the hive, decreasing honey production, and resulting in significant economic losses to beekeepers. Due to the inefficiency and/or low efficacy of some antibiotics, researches with nanotechnology represent, possibly, new therapeutic strategies. Nanostructure drugs have presented some advantagesover the conventional medicines, such as slow, gradual and controlled release, increased bioavailability, and reduced side-effects. In this study, different infected larvae were collected from two apiaries; the combs that had symptoms of American and European foulbrood were isolated. In vitro antimicrobial activity of camphor tree silver nano-particles against foulbrood diseases were characterized using UV–Vis spectrophotometry and scanning electron microscope (SEM) that proves the formation of silver nanoparticles with size range 160–660 nm. The antimicrobial activity of the silver nanoparticles was tested using agar diffusion assay and proved their ability to effectively cease the pathogenic bacterial growth in both AFB and EFB. DGGE-PCR technique has been applied for the identification of un-common bacterial infections honeybees depending on 16S rRNA amplification from their total extracted DNA and has been identified as Serratia marcescens (TES), deposited in GenBank with a new accession number (MT240613). The results were confirmed strain has been detected by DGGE-PCR analysis causing uniquely infected brood that was attacked by the American Foulbrood It could be concluded that greenly synthesized silver nanoparticles is projected to be used as effective treatment for honeybee bacterial diseases. These material need more investigations under field conditions and study the possibility of its residues in honeybee products such as honey, and beeswax.     

The effect of temperature on antibacterial activity of biosynthesized silver nanoparticles

The purpose of this study was the evaluation of two different temperatures on antibacterial activity of the biosynthesized silver nanoparticles. 38 silver nanoparticles-producing bacteria were isolated from soil and identified. Biosynthesis of silver nanoparticles by these bacteria was verified through visible light spectrophotometry. Two strains were relatively active for production of silver nanoparticles. These strains were subjected for molecular identification and recognized as Bacillus sp. and Acinetobacter schindleri. In the present study, the effect of temperatures was evaluated on structure and antimicrobial properties of the silver nanoparrticles by transmission electron microscopy (TEM), X-ray diffraction (XRD) analysis and antimicrobial Agar well diffusion methods. The silver nanoparticles showed antibacterial activity against all the pathogenic bacteria; however, this property was lost after treatment of the silver nanoparticles by high temperatures (100 and 300 °C). TEM images showed that the average sizes of heated silver nanoparticles were >100 nm. However, these were <100 nm for non-heated silver nanoparticles. Although, XRD patterns showed the crystalline structure of heated silver nanoparticles, their antibacterial activities were less. This was possible because of the sizes and accordingly less penetration of the particles into the bacterial cells. In addition, elimination of the capping agents by heat might be considered another reason.     

Antimicrobial activity of metal based nanoparticles against microbes associated with diseases in aquaculture

The emergence of diseases and mortalities in aquaculture and development of antibiotics resistance in aquatic microbes, has renewed a great interest towards alternative methods of prevention and control of diseases. Nanoparticles have enormous potential in controlling human and animal pathogens and have scope of application in aquaculture. The present investigation was carried out to find out suitable nanoparticles having antimicrobial effect against aquatic microbes. Different commercial as well as laboratory synthesized metal and metal oxide nanoparticles were screened for their antimicrobial activities against a wide range of bacterial and fungal agents including certain freshwater cyanobacteria. Among different nanoparticles, synthesized copper oxide (CuO), zinc oxide (ZnO), silver (Ag) and silver doped titanium dioxide (Ag–TiO₂) showed broad spectrum antibacterial activity. On the contrary, nanoparticles like Zn and ZnO showed antifungal activity against fungi like Penicillium and Mucor species. Since CuO, ZnO and Ag nanoparticles showed higher antimicrobial activity, they may be explored for aquaculture use.     

Green synthesis of ZnO nanoparticles for antimicrobial and vegetative growth applications: A novel approach for advancing efficient high quality health care to human wellbeing

The present work aims to synthesize zinc oxide (ZnO) nanoparticles via green approaches using leaf extract of Parthenium hysterophorus. UV–vis and FT-IR tests confirmed the existence of biomolecules, active materials, and metal oxides. The X-ray diffraction structural study exposes the ZnO nanoparticles formation with hexagonal phase structures. SEM and TEM analysis reveal surface morphologies of ZnO nanoparticles and most of them are spherical with a size range of 10 nm. ZnO nanoparticles were revealed strong antimicrobial activity against both bacterial and fungal strains. The germination of seeds and vegetative growth of Sesamum indicum has been greatly improved.     

A novel mechanism for the antibacterial effect of silver nanoparticles on Escherichia coli

Silver nanoparticles are known to have antimicrobial properties and have been used extensively in medicine, although the mechanism(s) of action have not yet been clearly established. In the present study, the findings suggest a novel mechanism for the antibacterial effect of silver nanoparticles on Escherichia coli, namely, the induction of a bacterial apoptosis-like response. We propose a possible mechanism for the bacterial apoptosis-like response that includes the following: accumulation of reactive oxygen species (ROS) (detected with H₂DCFDA staining), increased intracellular calcium levels (detected with Fura-2 AM), phosphatidylserine exposure in the outer membrane (detected with Annexin V) which is the hallmarks of early apoptosis, disruption of the membrane potential [detected with DiBAC₄(3)], activation of a bacterial caspase-like protein (detected by FITC-VAD-FMK staining) and DNA degradation (detected with TUNEL assay) which is the hallmarks of late apoptosis in bacterial cells treated with silver nanoparticles. We also performed RecA expression assay with western blotting and observed activation of SOS response to repair the damaged DNA. To summarize, silver nanoparticles are involved in the apoptosis-like response in E. coli and the novel mechanisms which were identified in this study, suggest that silver nanoparticles may be an effective antimicrobial agent with far lower propensity for inducing microbial resistance than antibiotics.     

Antimicrobial activity of chemically and biologically synthesized silver nanoparticles against some fish pathogens

Pathogens isolated from fish appear to possess considerable antimicrobial resistance and represent a problem for the economy and public health. Natural antimicrobial substitutes to traditional antibiotics represent an essential tool in the fight against antibiotic resistance. Nanotechnology has shown considerable potential in different research fields, and the antimicrobial properties of silver nanoparticles are known. Silver has been used for medical purposes since ancient times because of its bactericidal properties, and the highly reactive surfaces of silver nanoparticles (AgNPs) indicate that they might have a function in antimicrobial applications. This work aimed to study the antimicrobial properties of biologically produced AgNPs from Origanum vulgare leaves compared to chemically produced AgNPs. Both types were characterized by UV–vis spectrophotometry, TEM, and dynamic light scattering and tested against three bacterial strains (Streptococcus agalactiae, and Aeromonas hydrophila, both isolated from Nile tilapia and Vibrio alginolyticus, isolated from sea bass) and three fungal strains (Aspergillus flavus, Fusarium moniliforme, and Candida albicans, all isolated from Nile tilapia). Disk diffusion test and evaluation of ultrastructure changes of tested microorganisms treated with AgNPs by transmission electron microscopy were performed. Moreover, the hemolytic properties of AgNPs were studied on chicken and goat red blood cells. The results obtained declare that the green biological production of silver nanoparticles is safer and more effective than the chemical one; moreover, AgNPs have interesting dose-dependent antimicrobial properties, with better results for biologically produced ones; their effectiveness against tested bacterial and fungal strains opens the way to their use to limit fish diseases, increase economy and improve human health.     

Rapid nanoparticle-mediated monitoring of bacterial metabolic activity and assessment of antimicrobial susceptibility in blood with magnetic relaxation

Considering the increased incidence of bacterial infections and the emergence of multidrug resistant bacteria at the global level, we designed superparamagnetic iron oxide nanoparticles as nanosensors for the assessment of antimicrobial susceptibility through magnetic relaxation. In this report, we demonstrate that iron oxide nanosensors, either dextran-coated supplemented with Con A or silica-coated conjugated directly to Con A, can be used for the fast (1) quantification of polysaccharides, (2) assessment of metabolic activity and (3) determination of antimicrobial susceptibility in blood. The use of these polysaccharide nanosensors in the determination of antimicrobial susceptibility in the clinic or the field, and the utilization of these nanoprobes in pharmaceutical R&D are anticipated.     

Rapid biosynthesis of PVP coated silver nanoparticles by <Emphasis Type="Italic">Kocuria rosea</Emphasis> and their antimicrobial activity

The need for more effective antimicrobial agent and propitious application of nanotechnology in therapeutics and diagnostics has prompted the research on ecofriendly synthesis of silver nanoparticles. The objective of present study was to investigate the antibacterial and antifungal activity of biologically synthesized silver nanoparticles. The silver nanoparticles were synthesized by extracellular method, using soil bacteria Kocuria rosea. The synthesized silver nanoparticles were characterized by UV-Visible spectroscopy, X-ray diffractometry (XRD), transmission electron microscopy (TEM) and fourier transformation infrared spectroscopy (FTIR). On the basis of TEM analysis, the synthesized nanoparticles were found to be spherical with an average size of 30–50 nm. The biologically synthesized silver nanoparticles showed significant antimicrobial activity against pathogens.     

Employment of Cassia angustifolia leaf extract for zinc nanoparticles fabrication and their antibacterial and cytotoxicity

The plant Cassia angustifolia belongs to Saudi Arabia, which is one of the native places and now cultured throughout the global countries. Medical care in the Arab world is an essential outlet for medicinal plants, both because they are crucial elements for prophetic medicine and due to their lengthy background in the Middle East. C.angustifolia is one of the medicinal plants used in the Saudi Arabia. The usage of plant extracts for synthesizing nanoparticles is conducive to other biological material, since it avoids the lengthy phase of cell culture maintenance. Silver nanoparticles attract further attention due to their strong conductivity, stability and antimicrobial activity across different metal nanoparticles. The present study was designed in the Saudi C. angustifolia leaves with the zinc synthesis of nanoparticles and its antibacterial ability. The plant extracts of C. angustifolia was used for synthesis of zinc nanoparticles, antimicrobial activities against bacterial strains have been tested along with transmission electron microscope (TEM), UV spectroscopy and antimicrobial activities have been conducted. This study showed that silver ions may be transferred from the plant extract to silver nanoparticles. AgNPs biogenic capacity to antibacterial with lovo cell with IC₅₀ ranged from 33.5 ± 0.2 μg/mL demonstrated strong antibacterial capacity to antibody. The overall absorption value for the extract was between 420 and 440 nm and the color transition to green was the plasma absorption of the AgNPs. TEM results was showed in 200,000 magnification. The uniqueness of the current study is that Cassia angustifolia leaf extract from Saudi Arabia was used to prepare the metallic nanoparticles. Additionally, ZnCl₂ may also be used as nanoparticles of mineral salt and zinc, which, since their application has been confirmed, are antimicrobial.     

Biogenic synthesis of silver nanoparticles using Brazilian propolis

Biological methods have been used to synthesize silver nanoparticles through materials such as bacteria, fungi, plants, and propolis due to their reducing properties, stabilizer role and environmentally friendly characteristic. Considering the antimicrobial activity of propolis as well as the broad-spectrum antibacterial effects of silver nanoparticles, this study aim to describe the use of Brazilian propolis to synthesize silver nanoparticles (AgNP-P) and investigate its antimicrobial activity. The synthesis was optimized by factorial design, choosing the best conditions for smaller size particles. AgNP-P demonstrated a maximum absorbance at 412 nm in ultraviolet-visible spectra, which indicated a spherical format and its formation. Dynamic light scattering demonstrated a hydrodynamic size of 109 nm and polydispersity index less than 0.3, showing a good size distribution and stability. After its purification via centrifugation, microscopy analysis corroborates the format and showed the presence of propolis around silver nanoparticle. X-ray diffraction peaks were attributed to the main planes of the metallic silver crystalline structure; meanwhile infrared spectroscopy demonstrated the main groups responsible for silver reduction, represented by ∼22% of AgNP-P indicates by thermal analysis. Our product revealed an important antimicrobial activity indicating a synergism between propolis and silver nanoparticles as expected and promising to be an effective antimicrobial product to be used in infections.     

Mycoendophytes as efficient synthesizers of bionanoparticles: nanoantimicrobials,mechanism, and cytotoxicity

Mycoendophytes are the fungi that occur inside the plant tissues without exerting any negative impact on the host plant. They are most frequently isolated endophytes from the leaf, stem, and root tissues of various plants. Among all fungi, the mycoendophytes as biosynthesizer of noble metal nanoparticles (NPs) are less known. However, some reports showing efficient synthesis of metal nanoparticles, mainly silver nanoparticles and its remarkable antimicrobial activity against bacterial and fungal pathogens of humans and plants. The nanoparticles synthesized from mycoendophytes present stability, polydispersity, and biocompatibility. These are non-toxic to humans and environment, can be gained in an easy and cost-effective manner, have wide applicability and could be explored as promising candidates for a variety of biomedical, pharmaceutical, and agricultural applications. Mycogenic silver nanoparticles have also demonstrated cytotoxic activity against cancer cell lines and may prove to be a promising anticancer agent. The present review focuses on the biological synthesis of metal nanoparticles from mycoendophytes and their application in medicine. In addition, different mechanisms of biosynthesis and activity of nanoparticles on microbial cells, as well as toxicity of these mycogenic metal nanoparticles, have also been discussed.     

Investigation of antimicrobial activity of photothermal therapeutic gold/copper sulfide core/shell nanoparticles to bacterial spores and cells

Background

Au/CuS core/shell nanoparticles (NPs) were designed as a new type of transducer agent for photothermal therapy (PTT), with attractive features of easy preparation, low cost and small size for targeting. This paper studied for the first time the intrinsic antimicrobial activity of Au/CuS NPs to B. anthracis spores and cells in addition to its PTT effect.

Results

It was found that Au/CuS NPs were highly efficient in inactivating B. anthracis cells, but not effective to the spores. Treatment with NPs at ~0.83 μM for 30 min achieved a 7 log reduction in viable cells. The antimicrobial effect was both NPs concentration and treatment time dependent. SEM imaging and the efflux of DNA test demonstrated the damage of cell membrane after NPs treatment, yet further research is necessary to fully understand the precise inactivation mechanism.

Conclusions

The Au/CuS NPs had strong antimicrobial activity to B. anthracis cells, which showed a great potential to be an effective antimicrobial agent to bacterial cells.

     

Antibiofilm and antimicrobial activities of green synthesized silver nanoparticles using marine red algae Gelidium corneum

In our study, green synthesis of silver nanoparticles was carried out using a red algae Gelidium corneum extract as reducing agent. The obtained silver nanoparticles were characterized by UV–vis, TEM, XRD, FTIR and ICP-MS measurements. FTIR measurements indicated the possible functional groups responsible for the stabilization and reduction of nanoparticles, while XRD analysis results explained the crystalline structure of the particles with centric cubic geometry. TEM micrographs showed that the size of the nanoparticles was between 20–50 nm. According to the broth microdilution test results, AgNPs showed a high antimicrobial activity with very low MIC values (0.51 μg/ml for Candida albicans yeast and 0.26 μg/ml for Escherichia coli bacteria). The different ultrastructural effects of silver nanoparticles on yeast and bacterial cells were observed by TEM. Antibiofilm efficacy studies were also examined in two stages as prebiofilm and postbiofilm effect. In prebiofilm effect studies, AgNPs (0.51 μg/ ml) exhibited 81% reducing effect on biofilm formation. The highest reduction rate in postbiofilm studies was 73.5% and this was achieved with 2.04 μg/ml AgNPs. Our data support that the silver nanoparticles obtained by this environmentally friendly process have potential to be used for industrial and therapeutic purposes.     

Fabrication of nanofibers with antimicrobial functionality used as filters: protection against bacterial contaminants

A comparative study of antimicrobial activity is done using three different electrospun nanofibers namely-CA, PAN, and PVC used as control and with various amounts of AgNO(3) being treated with UV-irradiation leading to the enhancement of silver nanoparticles. DMF is used as the common solvent which helps to undergo spontaneous slow reduction at room temperature to form silver nanoparticles followed by UV-irradiation using a 400 W source. The time required for the formation of silver nanoparticles is short and they are more or less well dispersed with few such aggregates. The presence of silver nanoparticles is confirmed using various characterization techniques. The antimicrobial activity is studied using nanofibers with fabricated functionality.     

Development of a Standardized and Safe Airborne Antibacterial Assay,and Its Evaluation on Antibacterial Biomimetic Model Surfaces

Bacterial infection of biomaterials is a major concern in medicine, and different kinds of antimicrobial biomaterial have been developed to deal with this problem. To test the antimicrobial performance of these biomaterials, the airborne bacterial assay is used, which involves the formation of biohazardous bacterial aerosols. We here describe a new experimental set-up which allows safe handling of such pathogenic aerosols, and standardizes critical parameters of this otherwise intractable and strongly user-dependent assay. With this new method, reproducible, thorough antimicrobial data (number of colony forming units and live-dead-stain) was obtained. Poly(oxonorbornene)-based Synthetic Mimics of Antimicrobial Peptides (SMAMPs) were used as antimicrobial test samples. The assay was able to differentiate even between subtle sample differences, such as different sample thicknesses. With this new set-up, the airborne bacterial assay was thus established as a useful, reliable, and realistic experimental method to simulate the contamination of biomaterials with bacteria, for example in an intraoperative setting.     

Antibacterial and antibiofilm effects of silver nanoparticles against the uropathogen Escherichia coli U12

The drug-resistant bacterial strains' emergence increases day by day. This may be a result of biofilm presence, which protects bacteria from antimicrobial agents. Thus, new approaches must be used to control biofilm-related infections in healthcare settings. In such a study, biological silver nanoparticles were introduced in such a study as an anti-biofilm agent against multidrug-resistant E. coli U12 on urinary catheters. Seven different silver nanoparticles concentrations were tested for their antimicrobial activities. Also, anti-biofilm activities against E. coli U12 were tested. Using the dilution method, the silver nanoparticles concentration of 85 μg/ml was the MIC (Minimum Inhibitory Concentration) that had excellent biocompatibility and showed significant antibacterial activity against E. coli U12. Scanning electron microscopy (SEM) confirmed that the highest efficient dose of silver nanoparticles was 340 μg/ml at 144 h that reduced adhesion of E. coli U12 to the urinary catheter. E. coli U12 cells ruptured cell walls and cell membranes after being examined using transmission electron microscopy (TEM). Thus, biologically prepared silver nanoparticles could be used to coat medical devices since it is effective and promising to inhibit biofilm formation by impregnating urinary catheters with silver nanoparticles.     

Characterization of a Gloverin-Like Antimicrobial Peptide Isolated from Muga Silkworm, <Emphasis Type="Italic">Antheraea assamensis</Emphasis>

Antimicrobial peptides (AMPs) are produced in all living organisms including insects in a non-specific manner, and act as innate immune defense arsenal against the invading pathogens. Muga silkworm (Antheraea assamensis) larvae were injected with Candida albicans and AMPs were isolated from the hemolymph after extracting with methanol, acetic acid and water mixture (90:1:9) and evaluated for antimicrobial activity against fungal and bacterial pathogens. Further purification was done through successive semipreparative and analytical reversed phase HPLC using C-18 column. The obtained fractions were collected, lyophilized and tested for antimicrobial activity. Among the HPLC fractions, one showed highest activity with MIC value of 64 µg/ml against Gram-negative bacteria, Escherichia coli and Enterobacter cloacae. Purity of this isolated peptide was confirmed by SDS-PAGE and TLC, and its molecular mass was determined as 9.052 kDa by MALDI-TOF mass spectrometry. From the mass fingerprinting analysis of this peptide after trypsin digestion a peptide fragment with molecular mass of 2622.7 Da was obtained. De novo sequencing of this peptide fragment following MS/MS analysis identified few amino acid residues as “KSGGGGWGS” with a total score of 46.9 with gloverin peptide of A. mylitta. The peptide inhibited biofilm formation of the Gram-negative bacterial pathogens. SEM study revealed that peptide disrupted bacterial cell wall to leach out intracellular materials and may be the major target for its antimicrobial activity.     

Soil myxobacteria as a potential source of polyketide-peptide substances

Myxobacteria, a group of antimicrobial producing bacteria, have been successfully cultured and characterized from ten soil samples collected from different parts of Slovakia. A total of 79 myxobacteria belonging to four genera (Myxococcus, Corallococcus, Sorangium, and Polyangium) were isolated based on aspects of their life cycle. Twenty-five of them were purified, fermented, and screened for antimicrobial activities against 11 test microorganisms. Results indicated that crude extracts showed more significant activities against Gram-positive than against Gram-negative bacteria or fungi. Based on a higher degree and broader range of antimicrobial production, the two most potential extracts (K9-5, V3-1) were selected for HPLC fractionation against Micrococcus luteus and Staphylococcus aureus and LC/MS analysis of potential antibiotic metabolites. The analysis resulted in the identification of polyketide-peptide antibiotics, namely corallopyronin A and B (K9-5) and myxalamid B and C (V3-1), which were responsible for important Gram-positive activity in the observed strains. A sequence similarity search through BLAST revealed that these strains showed the highest sequence similarity to Corallococcus coralloides (K9-5, NCBI accession number KX256198) and Myxococcus xanthus (V3-1, NCBI accession number KX256197). Although screening of myxobacteria is laborious, due to difficulties in isolating cultures, this research represented the first report covering the isolation and cultivation of this challenging bacterial group from Slovakian soils as well as the screening of their antimicrobial activity, cultural identification, and secondary metabolite identification.     

Design and characterization of short antimicrobial peptides using leucine zipper templates with selectivity towards microorganisms

Design of antimicrobial peptides with selective activity towards microorganisms is an important step towards the development of new antimicrobial agents. Leucine zipper sequence has been implicated in cytotoxic activity of naturally occurring antimicrobial peptides; moreover, this motif has been utilized for the design of novel antimicrobial peptides with modulated cytotoxicity. To understand further the impact of substitution of amino acids at ‘a’ and/or ‘d’ position of a leucine zipper sequence of an antimicrobial peptides on its antimicrobial and cytotoxic properties four short peptides (14-residue) were designed on the basis of a leucine zipper sequence without or with replacement of leucine residues in its ‘a’ and ‘d’ positions with d-leucine or alanine or proline residue. The original short leucine zipper peptide (SLZP) and its d-leucine substituted analog, DLSA showed comparable activity against the tested Gram-positive and negative bacteria and the fungal strains. The alanine substituted analog (ASA) though showed appreciable activity against the tested bacteria, it showed to some extent lower activity against the tested fungi. However, the proline substituted analog (PSA) showed lower activity against the tested bacterial or fungal strains. Interestingly, DLSA, ASA and PSA showed significantly lower cytotoxicity than SLZP against both human red blood cells (hRBCs) and murine 3T3 cells. Cytotoxic and bactericidal properties of these peptides matched with peptide-induced damage/permeabilization of mammalian cells and bacteria or their mimetic lipid vesicles suggesting cell membrane could be the target of these peptides. As evidenced by tryptophan fluorescence and acrylamide quenching studies the peptides showed similarities either in interaction or in their localization within the bacterial membrane mimetic negatively charged lipid vesicles. Only SLZP showed localization inside the mammalian membrane mimetic zwitterionic lipid vesicles. The results show significant scope for designing antimicrobial agents with selectivity towards microorganisms by substituting leucine residues at ‘a’ and/or ‘d’ positions of a leucine zipper sequence of an antimicrobial peptide with different amino acids.