# **ANTIMICROBIAL COMPOUNDS FROM NATURAL SOURCES**

**Topic Editors Mirian A. Hayashi, Fernando C. Bizerra and Pedro Ismael Da Silva Jr**

#### *FRONTIERS COPYRIGHT STATEMENT*

© Copyright 2007-2014 Frontiers Media SA. All rights reserved.

All content included on this site, such as text, graphics, logos, button icons, images, video/audio clips, downloads, data compilations and software, is the property of or is licensed to Frontiers Media SA ("Frontiers") or its licensees and/or subcontractors. The copyright in the text of individual articles is the property of their respective authors, subject to a license granted to Frontiers.

The compilation of articles constituting this e-book, wherever published, as well as the compilation of all other content on this site, is the exclusive property of Frontiers. For the conditions for downloading and copying of e-books from Frontiers' website, please see the Terms for Website Use. If purchasing Frontiers e-books from other websites or sources, the conditions of the website concerned apply.

Images and graphics not forming part of user-contributed materials may not be downloaded or copied without permission.

Individual articles may be downloaded and reproduced in accordance with the principles of the CC-BY licence subject to any copyright or other notices. They may not be re-sold as an e-book.

As author or other contributor you grant a CC-BY licence to others to reproduce your articles, including any graphics and third-party materials supplied by you, in accordance with the Conditions for Website Use and subject to any copyright notices which you include in connection with your articles and materials.

All copyright, and all rights therein, are protected by national and international copyright laws.

The above represents a summary only. For the full conditions see the Conditions for Authors and the Conditions for Website Use.

**ISSN** 1664-8714 **ISBN** 978-2-88919-259-5 **DOI** 10.3389/978-2-88919-259-5

### *ABOUT FRONTIERS*

Frontiers is more than just an open-access publisher of scholarly articles: it is a pioneering approach to the world of academia, radically improving the way scholarly research is managed. The grand vision of Frontiers is a world where all people have an equal opportunity to seek, share and generate knowledge. Frontiers provides immediate and permanent online open access to all its publications, but this alone is not enough to realize our grand goals.

### *FRONTIERS JOURNAL SERIES*

The Frontiers Journal Series is a multi-tier and interdisciplinary set of open-access, online journals, promising a paradigm shift from the current review, selection and dissemination processes in academic publishing.

All Frontiers journals are driven by researchers for researchers; therefore, they constitute a service to the scholarly community. At the same time, the Frontiers Journal Series operates on a revolutionary invention, the tiered publishing system, initially addressing specific communities of scholars, and gradually climbing up to broader public understanding, thus serving the interests of the lay society, too.

### *DEDICATION TO QUALITY*

Each Frontiers article is a landmark of the highest quality, thanks to genuinely collaborative interactions between authors and review editors, who include some of the world's best academicians. Research must be certified by peers before entering a stream of knowledge that may eventually reach the public - and shape society; therefore, Frontiers only applies the most rigorous and unbiased reviews.

Frontiers revolutionizes research publishing by freely delivering the most outstanding research, evaluated with no bias from both the academic and social point of view.

By applying the most advanced information technologies, Frontiers is catapulting scholarly publishing into a new generation.

### *WHAT ARE FRONTIERS RESEARCH TOPICS?*

Frontiers Research Topics are very popular trademarks of the Frontiers Journals Series: they are collections of at least ten articles, all centered on a particular subject. With their unique mix of varied contributions from Original Research to Review Articles, Frontiers Research Topics unify the most influential researchers, the latest key findings and historical advances in a hot research area!

Find out more on how to host your own Frontiers Research Topic or contribute to one as an author by contacting the Frontiers Editorial Office<EMAIL_ADDRESS>
# **ANTIMICROBIAL COMPOUNDS FROM NATURAL SOURCES**

#### Topic Editors:

**Mirian A. Hayashi,** Universidade Federal de São Paulo (UNIFESP), Brazil **Fernando C. Bizerra,** Universidade Federal de São Paulo (UNIFESP), Brazil **Pedro Ismael Da Silva Jr,** Instituto Butantan, Brazil

Structural model for Juruin, an antifungal peptide from the venom of the Amazonian Pink Toe spider, which contains the inhibitory cystine knot motif.

Comparison of the structures of Juruin from Avicularia juruensis and U1-theraphotoxin-Ba1a (PDB ID: 2KGH) from Brachypelma ruhnaui, after homology modeling of Juruin. Images taken from: Ayroza G, Ferreira ILC, Sayegh RSR, Tashima AK and da Silva PI (2012) Juruin: an antifungal peptide from the venom of the Amazonian Pink Toe spider, Avicularia juruensis, which contains the inhibitory cystine knot motif. *Front. Microbio*. 3:324. doi: 10.3389/ fmicb.2012.00324

The nature is a generous source of a number of compounds with potential application for the treatment of several diseases including the infectious diseases, which is of utmost concern for the modern medicine due to the observed striding antimicrobial resistance. A number of sources of natural compounds with valuable and clinical antimicrobial activity can be listed, comprising medicinal plants, marine and terrestrial organisms, which includes fungi and bacteria. Nevertheless, there is still a vast fauna and flora that, once systematically explored, could provide additional antimicrobial leads and drugs.

Investigators were invited to contribute with original research and/or review articles on this area, specifically with studies exploiting the mechanism of action and the structureactivity aspects of natural compounds with antimicrobial activity that provides insights on potential ways to overcome the antimicrobial resistance.

Therefore, thanks to the contribution of active researchers in the field, several scientific studies mainly focused on natural products with antimicrobial activity are presented in this Research Topic Ebook.

# Table of Contents

*117 Antifungal, Cytotoxic, and Immunomodulatory Properties of Tea Tree Oil and Its Derivative Components: Potential Role in Management of Oral Candidosis in Cancer Patients*

Gordon Ramage, Steven Milligan, David F. Lappin, Leighann Sherry, Petrina Sweeney, Craig Williams, Jeremy Bagg and Shauna Culshaw

Jong H. Kim, Natália C. G. Faria, M. De L. Martins, Kathleen L. Chan and Bruce C. Campbell

*140 Juruin: An Antifungal Peptide From the Venom of the Amazonian Pink Toe Spider, Avicularia Juruensis, Which Contains the Inhibitory Cystine Knot Motif* Gabriela Ayroza, Ivan L. C. Ferreira, Raphael S. R. Sayegh, Alexandre K. Tashima and Pedro I. da Silva Jr

**EDITORIAL** published: 15 July 2013 doi: 10.3389/fmicb.2013.00195

#### *Mirian A. Hayashi 1 \*, Fernando C. Bizerra2 and Pedro Ismael Da Silva Jr. 3*

1 Departamento de Farmacologia, Federal University of São Paulo - UNIFESP, São Paulo, Brazil

2 Departamento de Medicina, Federal University of São Paulo - UNIFESP, São Paulo, Brazil

3 Centro de Toxinologia Aplicada, Butantan Institute, São Paulo, Brazil

\*Correspondence<EMAIL_ADDRESS>
#### *Edited by:*

Rustam I. Aminov, University of the West Indies, Jamaica

#### *Reviewed by:*

David W. Graham, Newcastle University, UK Fiona Walsh, Agroscope Changins Wädenswil, Switzerland

Infectious diseases are one of the main causes of morbidity and mortality worldwide. Nowadays many infections are often caused by multi-resistant microorganisms resulting in difficult to treat diseases and, consequently, substantial increases in healthcare costs. The relative easy access to the antimicrobials and also the massive employment of these compounds for industrial purposes, including food production, have both strongly contributed to the progressive increase of resistant microorganisms. As a result, these multi-resistant microorganisms are reasserting themselves as worldwide threats.

Research into natural products has demonstrated significant progress in the discovery of new compounds with antimicrobial activity. In fact, nature is a generous source of compounds with the potential to treat diseases, including infectious diseases. Among the known sources of natural compounds with valuable antimicrobial activity, we highlighted the medicinal plants and marine and terrestrial organisms, including fungi and bacteria. Nevertheless, there is still a vast fauna and flora that once systematically explored, could provide additional antimicrobial leads and new drugs.

Thousands of natural products with the potential to act as antimicrobial compounds or as a structural lead compound still await further investigation.

In this Research Topic Ebook, we present several scientific studies mainly focused on natural products with antimicrobial activity, which are the case of the natural antimicrobial peptides (AMPs) and host defense peptides (HDPs). This topic also includes recent studies on the roles of honey hydrogen peroxide

**REFERENCES**

Ayroza, G., Ferreira, I. L. C., Sayegh, R. S. R., Tashima, A. K., and da Silva Junior, P. I. (2012). Juruin: an antifungal peptide from the venom of the Amazonian Pink Toe spider, *Avicularia juruensis,* which contains the inhibitory cystine knot motif. *Front. Microbiol.* 3:324. doi: 10.3389/fmicb.2012.00324

Sherry, L., Jose, A., Murray, C., Williams, C., Jones, B., Millington, O., et al., (2012). Carbohydrate derived fulvic acid: an *in vitro* investigation of a novel membrane active antiseptic agent against *Candida albicans* biofilms. *Front. Microbiol.* 3:116. doi: 10.3389/fmicb. 2012.00116

in antimicrobial activity against resistant microbial strains, as well as the use of essential oils for food preservation. Such a wide and interesting topic also gave us an opportunity to include diverse sources, including plants, terrestrial and sea animals. Not to mention the interesting and unusual sources such as coal or lignite, which may provide future antimicrobial compounds candidates. The recent development of a patented process to GMP standards (PA107470/GB), rendering the obtainment of carbohydrate derived fulvic acid (CHD-FA), stimulated Sherry et al. (2012) to study and describe for the first time a highly effective novel antiseptic effect of fulvic acid with exquisite biofilm activity that acts by disrupting cell membranes. The antifungic peptide from Amazonian Pink Toe spider Juruin, described by Ayroza et al. (2012) is another outstanding example of the potential contribution of a systematic exploration of nature aiming to provide additional antimicrobial leads and drugs.

In other words, nature is a generous source of compounds, with the potential to treat diseases, including infectious diseases. Studies exploiting the mechanism of action and the structureactivity aspects of these natural compounds may provide both additional antimicrobial leads and drugs, and also significant insight into potential possibilities to overcome the antimicrobial resistance.

#### **ACKNOWLEDGMENTS**

We are grateful to FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the continuous support.

*Received: 28 May 2013; accepted: 24 June 2013; published online: 15 July 2013. Citation: Hayashi MA, Bizerra FC and Da Silva PI Jr (2013) Antimicrobial compounds from natural sources. Front. Microbiol. 4:195. doi: 10.3389/fmicb.2013.00195*

*This article was submitted to Frontiers in Antimicrobials, Resistance and Chemotherapy, a specialty of Frontiers in Microbiology.*

*Copyright © 2013 Hayashi, Bizerra and Da Silva. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.*

# Re-examining the role of hydrogen peroxide in bacteriostatic and bactericidal activities of honey

#### *Katrina Brudzynski 1,2\*, Kamal Abubaker 2, Laurent St-Martin2 and Alan Castle2*

1 Bee-Biomedicals Inc., St. Catharines, ON, Canada

2 Department of Biological Sciences, Brock University, St. Catharines, ON, Canada

#### *Edited by:*

Mirian A. F. Hayashi, Universidade Federal de São Paulo, Brazil

#### *Reviewed by:*

Jun Liu, Mount Sinai School of Medicine, USA Dmitri Debabov, NovaBay Pharmaceuticals, USA

#### *\*Correspondence:*

Katrina Brudzynski, Department of Biological Sciences, Brock University, 500 Glenridge Avenue, St. Catharines, ON, Canada L2S 3A1. e-mail<EMAIL_ADDRESS>
The aim of this study was to critically analyze the effects of hydrogen peroxide on growth and survival of bacterial cells in order to prove or disprove its purported role as a main component responsible for the antibacterial activity of honey. Using the sensitive peroxide/peroxidase assay, broth microdilution assay and DNA degradation assays, the quantitative relationships between the content of H2O2 and honey's antibacterial activity was established. The results showed that: (A) the average H2O2 content in honey was over 900-fold lower than that observed in disinfectants that kills bacteria on contact. (B) A supplementation of bacterial cultures with H2O2 inhibited E. coli and B. subtilis growth in a concentration-dependent manner, with minimal inhibitory concentrations (MIC90) values of 1.25 mM/107 cfu/ml and 2.5 mM/107 cfu/ml for E. coli and B. subtilis, respectively. In contrast, the MIC90 of honey against E. coli correlated with honey H2O2 content of 2.5 mM, and growth inhibition of B. subtilis by honey did not correlate with honey H2O2 levels at all. (C) A supplementation of bacterial cultures with H2O2 caused a concentration-dependent degradation of bacterial DNA, with the minimum DNA degrading concentration occurring at 2.5 mM H2O2. DNA degradation by honey occurred at lower than ≤2.5 mM concentration of honey H2O2 suggested an enhancing effect of other honey components. (D) Honeys with low H2O2 content were unable to cleave DNA but the addition of H2O2 restored this activity. The DNase-like activity was heat-resistant but catalase-sensitive indicating that H2O2 participated in the oxidative DNA damage. We concluded that the honey H2O2 was involved in oxidative damage causing bacterial growth inhibition and DNA degradation, but these effects were modulated by other honey components.

**Keywords: oxidative stress, hydrogen peroxide, bacteriostatic activity, honey, DNA degradation**

#### **INTRODUCTION**

Hydrogen peroxide is generally thought to be the main compound responsible for the antibacterial action of honey (White et al., 1963; Weston, 2000; Brudzynski, 2006). Hydrogen peroxide in honey is produced mainly during glucose oxidation catalyzed by the bee enzyme, glucose oxidase (FAD-oxidoreductase, EC <IP_ADDRESS>; White et al., 1963). The levels of hydrogen peroxide in honey are determined by the difference between the rate of its production and its destruction by catalases. Glucose oxidase is introduced to honey during nectar harvesting by bees. This enzyme is found in all honeys but its concentration may differ from honey to honey depending on the age and health status of the foraging bees (Pernal and Currie, 2000) as well as the richness and diversity of the foraged diet (Alaux et al., 2010). Catalases on the other hand, are of pollen origin. Catalase efficiently hydrolyzes hydrogen peroxide to oxygen and water due to its high turnover numbers. The total concentration of catalase depends on the amount of pollen grains in honey (Weston, 2000), and consequently, the hydrogen peroxide levels in different honeys may vary considerably (Brudzynski, 2006).

A substantial correlation has been found between the level of endogenous hydrogen peroxide and the extent of inhibition of bacterial growth by honey (White et al., 1963; Brudzynski, 2006). We have observed that in honeys with a high content of this oxidizing compound, bacteria cannot respond normally to proliferative signals and their growth remains arrested even at high honey dilutions. Pre-treatment of honey with catalase restored, to a certain extent, the bacterial growth, thus suggesting that endogenous H2O2 was implicated in the growth inhibition (Brudzynski, 2006).

Most of the conclusions on the H2O2 oxidizing action on bacteria are drawn from the simplified *in vitro* models, where direct effects of hydrogen peroxide on bacterial cells were analyzed. In contrast, honey represent complex chemical milieu consisting of over 100 different compounds (including antioxidants and traces of transition metals), where the interaction between these components and hydrogen peroxide may influence its oxidative action. We have recently unraveled that honey is a dynamic reaction mixture which facilitates and propagates the Maillard reaction (Brudzynski and Miotto, 2011b). The Maillard reaction which initially involves reaction between amino groups of amino acids or proteins with carbonyl groups of reducing sugars leads to a cascade of redox reactions in which several bioactive molecules are continuously formed and lost due to their cross-linking to other molecules (gain or loss of function; Brudzynski and Miotto, 2011b). We have shown that polyphenol-based melanoidins are a major group of Maillard reaction products possessing radicalscavenging activity (Brudzynski and Miotto, 2011a,b). These compounds are likely to interact with hydrogen peroxide and, depending of their concentration and redox capacity, either enhanced or diminished the oxidative activity of honey's H2O2. In view of these facts, we hypothesized that the oxidizing action of honey's hydrogen peroxide on bacterial cells may be modulated by the presence of other bioactive molecules in honey and therefore, may differ from the action of hydrogen peroxide alone.

Hydrogen peroxide is commonly used to disinfect and sanitize medical equipment in hospitals. For this purpose, the high concentrations of H2O2 in these disinfectants have to be maintained to overwhelmed defense systems of bacteria. At high concentrations, ranging from 3 to 30% (0.8 to 8 M), its bactericidal effectiveness has been demonstrated against several microorganisms including *Staphylococcus*-, *Streptococcus*-, *Pseudomonas-*species, and *Bacillus* spores (Rutala et al., 2008).Under these conditions, the bacterial cell death results from the accumulation of irreversible oxidative damages to the membrane layers, proteins, enzymes, and DNA (**?**Davies, 2000; Rutala et al., 2008; Finnegan et al., 2010).

However, the hydrogen peroxide content in honey is about 900-fold lower (Brudzynski, 2006). Moreover, the literature data indicate that the cell death of cultured mammalian, yeast, and bacterial cells required H2O2 concentrations higher than 50 mM and was associated with chromosomal DNA degradation (Imlay and Linn, 1987a,b; Brandi et al., 1989; Davies, 1999; Bai and Konat, 2003; Ribeiro et al., 2006), which is still five to 10-fold higher then that observed in honeys. Therefore, we have undertaken this study to re-examined the role of hydrogen peroxide in antibacterial activity of honeys.

The hydrogen peroxide efficacy as an oxidative biocide is related to the bacterial sensitivity to peroxide stress. Defense mechanisms to oxidative stress varies between bacterial species such as Gram-negative *E. coli* and Gram-positive *B. subtilis* used in this study and depend on the growth phase (exponential- versus stationary-phase of growth), and on the adaptive and survival mechanisms (non-spore forming versus spore-forming bacteria; Dowds et al., 1987; Chen et al., 1995; Storz and Imlay, 1999; Cabiscol et al., 2000). In honey, the effects of H2O2 on the growth and survival of microorganisms may be mitigated or enhanced due to the presence of honey compounds. On one hand, a high content of sugars in honey that abstracts free water molecules from milieu inhibits bacterial growth and proliferation, but honey dilutions may create growth-supportive conditions due to the abundance of sugars as a carbon source for the growing cells. Hydrogen peroxide has deleterious effects on the growth and survival of bacterial cells but honey antioxidants such as catalases, polyphenols, Maillard reaction products, and ascorbic acid may lower the oxidative stress to cells and may have a protective effect against endogenous H2O2 (Brudzynski, 2006). Even less information exists on the mechanism of bactericidal action of honey's hydrogen peroxide. The most fundamental and unsolved questions concerns the molecular targets of H2O2 cytotoxicity: does molecular hydrogen peroxide at concentrations present in honey cause DNA degradation?

During last decades, several honey compounds were identified as those implicated in honey antibacterial activity (for review,Irish et al., 2011). Despite this knowledge, the mechanisms by which these compounds lead to bacterial growth inhibition and bacterial death have never been explained or proven in biochemical terms. Since there is a persistent view that hydrogen peroxide is a main player in these events, the aim of this study was to critically analyze the effects of hydrogen peroxide on growth and survival of bacterial cells in order to prove or disprove its purported role as a main component responsible for the antibacterial activity of honey.

#### **MATERIALS AND METHODS HONEY SAMPLES**

Honey samples included raw, unpasteurized honeys donated by Canadian beekeepers and two samples of commercial Active Manuka honey (Honey New Zealand Ltd., New Zealand, UMF 20+, and 25+; M and M2, respectively; **Table 1**) that were used as a reference in this study. During the study, honey samples were kept in the original packaging, at room temperature (22 ± 2˚C) and in the dark.

A stock solution of 50% (w/v) honey was prepared by dissolving 1.35 g honey (average density 1.35 g/ml) in 1 ml of sterile, distilled water warmed at 37˚C. The stock solution was prepared immediately before conducting the antibacterial assays.

#### **PREPARATION OF ARTIFICIAL HONEY**

Artificial honey was prepared by dissolving 76.8 g of fructose and 60.6 g of glucose separately in 100 ml of sterile, deionized water, and by mixing these two solutions in a 1:1 ratio. The osmolarity of the artificial honey was adjusted to that of the honey samples (BRIX) using refractometric measurements.

#### **BACTERIAL STRAINS**

Standard strains of *Bacillus subtilis* (ATCC 6633) and *Escherichia coli* (ATCC 14948; Thermo Fisher Scientific Remel Products, Lenexa, KS 66215) were grown in Mueller–Hinton broth (MHB; Difco Laboratories) overnight in a shaking water-bath at 37˚C.

Overnight cultures were diluted with broth to the equivalent of the 0.5 McFarland Standard (approx.108 cfu/ml) which was measured spectrophotometrically at *A*600 nm.

#### **ANTIBACTERIAL ASSAY**

The antibacterial activity of honeys was determined using a broth microdilution assay using a 96-well microplate format. Serial twofold dilutions of honey were prepared by mixing and transferring 110μl of honey with 110μl of inoculated broth (106 cfu/ml final concentrations for each microorganism) from row A to row H of a microplate. Row G contained only inoculum and served as a positive control and row H contained sterile MHB and served as a blank.

After overnight incubation of plates at 37˚C in a shaking waterbath, bacterial growth was measured at *A*595 nm using the Synergy HT multi-detection microplate reader (Synergy HT, Bio-Tek Instruments, Winooski, VT, USA).

The contribution of color of honeys to the absorption was corrected by subtracting the absorbance values before (zero time) incubation from the values obtained after overnight incubation.

**Table 1 | Hydrogen peroxide concentrations in different honeys. Relationship between antibacterial activities of honey and hydrogen peroxide concentrations.**

\*Hydrogen peroxide concentration was measured at honey dilution of 8× (25% v/v) and represent an average of three experimental trials, where each honey was tested in triplicate.

The absorbance readings obtained from the dose–response curves were used to construct growth inhibition profiles (GIPs). The minimal inhibitory concentrations (MIC) were determined from the GIPs and represented the lowest concentration of honey that inhibited the bacterial growth. The MIC end point in our experiments was honey concentration at which 90% bacterial growth reduction was observed as measured by the absorbance at *A*595 nm.

Statistical analysis and dose response curves were obtained using KC4 software (Synergy HT, Bio-Tek Instruments, Winooski, VT, USA).

#### **HYDROGEN PEROXIDE ASSAY**

Hydrogen peroxide concentration in honeys was determined using the hydrogen peroxide/peroxidase assay kit (Amplex Red, Molecular Probes, Invitrogen, Burlington, ON, Canada). The assay was conducted in the 96-well microplates according to the manufacturer's instruction. The fluorescence of the formed product, resorufin, was measured at 530 nm excitation and a 590 nm emission using the Synergy HT (Molecular Devices, BioTek Instruments, Winooski, VT, USA) multi-detection microplate reader, and the dose–response curves were generated using the KC4™data reduction software.

To calculate the hydrogen peroxide concentrations of the honeys, a standard curve was run alongside the honey serial dilutions. The standard curve was prepared from the 200 μM H2O2 stock solution. Each of the honey samples, and the standard curve, were tested in triplicate.

#### **CATALASE-TREATMENT OF HONEYS**

Honey were treated with catalase (13 800 U/mg solid; Sigma-Aldrich, Canada) at ratio of 1000 units per 1 ml of 50% honey solution in sterile water for 2 h at room temperature.

#### **INCUBATION OF BACTERIAL CULTURES WITH HONEY OR HYDROGEN PEROXIDE**

Overnight cultures of *E. coli* and *B. subtilis* (1.5 ml, adjusted to 107 cfu/ml in MHB) were treated with either the 50% honey solution in a 1:1 ratio (v/v), an artificial honey solution, or with hydrogen peroxide solutions containing 5, 2.5, 1.2, 0.62, and 0.3125 mM (final concentrations) H2O2 prepared from the 20 mM stock solution. After overnight incubation at 37˚C with continuous shaking, the cells were harvested by centrifugation at 3,000×*g* (Eppendorf) for 30 s and then their DNA was isolated.

#### **DNA ISOLATION**

The total genomic bacterial DNA was isolated from the untreated, control cells and from the honey- or hydrogen peroxide-treated cells using a DNA isolation kit (Norgen Biotek Corporation, St. Catharines, ON., Canada), according to the manufacturer's instructions.

#### **AGAROSE GEL ELECTROPHORESIS**

Agarose gel (1.3%) electrophoresis was carried out in 1× TAE buffer containing ethidium bromide (0.1μg/ml w/v). Ten microliters of DNA isolated from the untreated and treated bacterial cells was mixed with 5X loading dye (0.25% bromophenol blue, 0.25% xylene xyanol, 40% sucrose) and loaded into the gel. The DNA molecular weight markers selected were the HighRanger 1 kb DNA Ladder, MidRanger 1 kb DNA Ladder, and PCRSizer 100 bp DNA Ladder from Norgen Biotek (Thorold, Ontario). The gels were run at 85 V for 1 h and then visualized and photographed using the Gel Doc 1000 system and the Quantity One 1-D Analysis software (version 4.6.2 Basic) from Bio-Rad.

#### **RESULTS**

#### **DETERMINATION OF THE HYDROGEN PEROXIDE CONCENTRATIONS IN HONEYS**

Formation of H2O2 depends on the honey dilution since glucose oxidase is inactive in undiluted honey (White et al., 1963; Brudzynski, 2006). Honeys used in this study required a four to 16-fold dilution for the maximal production of hydrogen peroxide to be observed (**Figure 1**). At the peak, H2O2 concentrations ranged from 2.68 ± 0.04 to 0.248 ± 0.02 mM in the different honeys (**Table 1**), as measured by a sensitive, high-throughput hydrogen peroxide/peroxidase assay (Amplex Red assay).

**FIGURE 1 | Effect of honey dilutions on the production of hydrogen peroxide.** Honeys of buckwheat origin, H58 and H23, together with sweet clover (H20), and wildflower/clover (H11) produced distinctively higher amounts of H2O2 than Manuka (M2) or honey blends (H56 and H60). The H2O2 content was measured in twofold serially diluted honeys, the x axis represents a log2 values.

#### **CONCENTRATION-DEPENDENT EFFECT OF HYDROGEN PEROXIDE ON BACTERIAL GROWTH INHIBITION**

Throughout this study, we used terms: endogenous hydrogen peroxide to describe H2O2 produced in honey by glucose oxidase and exogenous hydrogen peroxide, which has been added as a supplement to the bacterial cultures. These terms were introduced in order to differentiate between the effects of honey's endogenous H2O2 whose action on bacterial cells could be modulated/obscured by other honey components as opposed to true, well-defined action of exogenous hydrogen peroxide directly added to bacterial culture.

In agreement with previous reports (Brudzynski, 2006), we found a strong correlation between the content of honey hydrogen peroxide and the growth inhibitory action of Canadian honeys; honeys with high MIC90 values (6.25 to 12.5% v/v) corresponding to16 to 8× dilution) also possessed a high content of H2O2 (**Table 1**). Since the minimum inhibitory concentration values and the hydrogen peroxide peak were both observed at the 4 to 16× honey dilutions, we hypothesized that the maximal hydrogen peroxide production is required to achieve the bacteriostatic activity of honey at the MIC90 level. To test this assumption, we first examined the dose–response relationship between the concentration of exogenous hydrogen peroxide, ranging from 10 to 0.312 mM, and its growth inhibitory activity against *E. coli* and *B. subtilis*. The dose–response curves and growth inhibitory profiles revealed very reproducibly that H2O2 concentrations of 1.25 mM (1.25μmoles/107 cfu/ml) and 2.5 mM (2.5μmoles/107 cfu/ml) were required to inhibit the growth of *E. coli* and *B. subtilis* by 90%, respectively (**Figure 2**).

#### **RELATIONSHIP BETWEEN THE ENDOGENOUS H2O2 CONTENT AND THE GROWTH INHIBITORY ACTIVITY OF HONEYS**

To investigate whether the content of honey H2O2 influences honey's bacteriostatic potency in a similar manner to that of exogenous H2O2, each honey was analyzed for growth inhibitory activity and the production of hydrogen peroxide in the same range of honey dilutions. When the profiles of hydrogen peroxide production were superimposed on the growth inhibitory profiles

of honeys against *E. coli,* it appeared that almost all of the bacteriostatic activity of honeys could be assigned to the effects of this compound (**Figure 3A**). In honeys, the endogenous H2O2 of 2.5 mM was of critical importance for the growth inhibition of *E. coli*; the dilutions that reduced H2O2 concentrations below this value showed a loss of honey potency to inhibit bacterial growth at the MIC90 level (**Figure 3A**). These data suggest that upon honey dilution, endogenous H2O2 mediates growth inhibition of *E. coli.* However, the concentrations required to reach MIC90 were twofold higher than that found for exogenous hydrogen peroxide (2.5 versus 1.25 mM, respectively; **Figure 3A**).

In contrast to *E. coli*, the inhibition of growth of *B. subtilis* seemed not to be due to the effect of the levels of honey H2O2 (**Figure 3B**). A rapid increase of *B. subtilis* growth with honey dilutions occurred despite the presence of high levels of H2O2 (honeys H58, H23, H20, and H11, **Figure 3B**). While exposure of the *B. subtilis* culture to exogenous H2O2 resulted in a concentrationdependent growth inhibition with MIC90 at 2.5 mM (**Figure 2**), comparable concentrations of H2O2 in honeys were ineffective. This indicated that other honey compounds/physical features were responsible for the growth inhibition, such as honey's high osmolarity. Moreover, higher honey dilutions, beyond 16-fold, had a stimulatory effect on *B. subtilis* growth (**Figure 3B**).

Thus, our results demonstrated for the first time that bacteriostatic effects of endogenous versus exogenous hydrogen peroxide are markedly different due to the presence of other honey components and, more importantly, that the effects of honey H2O2 on bacterial growth are markedly different in *E. coli* and *B. subtilis.*

#### **COMPARISON OF EFFECTS OF HONEY AND HYDROGEN PEROXIDE ON DNA DEGRADATION IN BACTERIAL CELLS**

To exert effectively its oxidative biocide action, the concentrations of hydrogen peroxide in various disinfectants are high ranging from 3 to 30% (0.8 to 8 M). In contrast, we have established that the average content of H2O2 in tested honeys ranged from 0.5 to 2.7 mM (**Table 1**). The concentrations of H2O2 measured in honeys, therefore was about 260–1600-fold lower than the effective bactericidal dose of H2O2 in disinfectants. Therefore, we asked the question whether hydrogen peroxide at concentrations present in honey can cause DNA degradation and ultimately bacterial cell death.

To examine the effects of honey and hydrogen peroxide on the integrity of bacterial DNA, *E. coli* cultures (107cfu/ml) were exposed to increasing concentrations of exogenous H2O2 (5– 0.3125 mM) or to honeys containing known amounts of H2O2. After 24 h incubation at 37˚C, bacterial DNA was isolated and its integrity examined on agarose gels. **Figure 4** shows that the exposure of *E. coli* cultures to hydrogen peroxide at concentrations of 5 and 2.5 mM caused DNA degradation, while H2O2 concentrations lower than 2.5 mM were ineffective.

In contrast, honeys of relatively high H2O2 concentrations but below 2.5 mM (H203, 204, 205; **Table 1**) exerted DNA degrading activity (**Figure 4**). The ability of honeys H2O2 to degrade DNA appeared to be concentration-dependent. Honey H200 containing 0.25 mM H2O2 was unable to cleave DNA (**Figure 5**). The differences in the concentrations of H2O2 between exogenous and honey's hydrogen peroxide that were required to effectively degrade chromosomal DNA may indicate that the action of honey H2O2 is enhanced by other honey components.

Manuka honey also possessed low concentration of H2O2 (0.72 mM), but efficiently degraded DNA (**Figures 4** and **5**). The antibacterial activity of manuka honey however is not regulated by the honey H2O2 content (Molan and Russell, 1988; Allen et al., 1991).

#### **DNA DEGRADATION IN** *E. COLI* **CELLS EXPOSED TO CATALASE-TREATED OR HEAT-TREATED HONEYS**

To gain more insight into the role of H2O2 in chromosomal DNA degradation, *E. coli* cultures were exposed to honeys which were treated with catalase. Removal of H2O2 by catalase abolished DNA degrading activity of honey H205 and had a protective effect on bacterial DNA (**Figure 5**). The short incubation of catalase-treated honey H204 with DNA (8 h instead of 24 h) also prevented DNA degradation (**Figure 5**). Inactive honey H200 remained unable to degrade DNA after catalase-treatment (**Figure 5**). However, when honey H200 was supplemented with 2 mM H2O2, and then incubated with *E. coli* culture at 37˚C for 8 h, it became active in degrading DNA and the extent of DNA degradation was comparable to that of honey H204 (**Figure 5**). On the other hand, catalase– treatment of manuka honey did not prevent DNA degradation, consistent with the notion, that manuka honey antibacterial activity is hydrogen peroxide-independent (Molan and Russell, 1988; Allen et al., 1991).

To investigate the potential involvement of DNases in DNA degradation, honeys were heat-treated under conditions which inactivate DNase activity (75˚C for 10 min). Unheated and heattreated honeys were then incubated with *E. coli* cultures at 37˚C for 8 h, followed by DNA isolation and its analysis on agarose gels. Heat-treatment of active honeys H205 and H23 did not prevent

**FIGURE 4 | Effect of exposure of***E. coli* **cultures to honey or exogenous hydrogen peroxide on the integrity of bacterial DNA**. The cells were treated with honeys (manuka, buckwheat honeys H203, H204, and H205) or with increasing concentrations of exogenous H2O2 (5–0.312 mM). Untreated cells and cells treated with the sugar solution (artificial honey, AH) served as the controls. The integrity of DNA was analyzed on agarose gels.

DNA degradation suggesting against the involvement of DNase in this process (**Figures 6** and **7**). Moreover, the fact that some honeys displayed DNA degrading activity (H23 or H205) in bacterial culture while others did not (H200 and H60) makes it unlikely that this process was mainly due to the contamination of honeys with DNases. On the other hand, inability of honeys H200 and H60 to degrade DNA was closely related to the very low concentration of H2O2 in these honeys.

Together, these results provided a strong support for the role of H2O2 in DNA degradation.

#### **DISCUSSION**

The findings described in this study revise the old views and provide novel information on the role of hydrogen peroxide in the regulation of bacteriostatic and bactericidal activities of honey.

Firstly, we found that the exponentially growing *E. coli* and *B. subtilis* cells were inhibited in a concentration-dependent manner by exogenous H2O2 reaching MIC90 at 1.25 mM (1.25μmoles/107 cfu/ml) and 2.5 mM (2.5μmoles/107 cfu/ml), respectively. The bacteriostatic efficacy of H2O2 however differed significantly from that of honey H2O2. The main factors that contributed to these differences were (a) bacterial susceptibility/resistance to the oxidative action of hydrogen peroxide and (b) interference from other honey components.

Endogenous H2O2 inhibited the growth of *E. coli* in a concentration-dependent manner, but its MIC90 was twofold higher than those of exogenous H2O2 (2.5 versus 1.25 mM, respectively). The honeys MIC90 levels against *E. coli* coincided with the dilutions at which a peak of hydrogen peroxide production occurred. Treatment of honeys with catalase led to a

**FIGURE 6 | Effect of exposure of** *E. coli* **cultures to heat-treated (h), catalase-treated (cat), and untreated (un) honeys on the integrity of chromosomal DNA.** AH-cell treated with artificial honey, or buckwheat honeys H200, and H205. Lane "H200 2 mM H2O2" represents the effect of honey H200 supplementation with hydrogen peroxide on the E. coli DNA integrity.

**FIGURE 7 | Effect of exposure of** *E. coli* **naked DNA to unheated and heat-treated honeys H60 and H23.**

significant reduction in their bacteriostatic activity (Brudzynski, 2006). Together, these data provide direct evidence that *E. coli* growth is sensitive to oxidative action of honey H2O2.

In contrast, growth inhibition of *B. subtilis* was not due to the action of honey H2O2. While exposure of *B. subtilis* cultures to H2O2 resulted in a concentration-dependent growth inhibition, the comparable concentrations of honey H2O2 in honey were ineffective in arresting *B. subtilis* growth*.* The rapid decrease in bacteriostatic activity of honey upon dilution was observed even in the presence of high concentrations of honey H2O2. These results suggest that other honey compounds were responsible for the inhibition of *B. subtilis* growth. As a consequence of growth arrest, the change in sensitivity of *B. subtilis* to honey H2O2 occurred. Instead of growth inhibition, we observed growth stimulation of *B. subtilis* at high honey dilutions (16-fold and over) and in the presence of high levels of H2O2. Literature data provides compelling evidence that transition from the exponential-phase growth to the stationary-phase growth evokes *B. subiltis* sporulation and with it, increased resistance to hydrogen peroxide. The transition to stationary-phase growth activates RNA polymerase σs transcriptional factor which regulates a stationary-phase gene expression of *rpoS* regulon. The expression of σs factor in *B. subtilis* evokes spore-formation to enhance bacterial survival (Dowds et al., 1987; Dowds, 1994; Loewen et al., 1998; Zheng et al., 1999; Chen and Schellhorn, 2003). Dowds et al. (1987) have shown that stationaryphase cultures of *B. subtilis* displayed viability even at the 10 mM concentration of H2O2. These data may explain, at least in part, an apparent insensitivity of *B. subtilis* to high levels of hydrogen peroxide in honey.

These results revealed significant differences in the sensitivities of *E. coli* and *B. subtilis* to oxidative stress caused by honey H2O2. As aerobic bacteria, both *E. coli* and *B. subtilis* are equipped with molecular machinery to cope with oxidative stress by activating several stress genes under *oxyR-* or *perR*-regulons, in *E. coli* and *B. subtilis* respectively (Dowds et al., 1987; Christman et al., 1989; Dowds, 1994; Bsat et al., 1998; Storz and Imlay, 1999). The *oxyR* and *perR* genes control expression of inducible forms of *katG* (catalase hydroperoxidase I, HP1), *ahpCF* (alkylhydroperoxide reductase) that function to reduce hydrogen peroxide to levels that are not harmful to growing cells (Hassan and Fridovich, 1978; Loewen and Switala, 1987; Storz et al., 1990; Seaver and Imlay, 2001). While these responses are similar in both bacteria, the main difference concerns their adaptive and survival mechanisms to oxidative stress.

Relatively little is known about the contribution of honey's hydrogen peroxide to bacterial cell death. The most important result obtained in this work is the demonstration that honey H2O2 participated in bacterial DNA degradation. Several lines of evidence support this finding. Firstly, the treatment of exponentialphase *E. coli* cultures with increasing concentrations of exogenous hydrogen peroxide (5–0.3125 mM) or honeys of different content of endogenous H2O2 led to a concentration-dependent DNA degradation. While the minimum DNA degrading activity of exogenous H2O2 occurred at 2.5 mM (2.5μmoles/107 cfu/ml), in contrast, honeys possessing H2O2 concentrations lower than 2.5 mM were still active in this process. Secondly, DNA degradation by active honeys was abolished by removal of H2O2 by catalase. Thirdly, honeys with the low content of H2O2 were unable to degrade DNA but the supplementation with 2 mM of hydrogen peroxide caused the appearance of this activity. The extent of DNA degradation by honey, which was supplemented with H2O2, was comparable to that of active honeys.

Heat-treatment of active honeys prior to incubation with *E. coli* cultures did not prevent DNA degradation, suggesting against the involvement of DNase in this process. Moreover, not all tested honeys displayed DNA degrading activity on *E. coli* cells. Given that bacterial cells are impermeable to DNase, the DNA degradation by honeys observed in this study could not be simply explained by the DNase contaminations. Rather, the close relationship between DNA degradation and H2O2 content in honeys advocates for the role of H2O2 in the mechanism of DNA cleavage.

DNA degradation is a lethal event which ultimately kills the cell. Literature data indicate that the concentration of hydrogen peroxide plays a decisive role in the type of cell death that follows H2O2 exposure. In simplified *in vitro* models, where direct effects of hydrogen peroxide on bacterial cells were analyzed, two separate modes of killing were observed for *E. coli.* At low concentrations of H2O2 (≤2.5 mM), *E. coli* cells were dying because of DNA damage inflicted on the metabolically active cells (Imlay and Linn, 1986; Imlay and Linn, 1987a,b; Brandi et al., 1989). At H2O2 concentrations of 10–50 mM, cell death resulted from cytotoxic effects due to hydroxyl radicals formed from hydrogen peroxide (Imlay and Linn, 1987a,b; Brandi et al., 1989). In a full agreement with these data, we established that the minimum DNA degrading activity of exogenous H2O2 on *E. coli* cells was 2.5 mM (2.5μmoles/107cfu/ml). In contrast to exogenous H2O2,

#### **REFERENCES**

multiple Fur homologues: identification of the iron uptake (Fur) and peroxide regulon (PerR) repressors. *Mol. Microbiol.* 29, 189–198.

the minimum DNA degrading activity of honey H2O2 was below 2.5 mM. The lower concentrations of honeys H2O2 required to effectively degrade chromosomal DNA strongly suggest that the oxidizing effect of H2O2 was augmented by other honey components such as transition metals (Fe, Cu) commonly present in honeys. In support of this notion, the recent literature evidence indicates that it is the hydroxyl radical (HO) that is produced in the metal-catalyzed Fenton reaction from H2O2 rather than molecular hydrogen peroxide that causes the oxidative damage to membrane structures, proteins, and DNA (Imlay et al., 1988; Storz and Imlay, 1999; Cabiscol et al., 2000; Imlay, 2003).

In conclusion, our study demonstrated that honey H2O2 exerted bacteriostatic and DNA degrading activities to bacterial cells. The extent of damaging effects of honey H2O2 was strongly influenced by the bacterial sensitivity to oxidative stress, the growth phase and their survival strategy (non-spore forming versus spore forming species) as well as by the modulation of other honey compounds.

#### **ACKNOWLEDGMENTS**

This research was supported by funds from the Agricultural Adaptation Council, Agriculture and Agri-Food Canada (ADV-380), and the Ontario Centres of Excellence (BM50849) awarded to Katrina Brudzynski.

*subtilis*. *FEMS Microbiol.* 124, 155–264.

products to the antibacterial activity of honey: a review. *Food Chem.* 71, 235–239.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 01 September 2011; accepted: 03 October 2011; published online: 25 October 2011.*

*Citation: Brudzynski K, Abubaker K, St-Martin L and Castle A (2011) Reexamining the role of hydrogen peroxide in bacteriostatic and bactericidal* *activities of honey. Front. Microbio. 2:213. doi: 10.3389/fmicb.2011.00213 This article was submitted to Frontiers in Antimicrobials, Resistance and Chemotherapy, a specialty of Frontiers in Microbiology.*

*Copyright © 2011 Brudzynski,Abubaker, St-Martin and Castle. This is an openaccess article subject to a non-exclusive license between the authors and Frontiers Media SA, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and other Frontiers conditions are complied with.*

# Exploring the pharmacological potential of promiscuous host-defense peptides: from natural screenings to biotechnological applications

#### *Osmar N. Silva1,2, Kelly C. L. Mulder 1, Aulus E. A. D. Barbosa1, Anselmo J. Otero-Gonzalez 3, Carlos Lopez-Abarrategui 3,Taia M. B. Rezende1, Simoni C. Dias 1 and Octávio L. Franco1,2\**

1 Programa de Pós-Graduação em Ciências Genômicas e Biotecnologia, Centro de Análises Proteômicas e Bioquímicas, Universidade Católica de Brasília, Brasília, Brazil

2 Programa de Pós-Graduação em Genética e Biotecnologia, Universidade Federal de Juiz de Fora, Juiz de Fora, Brazil

3 Centro de Estudios de Proteínas, Facultad de Biología, Universidad de La Habana, La Habana, Cuba

#### *Edited by:*

Mirian A. F. Hayashi, Universidade Federal de São Paulo, Brazil

#### *Reviewed by:*

Atte Von Wright, University of Eastern Finland, Finland Adam Paul Roberts, University College London, UK Sonia Alexandra Mendo, University of Aveiro, Portugal Jun Liu, Mount Sinai School of Medicine, USA Dmitri Debabov, NovaBay Pharmaceuticals, USA

#### *\*Correspondence:*

Octávio L. Franco, Pos-Graduação em Ciencias Genomicas e Biotecnologia, Centro de Analises Proteomicas e Bioquimicas, SGAN 916N, Modulo C, Avenue W5, Asa Norte, Sala 219, Distrito Federal, Brasilia 71837-360, Brazil.

e-mail<EMAIL_ADDRESS>
In the last few years, the number of bacteria with enhanced resistance to conventional antibiotics has dramatically increased. Most of such bacteria belong to regular microbial flora, becoming a real challenge, especially for immune-depressed patients. Since the treatment is sometimes extremely expensive, and in some circumstances completely inefficient for the most severe cases, researchers are still determined to discover novel compounds. Among them, host-defense peptides (HDPs) have been found as the first natural barrier against microorganisms in nearly all living groups. This molecular class has been gaining attention every day for multiple reasons. For decades, it was believed that these defense peptides had been involved only with the permeation of the lipid bilayer in pathogen membranes, their main target. Currently, it is known that these peptides can bind to numerous targets, as well as lipids including proteins and carbohydrates, from the surface to deep within the cell. Moreover, by using in vivo models, it was shown that HDPs could act both in pathogens and cognate hosts, improving immunological functions as well as acting through multiple pathways to control infections. This review focuses on structural and functional properties of HDP peptides and the additional strategies used to select them. Furthermore, strategies to avoid problems in large-scale manufacture by using molecular and biochemical techniques will also be explored. In summary, this review intends to construct a bridge between academic research and pharmaceutical industry, providing novel insights into the utilization of HDPs against resistant bacterial strains that cause infections in humans.

**Keywords: host-defense peptides, innate immunity, microbial infections, antimicrobials**

#### **INTRODUCTION**

Infectious diseases caused by fungi and bacteria have affected humanity since the early days of civilization. Nevertheless, the discovery of penicillin by Fleming (1929) provided a potent defense in mammalian survival against pathogens. Based on penicillin, several other molecules and different antibiotic classes have been developed. However, all these agents have lost efficiency and are becoming useless against resistant bacterial strains.

Antimicrobial peptides (AMPs) have arisen as an alternative strategy for the treatment of infections caused by super bugs (Arias and Murray, 2009). AMPs are natural antibiotics found in microorganisms, plants, and animals (Hancock and Chapple, 1999). They can be structurally classified, being formed by αhelices, β-sheets, extended structures, or disordered loops. Moreover, these peptides could also be classified by physical–chemical properties: cationic, anionic, and amphipathic (Peters et al., 2010). Mostly, cationic AMPs show their antimicrobial activity as a result of lipid bilayer disruption. Nevertheless they may also act on various cell targets, in some cases being considered promiscuous

molecules (Huang et al., 2010). In the last few years the conventional idea that peptides possess an unconditional structure directly related to a particular function clashes with the peptide's ability to change and develop new functions. Considering these contrasting ideas, the knowledge of peptide promiscuity, in which multiple functions may be associated with a sole structure, has been gaining consideration in several research fields including the development of antibiotics. Indeed, several AMPs have shown their wide range of functions that are able to control numerous target pathogens simultaneously and in different conditions (as reviewed by Franco, 2011). Moreover, some peptides, in addition to activity against pathogens, also have shown multiple activities related to host innate immunity, called host-defense peptides (HDPs; Hancock et al., 2006).

Host-defense peptides are relatively small compounds, with 12–50 amino acid residues, positive net charge (+2 to +9), and are isolated from single-celled microorganisms, invertebrates, plants, amphibians, birds, fishes, and mammals including humans (Hancock and Sahl, 2006). Furthermore, HDPs are classified

into various groups according to a three-dimensional structure arrangement that includes α-helices (magainin, cecropin, and cathelicidin), β-sheets (hepcidin, human α-defensin 1), a mixture of α-helices/β-sheets (human β-defensins 1), cyclic (cyclotides and catestatin), as well as extended and flexible loops (e.g., indolicidin; **Figure 1**; Hancock et al., 2006). In addition to their direct action against microorganisms, as previously described, HDPs also present activities related to innate immunity. These include the induction or modulation of pro-inflammatory cytokine and chemokine production, chemotaxis, apoptosis, inflammatory response inhibition, recruitment, and stimulation of proliferation of macrophages, neutrophils, eosinophils, T lymphocyte activation, and differentiation of dendritic cells (DCs; Bowdish et al., 2005; Nijnik et al., 2009). One property that makes HDPs extremely attractive molecules for therapeutic use is that they are, in general, non-toxic to mammalian cells. The basis for this selectivity appears to be related to the lipid composition of the target membrane (fluidity, negative charge, and the absence/presence of cholesterol; Nicolas, 2009). The negative charge of a bacterial outer membrane is an example of a typical HDP target. In contrast, zwitterionic membranes, commonly found in plants and animals are not normally accessible to HDPs (Matsuzaki, 1999; Zasloff, 2002). Furthermore, the presence of cholesterol in the membrane may usually reduces HDP activity, since cholesterol helps in lipid bilayer stabilization, thus reducing membrane fluidity and flexibility (Matsuzaki, 1999).

This concept, in addition to peptide promiscuity, adds remarkable value to peptide antibiotic compounds, which have been shown to be much more useful for an organism's protection than merely membrane disruptors. The phenomenon of protein promiscuity, in which several functions are associated with a single peptide structure, has gained attention in several research fields, especially in the area of drug design (Franco, 2011). From this perspective, this review focuses on these compounds from various sources in order to shed some light on the structure and the observed mechanism of action. Furthermore, biotechnological and pharmaceutical potential will also be evaluated and discussed, as well as isolation and production in large-scale.

#### **PLANT HOST-DEFENSE PEPTIDES**

Plants have an arsenal of peptides in their own primitive innate immune system that is completely different from that of animal systems. Since plants are unable to synthesize antibodies, these peptides form an efficient barrier against bacteria, fungi, and insects. Furthermore, along with these peptides, plants have a set of physical and chemical barriers that act together to prevent infection (Sels et al., 2008). The plant cell wall is the first level of defense, formed by a complex network of proteins and polysaccharides. Even during a pathogen attack, plants are able to deposit callose, otherwise known as the polysaccharide β-1,3-glucan, at the infection site and further synthesize lignin-like polymers, thus reinforcing the wall (Hématy et al., 2009). Callose is a polysaccharide that acts in response to multiple biotic and abiotic stresses, as well as during a variety of processes in plant development (Chen and Kim, 2009). When a pathogen crosses through the cell wall, plants can induce a hypersensitive response (HR) or synthesize antibacterial and antifungal compounds for infection

control. HR is characterized by an induced cell death at the point of pathogen contact and can be related to resistance (Mur et al., 2008). The antimicrobials produced are: ROS, secondary metabolites, proteins, and peptides were found (Benko-Iseppon et al., 2010).

In addition to providing a primary defense function, several plant peptides have shown a side biological property that could be exploited for biotechnological applications. This ability, in which a single structure provides multiple functions, has been denominated peptide promiscuity (Franco, 2011). Several plant peptides that show medicinal properties include defensins, thionins, and cyclotides. These peptides have shown a wide diversity of simultaneous activities, in addition to antibacterial and antifungal properties, which includes anti-HIV (Wong and Ng, 2006), antitumor (Lin et al., 2010), leishmanicidal (Berrocal-Lobo et al., 2009), uterotonic (Gran, 1973), anthelmintic (Colgrave et al., 2009), and neurotensin blocking activities (Witherup et al., 1994). However, no plant peptide was found to have both simultaneous antimicrobial and immunomodulatory activities in mammals, clearly limiting the classification of HDPs into the plant kingdom. Nevertheless,we believe that this lack of immunomodulatory activity is related to the low quantity of studies in this field and not to the intrinsic properties of these promiscuous peptides in plants.

Plant AMPs show common properties that are characterized by a low molecular mass (around 10 kDa), cationic charge, and the presence of cysteine residues that are linked through disulfide bonds, thus stabilizing the molecular structure (Padovan et al., 2010). However, defensins, cyclotides, and thionins have specific and typical structures that will be described below.

Defensins are ubiquitous peptides in plant species, showing several nuanced functions (Padovan et al., 2010). Defensins are cationic peptides with pI values around 9.0 and are 45–55 amino acids residues long, with molecular masses ranging between 5 and 7 kDa (Carvalho Ade and Gomes, 2009). The common tertiary structure consists of a triple-stranded antiparallel β-sheet, and one α-helix which in most cases is stabilized by either three, four, or five disulfide bonds (**Figure 2A**; Garcia-Olmedo et al., 1998). The primary sequence is low conserved between different defensins, and this may explain the diverse biological activities reported (Padovan et al., 2010). Some of the defensins can be classified as promiscuous peptides (Franco, 2011) having multiple-biological functions (**Table 1**). In addition to defensins, thionins are peptides with low molecular masses (5 kDa), 45–47 amino acid residues in length and can present three or four conserved disulfide bonds that stabilize the structural fold (Padovan et al., 2010). Due to their sequences and disulfide bonds patterns, thionins are classified into five structural types (I–V). The conserved structure is formed by two antiparallel α-helices, one β-turn, and one antiparallel β-sheet (**Figure 2B**; Padovan et al., 2010). Some thionins demonstrated unusual biological functions (**Table 1**).

Finally, cyclotides are small peptides characterized by 28–37 amino acids long, presenting three disulfide bonds, and a cyclic backbone formed through the linking of amino and carboxylterminus (Craik, 2010). This structure confers to the cyclotides a strong and stable molecular structure (**Figure 2C**). Although *M. cochinensis* trypsin inhibitors have been classified as cyclotides due

**α-helices/β-sheets structures, (D) cyclic, and (E) extended structures.** Disulfide bonds are represented in ball and stick.

to their structural similarities, they do not display sequence homology to the previously identified plant cyclotides (Felizmenio-Quimio et al., 2001). Until now, cyclotides were found inViolaceae, Rubiaceae, Apocynaceae, Cucurbitaceae, Poaceae, and Fabaceae families (Craik, 2010). Despite their conserved molecular structures, these cyclic peptides have shown diverse biological functions in addition to insecticidal activity (**Table 1**).

#### **HOST-DEFENSE PEPTIDES AND THEIR MULTIPLE ROLES IN ANIMAL IMMUNE DEFENSE**

Previously, it was believed that the direct antimicrobial activity of AMPs was essential for their role in innate immunity and hostdefense. However, some studies have shown that peptide concentrations found in various body parts, such as mucous for example, are relatively low when compared with those used in *in vitro* studies (Hirsch et al., 2008). It has been known that, in addition to this

activity against pathogens, HDPs may also have an indirect antimicrobial effect, as previously cited (Auvynet and Rosenstein, 2009). The human cathelicidin LL-37 (hCAP18), a well-characterized multifunctional HDP, is constitutively produced by leukocytes and induced in barrier organs upon inflammation and infection, thus being essential for immune response to injury and tissue infection (Bowdish et al., 2005). Another well-characterized HDP is the human β-defensin-2 (hBD2). It is known that this peptide plays an important role in immune response at different mucosal surfaces and also in the skin barrier (Ganz, 2003). Among the LL-37 and hBD2 wide activities spectrum, the induction of histamine and prostaglandin D2 released by mast cells after stimulating HDPs have important roles in microbial invasion response (Niyonsaba et al., 2001). Concomitantly to these activities, LL-37 induces keratinocytes to release IL-1β, IL-8, TNF-α, IL-6, colony-stimulating factor and granulocyte–macrophage (GM-CSF), and immature DCs to release TNF-α and IL-6 (Braff et al., 2005). However, the peptide response is dependent on the cell type that is studied. For example, in human peripheral blood mononuclear cells (PBMCs), LL-37 inhibits the expression of pro-inflammatory molecules, such as TNF-α and IL-6, and nuclear translocation of NFκB p50/p65 induced by toll-like receptor (TLR)-2 and TLR-4 in response to lipoic acid (LA) and lipopolysaccharide (LPS), respectively. This inhibition is an important event in severe microbial infections, such as sepsis (Mookherjee et al., 2006). In addition, the hBD2 also acts on TLRs, functioning as an endogenous TLR-4 ligand, further activating immature DCs, and triggering a strong Th1 response (Biragyn et al., 2002).

Moreover, other not-so-well-studied peptides also act significantly on the mammal immune system. Human α-defensins (HNP-1, HNP-2, and HNP-3) and β-defensins (hBD3 and hBD4) are involved in neutrophil and monocyte recruitment, while mast cells may be attracted to the infection site by LL-37, HNP-1–3, and hBD2 (Chen et al., 2007). Besides, human α-defensins and βdefensins are chemotactic for immature DCs and memory T cells

(Yang et al., 2002), where human α-defensins selectively induce the migration of human cells CD4+, CD45+, and CD8+ naive, suggesting that HDPs play an important role in mobilizing and amplifying the innate and adaptive immunity against microbial invasions (Yang et al., 2001). Indolicidin, for example, inhibits TNF-α secretion by macrophages in response to LPS treatment (Bowdish et al., 2005). Similar to the effects of LL-37, indolicidin induces the production of IL-8 in human bronchial epithelial cells 16HBE14o−. They also have demonstrated that the peptide bactenecin 2A, a member of bactenecins family, presented chemotactic activity over TH1 cells. Dermcidin-1L, another HDP example, stimulates keratinocytes to secrete TNF-α, IL-8, CXCL10, and CCL20 (Niyonsaba et al., 2009). Another group of peptides with strong antimicrobial activity and immunomodulatory activity are protegrins. Protegrins-1 and -3 promote rapid and efficient mature IL-1 beta release. Both peptides also promote modification in the morphology of monocytes and a loss of latency of the cell membranes (Perregaux et al., 2002).

Recent data have shown that HDPs are multifunctional molecules at different tissue levels, acting in the neuroendocrine system. Cathelicidins and defensin mRNA were widely found in the brain (Su et al., 2010), suggesting that HDPs could be involved in innate immune brain defense. Recently, it has been discovered that some neuropeptides play an important role in antimicrobial activity. Enkelytin, a pro-enkephalin-derived peptide, has shown strong activity against Gram-positive bacteria and also has acted as a link of communication and interaction between nervous, endocrine, and immune systems (Goumon et al., 1996). Met5-enkephalin peptide stimulated the proliferation of natural killer cells, B- and T-lymphocytes, migration of monocytes, lymphocytes, and neutrophils, and also stimulated the secretion of IL-6 by monocytes (Kamphuis et al., 1998). Recently, Shan et al. (2011) suggested that Met5-enkephalin appeared to be involved in the regulation between the neuroendocrine and immune systems, modulating several cell functions related to innate and adaptive immunity. They also have found that Met5-enkephalin activated CD4 + T cells by increasing the expression of delta receptors. Moreover, this peptide also induced DC maturation through the expression of surface molecules (MHC class II, CD86, CD40), stimulated the production of IL-12, and down-regulated the intracellular acid phosphatases (ACP) in DCs. In addition, Met5-enkephalin (alone or in combination with either IL-2 or IFN-γ) up-regulated the proliferation of CD4 + T cells and increased production of interferon-γ in these cells. The peptide leucine-enkephalin stimulated the production of T helper cells and cytotoxic T cells (Sizemore et al., 1991).

Among peptides with antimicrobial activity described in the neuroendocrine system, those derived from chromogranins have been highlighted. They are members of the granin family, which are acidic proteins present in secretory vesicles in the endocrine and immune systems (Shooshtarizadeh et al., 2010). Chromogranins have shown multiple-biological activities including neuroendocrine activity, modulation of homeostatic processes such as regulation of calcium metabolism and glucose, gastrointestinal motility, nociception, strong activity in tissue repair, and inflammatory response mediation. More recently, peptides derived from chromogranins could act as defense agents during infections of bacteria and fungi. Moreover, chromogranin-derived peptides have shown activity in innate immunity, being able to activate polymorphonuclear neutrophils, mediate the monocyte chemotaxis, and induce microglial cell activation (Shooshtarizadeh et al., 2010). Peptides derived from chromogranins have shown special activity against pathogens commonly found in the skin, revealing that the expression of these peptides may be performed by keratinocytes. In these cells, chromogranin A fragments generated catestatin: a broad-spectrum antimicrobial peptide. These fragments induce monocyte chemotaxis and act on the production of cytokines and chemokines by mast cells (granulocyte– macrophage colony-stimulating factor, monocyte chemotactic protein-1, macrophage inflammatory protein-1α, macrophage inflammatory protein-1β, and chemokine – C-C motif – ligand 2, 3, and CCL2, CCL3, CCL4). Furthermore, it also stimulated keratinocyte interleukin-8 production through the activation of mitogen-activated protein kinases (Radek et al., 2008; Shooshtarizadeh et al., 2010). Other peptides derived from chromogranins, termed vasostatins, have shown deleterious activity against Grampositive bacteria, filamentous fungi, and yeasts (Dondossola et al., 2010). These studies have demonstrated that a neuroendocrine peptide may have antimicrobial activity against a wide variety of skin pathogens and may be up-regulated in the lesion. These data have demonstrated a direct link between the neuroendocrine and immune system (Shooshtarizadeh et al., 2010). Finally, the expression of catestatin in mice skin resulted in higher protection against infection and injury. Catestatin is a 21-amino acid residue,which is made endogenously from the proteolytic cleavage of chromogranin A (Radek et al., 2008).

Studies have shown that members of the neuropeptide bombesin family,in addition to direct activity against microorganisms, mediate a variety of biological activities in the gastrointestinal tract and central nervous system (CNS) of mammals,including smooth muscle contraction, hormone secretion, cell proliferation stimulation, and central-homeostatic mechanism regulation (Su et al., 2010). Another HDP family found in mammalian brains are hepcidins, which besides having direct antimicrobial activity, also reduce secretion of TNF-α, IL-1α, IL-1β, and IL-6 by regulating the COX-2 gene and PDE4D by using mechanisms dependent on pERK1/2 in macrophage cells (Su et al., 2010). In this view, due to LPS stimulation, a significant increase in hepcidin mRNA expression was observed in cortex and *substantia nigra*. These findings suggest that the CNS can use many peptides as anti-infective and immunomodulatory agents by delivering them quickly and accurately to innervated sites, indicating a new dimension for an immunomodulatory role for neuropeptides in inflammatory and immune responses (El Karim et al., 2008).

#### **HOST-DEFENSE PEPTIDES: FUNDAMENTAL MOLECULES IN RESPONSE TO HUMAN MICROBIAL INFECTIONS**

HDPs are evolutionarily ancient molecules that reinforce the human innate immune system against microbial invasion (Nijnik et al., 2009). A constitutive HDP expression has been observed in phagocytic cells and in body tissues (**Figure 3**). This occurrence

happens particularly at human interfaces between the environment and the host, such as skin and mucosal membranes (Hirsch et al., 2008). HDP synthesis and release are regulated by microbial stimuli, cytokines, neuroendocrine signals, and epigenetic regulation (Ganz, 2003).

Along these lines, intestinal mucosal pathologies, such as Crohn's disease and intestinal inflammation, result from the reduction of α-defensin expression in the small intestine (Inaba et al., 2010). The protective role of α-defensins against pathogenic bacteria in intestinal infections has been certified in αdefensin-deficient mice, since mice became more susceptible to *Salmonella typhimurium* infections (Inaba et al., 2010). Moreover, hBD1 and hBD2 are important HDPs in gastric-intestinal mucosa. The expression of both peptides increased in the gastric antrum mucosa, which is commonly induced by gastritis caused via *Helicobacter pylori* (Bajaj-Elliott et al., 2002).

Regarding HDPs produced in human skin, patients with infectious cellulitis presented an increased expression of LL-37 and β-defensin in comparison with those normal skins (Stryjewski et al., 2007). This datum suggests that HDPs may provide additional protection against microbial dissemination, confining the focus of infectious cellulitis to a single location. However, patients with atopic dermatitis presented an absence or reduced expression of LL-37, hBD2, hBD3, and dermicidin (Bernard and Gallo, 2011). These findings suggest that patient susceptibility to atopic dermatitis in skin infections could be higher due to a significant increase in either bacterial colonization, fungal, or viral infections (Bernard and Gallo, 2011). Recent studies have detected high levels of human β-defensin, hBD2, hBD3, and psoriasin in cutaneous infections after skin surgery (Kesting et al., 2010). Expression of hBD2-3 is reduced in burn wounds, while the expression hBD1 is unchanged (Milner and Ortega, 1999). However, the skin's lower layers continue to express hBD1-3 after wound recovery, maintaining a barrier against infection and preventing burn sepsis (Poindexter et al., 2006).

Sepsis is a major cause of mortality and high hospital costs and, despite the efforts that have been made by public health authorities in recent decades, the incidence of this syndrome continues to increase year after year due in particular to the increasing emergence of microorganisms resistant to the available antibiotics (Barnato et al., 2008). Several HDPs have been reported to modulate the host response to bacterial sepsis. LL-37 acted as a potent inflammation molecule mediator, protecting mice and rats from the lethal effects of endotoxemia and intra-abdominal sepsis. This protection occurs due to a reduction in the levels of TNF-α and LPS in plasma, which inhibit the activation of macrophages by LPS, lipoteichoic acid (LTA), and non-mannose-capped lipoarabinomannan, as well as regulating the expression of coding genes and receptors (CXCR-4, CCR2, and IL-8RB) of chemokines in macrophages (Torossian et al., 2007). The main mechanism of TNF-α expression inhibition by LL-37 in mice is believed to occur by the blocking of LBP-mediated transport of LPS in CD14+ cells (Nagaoka et al., 2001). HDPs buforin II, indolicidin, and cecropin B have shown effectiveness in the treatment of rats with septic/endotoxic shock caused by multi-resistant *Escherichia coli* and LPS, significantly reducing the plasma levels of endotoxin and TNF-α when compared to animals treated with piperacillin (Vallespi et al., 2003). Limulus anti-LPS factor (LALF), a peptide isolated from the horseshoe crab, could increase the survival of mice after administration of a lethal *P. aeruginosa* dose. This activity may be correlated with a decreased TNF-α mRNA synthesis and elevation of systemic IL-2, IL-12, and IL-13 into animal spleen and livers (Vallespi et al., 2003). Mice with endotoxic shock derived from an LPS and treated with S-thanatin, an insect peptide, showed reduced levels of TNF-α accompanied by a significant increase (80%) of animal survival (Wu et al., 2011a). In another study, animals infected with multi-resistant *P. aeruginosa* and further treated with a single dose of tachyplesin III isolated from horseshoe crabs has shown reduced TNF-α levels in contrast to a clear survival increase (Cirioni et al., 2007). Treatment of amphibian source magainins (I, II, and III) in septic/endotoxic shock significantly reduced the concentrations of endotoxin and TNF-α in plasma rats, thus increasing host survival (Cirioni et al., 2002). These data suggest that peptides that could act against septic shock could be found at different sources and their activities are clearly related to TNF-α inhibition and endotoxin neutralization, which seem to be important mechanisms that increase the survival of infected animals.

#### **NOVEL AND CLASSICAL STRATEGIES FOR HDPs ISOLATION**

Currently, several limitations have driven the area in the development and production of HDP molecules away from natural peptides to the shorter and more stable synthetic forms. Libraries, synthetic strategies, and peptidomimetic technologies are being refined in order to overcome current limitations (Yeung et al., 2011).

Traditionally, since the beginning in 1928, with the discovery of lantibiotic Nisin produced by a *Lactococcus lactis*, until nearly the end of the twentieth century, the discovery and identification of peptide antimicrobial molecules from natural sources with pharmaceutical anti-infectious purposes have been carried out by conventional biochemical procedures. Both goals frequently started from an expected and confirmed biological activity: generally a direct inhibition of microbial growth of bacteria, virus,fungi, and parasites (using a medical concept of microbes). This primary evaluation of crude extracts was the starting point for combined steps of clarification, salt or acid precipitation, ultrafiltration, chromatographic purification, and the eventual available structural characterization. This approach, on the other hand, required an important volume of specimen, whole bodies or parts, to assure a significant level of antimicrobial activity. These procedures were achieved representing a real ecological challenge for the different life kingdoms if, finally, a large-scale pharmaceutical industry could be implemented for sustaining permanent production based on such an animal source (Bulet, 2008). Such natural sources were, in principle, associated with invertebrates, since HDPs are their major humoral tool to defeat a vast microbial challenge and must be effective in the absence of acquired immunity (Otero-Gonzalez et al., 2010).

New trends in research are currently categorizing the discovery of new HDPs through the use of biological peptide libraries, thus providing a larger and more diverse quantity of molecules for evaluation. Such libraries based on phage (Pini et al., 2005), bacterial (Betscheider and Zangen, 2005), or ribosome display (Xie et al., 2006) are nevertheless time-consuming procedures and difficult to handle. Infact, a novel methodfor the identification of AMPs using a phage library and bacterial magnetic particles has been reported (Betscheider and Zangen, 2005; Pini et al., 2005). Using this library, six antibacterial peptides specifically active against *B. subtilis* have been identified. It was the first attempt that showed the functionality of a magnetic selection of HDPs targeting the bacterial inner membrane. Furthermore, these methodologies produce fusion instead of individual peptides and, additionally, only amino acids encoded in the genome can be used with a reasonable support of biological diversity. A clear advantage of these approaches is that peptides are biologically synthesized, avoiding the use of expensive chemicals and facilities (Mcphee et al., 2005). Furthermore, the advances in the genomic area including next generation sequencing and improvements in databank and bioinformatics tools have permitted the identification of many different compounds including HDPs. In a well-designed genetic approach reported in flatfish, the identification of active, novel antimicrobial molecules has been determined by screening both the genomic information and the mRNA transcripts from a number of different sequences encoding AMPs. Predictions of active peptide sequences from the genetic information were the starting point for the production of chemically synthesized peptides that have been tested for their cognate activities. A very active peptide was found to show inhibitory activity against a test panel of pathogens including antibiotic-resistant bacteria and fungi (Patrzykat et al., 2003).

As an attempt to expand the efficiency of discovering novel and effective HDPs, including unnatural HDPs, chemical peptide libraries are open to amino acids not encoded in the genome, damino acids, and even non-canonical amino acids. Screening large numbers of peptides using a synthesis of peptide arrays on cellulose membranes (SPOT technology) provides an extraordinary tool for characterizing large quantities of peptides for a particular biological activity (Hilpert, 2010). This improved method using hydroxymethyl phenoxyacetic acid (HMPA) for 19 amino acids and 4-(4-hydroxymethyl-3-methoxyphenoxy)-butyric acid (HMPB) for proline, since acidic labile linkers in SPOT synthesis have been developed. Using this approach, reduced side-chain reactions, normally occurring during conventional alkaline peptide cleavage of cellulose membranes, was achieved. This method is suitable for synthesizing many thousands of different peptides subsequently used for direct multiple-biological assays (Ay et al., 2008).

Since the search for novel classes includes the purification process, the properties of target peptides such as hydrophobicity, cationicity, and small size have been broadly used in their purification. As follows, reversed-phase chromatography, ion exchange, and gel filtration are commonly included in the purification procedures for these molecules (Schroder, 2010). Although direct interaction of HDPs with different target molecules such as LPS (Gustafsson et al.,2010),lipid II (Schneider et al.,2010), and sphingolipid receptors are widely known (de Medeiros et al., 2010), affinity chromatography has not been widely used for their isolation. The use of this type of chromatography may improve the isolation of AMPs with desired affinities for biomolecules. For example, HDPs with the ability to bind LPS are very important in the treatment of sepsis, as previously described (Giuliani et al., 2010). However, the majority of molecules capable of inhibiting LPS have been synthesized on the basis of the Limulus antilipopolysaccharide factor (Andra et al., 2004). There is little doubt that the rational use of the LPS-affinity chromatography could enhance the number of natural HDPs with anti-LPS activity. In this case, affinity chromatography also acts as a screening procedure. In fact, it has isolated lipopolysaccharide-binding proteins from porcine milk and from the invertebrate hemolymph *Tachypleus tridentatus* using a similar chromatographic principle (Shahriar et al., 2006). Furthermore, HDPs are able to be induced, being another HDP property that could be exploited in their purification (Lavine et al., 2005). In fact, the microbial challenge test of different vertebrates and invertebrates permitted the isolation of augmented HDPs (Isobe et al., 2009). Thus, it could be possible to induce HDPs with a potent anti-proliferative activity against a particular group of pathogens (Lemaitre et al., 1997). The induction of these molecules may reveal basal HDPs and facilitate their purification by augmenting the concentration levels for further purification. Traditionally, once a peptide sequence had been identified and/or partially purified from a natural source, a structural approach had been applied with the aim to improve its antimicrobial activity and/or decrease its toxic or antigenic implications for experimental tests in animals or initial human trials. In this sense, amino acid substitutions based on the sequence data or on the three-dimensional structure such as the replacing of arginine residues with α-amino-3-guanidino-propionic acid of the natural peptides may help to reduce serum AMPs degradation. The modified peptide stability was increased by nearly 80% without affecting the antimicrobial activity (Knappe et al., 2010). In a completely different but valid approach (based on the membrane-binding activity of most AMPs), cell membrane affinity chromatography has been reported to screen antibacterial peptide molecules from

plant extracts. In this case, cell membrane affinity chromatography was useful for primary screening of cell membrane receptors according to its chromatographic retention. This constitutes one of the first reports on a high-throughput screening (HTS) of AMPs by such a principle (Xiao et al., 2011).

High-throughput screening is a well-established method in the bio-industry and is now used for basic and applied scientific research (**Figure 4**). It involves the screening of large biological and chemical libraries for activity against biological targets by automation, miniaturized assays, and large-scale data processing (Mayr and Bojanic, 2009). While HTS was initially applied to relatively non-complex simple assays concerning single molecular objectives or using affinity interactions relationships, HTS has also been applied to more integrated complex biological multicomponent systems, such as whole-cell-based tests. The intense need for novel and efficient antimicrobial molecules highlights the existence of multi-drug-resistant microorganisms, where multipletarget or cell-based assays are frequently desired, and thus is obliging researchers to reflect on multi-target, high-throughput technologies (Blondelle and Lohner, 2010). Clearly, an improved understanding of non-lytic modes of HDP interaction with target microbes will lead to the design and discovery of more effective antimicrobial molecules (Marcos and Gandía, 2009). In the pharmaceutical industry,HTS means challenging millions of molecules in massive automated applications. On the other hand, in a current academic laboratory, it may imply screening only a small quantity of peptides, mainly by conventional artisanal techniques. HDP activity may be best described by the concept of "interfacial activity" (Wimley, 2010). Structure–function relationships are infrequently utilized. The next challenge is not the generation of an extensive repertoire of molecules, but HTS employed to efficiently identify the active members from a massive source. In general, HTS could be divided into two categories: biological and nonbiological procedures (Rathinakumar and Wimley, 2010), which will be discussed next.

#### *IN VITRO* **AND** *IN SILICO* **BIOLOGICAL ASSAYS FOR THE FUNCTIONAL EVALUATION OF HDPs**

Broth dilution and agar diffusion methods, in which the activity of HDPs are directly estimated for the inhibition of microbial growth in a nutritive broth, could be in principle amended for HTS (Wiegand et al., 2008). Automated and massive variations of such procedures have been established in order to scale the number and diversity of HDPs to be verified. The use of luminescent bacteria for fast screening and characterization of short cationic AMPs synthetically immobilized in cellulose using peptide array technology has been developed (Hilpert et al., 2005). In this unusual approach, the authors used bacteria expressing the luciferase gene cassette (luxCDABE). Any synthetic peptide decreasing the energy level within the microbial cell will cause a quantifiable decrease in luminescence. The effectiveness of a tested molecule, at different concentrations, is reflected by the decreasing rate in luminescence. The assay is rapid and high-throughput and has shown significant correlation when compared with conventional suspension inhibitory culture assays performed with the same peptides synthesized by standard solid-phase peptide synthesis. Using this approach, 277 variants of the antimicrobial peptide

Bac2A (a 12-mer linear variant of the HDP bactenecin) were studied. The antimicrobial activity of Bac2A derivatives ranged from superior to inactive compared to Bac2A (Hilpert et al., 2006). This technology allowed for a decreased amount of time required to convert leaded antimicrobial molecules into drugs.

In another interesting approach, a novel assay that had exploited fluorescence resonance energy transfer (FRET) was set up for antimicrobial peptide screening. It took advantage of changes in pH that occur in FRET efficiency due to the instability of increased yellow fluorescent protein against the stability of increased cyan fluorescent protein in a reduced-pH environment. Evaluation of antimicrobial activity was achieved through a difference of FRET efficiency between fusion molecules released from disrupted bacteria. It represented a clear example applicable to HTS of candidate peptide libraries (Kim and Cha, 2006). In this line of thought, a high-throughput method for screening cDNA libraries has been developed to detect possible AMPs. It was based on a fast dye inclusion assay for determining a bacterial viability loss. Colonies were grown on a membrane on a suitable medium until full colony development. The membrane containing the array of colonies is transferred onto an inductive medium containing a vital dye. Upon expression of any active peptides, the cell membrane becomes susceptible and permits dye infusion to induce visual identification of positive peptides (Loit et al., 2008). In this way, a random oligonucleotide library containing a potential pool of 100,000 peptides was screened. The authors found three novel antibacterial peptides. One of them has shown membrane disruption and bacterial aggregation activity (Loit et al., 2010). Unfortunately, these HTSs are not available yet for other HDP activities like immunomodulation or wounding repair. The development of techniques in these areas will boost the insertion of HDPs into the drug industry.

An early attempt for automating the screening of membranerelatedAMPs was reported in which a colorimetric sensor by which particles composed of phospholipids and polymerized polydiacetylene lipids were shown to exhibit clearly visible color changes upon interactions with antimicrobial membrane peptides. The color changes in the system are due to a structural perturbation of the lipids after their interactions with AMPs. The assay was also valuable for detecting functionally related peptide analogs (Kolusheva et al., 2000). Alamethicin, gramicidin, and valinomycin have been investigated in such planar film systems (Volinskya et al., 2006). Otherwise, the *in silico*-based screening systems include computerized methods as well as simulation methods that mimic the interaction between peptides and membranes (Raventos et al., 2005). A mathematical model has been developed to predict, before synthesis, a peptide with an activity against *P. aeruginosa* (Jenssen et al., 2008). Using original descriptors for assessing the contact energy between spatially closed amino acids, as well as a set of inductive and conventional quantitative structure–activity relationship (QSAR) descriptors, it has been possible to model the antibacterial activity of peptides before the synthesis step. Cross-correlation and optimization of the selected descriptor values permitted these authors to build two models, using very limited number of peptides. Even when such models were significantly different in size, no significant difference was demonstrated in their predictive power, meaning that it was possible to use this tool to obtain useful and potent predictive models, even when using small sets of peptides with different structures. The development of biological assays in HTS has increased the number of known antimicrobial structures allowing the development of newer inductive QSAR descriptors, which in combination with more complex mathematical algorithms, improve the *in silico* identification of novel HDPs (Hilpert et al., 2008). Also, QSAR

descriptors have been developed for predicting antimicrobial peptide toxicity against host cells (Langham et al., 2008). Additionally, recent advances in molecular dynamics simulations of AMPs and membrane mimics offer new data to help us to understand the mechanism of action of these molecules (Langham and Kaznessis, 2010).

In a recent chemical approach, novel cationic AMPs were proposed with a simple strategy using acid-amide substitution to add a net positive charge to natural non-antibacterial amino acid sequences showing structures that were differentfrom the reported cationic AMPs. Alterations produced in membranes by these modified peptides were evaluated and conformational changes were estimated from far-ultraviolet circular dichroism spectra. These novel cationic AMPs showed sequences completely distant from any other AMPs described before, suggesting that such modified natural sequences could be an interesting source of novel frames for cationic AMPs (Ueno et al., 2011).

#### **LARGE-SCALE HDP PRODUCTION: OVERCOMING A MAIN PROBLEM**

Structural and functional features, as well as several other significant physiological characteristics of these molecules as described in this review, are reasons why HDPs have received much attention from the scientific community for the development of new pharmaceuticals. However, there are real barriers in developing and producing natural HDPs. The challenge begins with the understanding of the fundamental principles of action in HDPs, as well as their structure–function relationship and pharmaceutical potential (Wimley, 2010). The lack of this understanding has been one of the major reasons why the development of these peptides into new drugs has slowed. This is due to the fact that only a few heterologous systems are capable of yielding a sufficient amount (in the range of ∼50–690 mg L−1) of soluble and purified peptides in order to enlighten such extensive biological and structural questions (Xu et al., 2007; Li, 2009). Moreover, in order to accomplish pharmaceutical and biotechnological goals, it is necessary to produce these peptides in large-scale. This sort of production has been the biggest challenge in studies regarding screening, synthesis, and characterization of these molecules.

Despite two decades of continuous efforts, the limitation of small-scale production has impaired the successful use of HDPs in health care. For instance, only four peptides to date have reached global sales of over US\$ 1 billion as therapeutic drugs. These have been prescribed for treatments for multiple sclerosis as Glatiramer acetate (also known as Cop-1 or Copaxone; \$3.8 billion), for prostate cancer and breast cancer as leuprolide acetate (Lupron; \$2.12 billion) and goserelin acetate (Zoladex; \$1.14 billion), and for treatment of acromegaly and carcinoid syndrome as octreotide acetate (Sandostatin; \$1.12 billion; Peptide Therapeutics Foundation, 2010 Report Summary). Moreover, in the last 5 years, only one HDP, the human bactericidal/permeability-increasing protein rBPI23 (Neuprex – indicated for treatment of meningococcal sepsis) has been approved for marketing. Furthermore, only three HDPs have progressed to phase III of clinical-efficacy trials (Pexiganan, Iseganan, and Omiganan) and none of these have to date received FDA approval for clinical use (Hancock and Sahl, 2006; Yeung et al., 2011).

To change this scenario, current approaches need to be refined and new technologies are required to be employed in the development and production of HDPs. In order to recover and produce peptides, different methods can be employed such as the direct isolation from natural sources, chemical synthesis, or recombinant expression. The first strategy, despite extensive and efficient utilization at academic levels, it is extremely time-consuming and only low amounts of peptides are recovered from the host organism. This technique may even present environmental issues, especially for peptides isolated from species that occur in low numbers in nature (Li, 2011).

Chemical synthesis has been traditionally used to synthesize short and simple molecules and has allowed for the production of both natural and synthetic peptides (Zhou et al., 2010). However,it is tremendously costly and practically unfeasible for the synthesis of sequences larger than 15 residues of amino acids, running in the range of US\$ 100–600/g (Hancock and Sahl, 2006). Moreover, the cost may increase substantially due to difficulties that arise during synthesis of certain sequences such as those containing one or more disulfide bonds, or showing multiple post-translational modifications (Tay et al., 2011).

In view of the limits and inefficiencies of both methods described above, the most widely used method is the heterologous expression system. In the last few decades, various systems have been developed to reach a cost-effective and large-scale production of several proteins (Huber et al., 2005) and HDPs such as human defensins LL-37 and IDR-1 (Bommarius et al., 2010).

In most cases, the effectiveness of heterologous expression for the production of HDPs involves the fusion of carrier proteins containing an enzymatic or chemical cleavage site that allows the target peptide to be released. Some of these proteins act as stabilizing molecules due to their ability to neutralize the positive charge of the peptide, thus resulting in a non-toxic, efficient, and soluble expression of the target peptide (Rao et al., 2004). According to the *Recombinantly produced* AMPs *Database* (http://faculty.ist.unomaha.edu/; Li and Chen, 2008), the carrier protein most frequently used is the thioredoxin, representing more than 20% of all reported fusion proteins, followed by the use of GST (glutathione transferase –∼12%), PurF (amidophosphoribosyltransferase), and intein-mediated protein (∼8%). The SUMO protein (small ubiquitin-related modifier) has been a promising carrier protein and has been shown to increase expression levels as well as the solubility of peptides (Malakhov et al., 2004; Bommarius et al., 2010).

The heterologous expression system may also be composed of one or more epitope tags (Rubio et al., 2005). These are short and hydrophilic peptide sequences recognized by specific antibodies, thus offering efficient detection. The most used epitope tags are HA (YPYDVPDYA), FLAG (DYKDDDDK), cMyc (EQKLISEEDL), and 6xHis (HHHHHH). Besides being used for subcellular location, their small size provides efficient affinity purification and is more likely to interfere less with the peptide of interest regarding its folding and function, when compared to larger carrier proteins (Earley et al., 2006).

Among the available commercial options of heterologous expression vectors, the most commonly used are those which belong to the pET and pQE series, developed by Novagen and Qiagen, respectively. These vectors have been largely used for expression of HDP derived from several organisms such as defensins from humans (e.g., α-defensin 6), bovine (β-defensin 12) and defensin-like from fungus (plectasin; Wu et al., 2011b). In addition, new mutant strains of *E. coli* BL21 (DE3) have been developed to overcome the lethality caused by the expression of AMPs (Li et al., 2010).

Regarding the prokaryotic systems, *E. coli* is the most popular organism used to express recombinant proteins. Due to its features such as rapid growth rate, low cost, large availability for commercial expression vectors, and extensive knowledge about its genetics and physiology, *E. coli* has been applied to innumerable studies of synthesis and expression of AMPs and HDPs from different organisms (Canales et al., 2011) and has also been used in large-scale production of these peptides (Bommarius et al., 2010).

The limitation of the use of prokaryotic systems is to perform post-translational modifications required for stability and biological activity of certain peptides. To overcome this limitation, the use of eukaryotic systems is the appropriate approach. The yeast species *Pichia pastoris* and *Saccharomyces cerevisiae* represent the most common eukaryotic host system used for expression of heterologous proteins (Holz et al., 2002). The yeast *P. pastoris* has been used for expression of HDPs derived from humans and plants, although none have reported large-scale production (Yang et al., 2007). Naturally, the disadvantage in using these organisms is their innate sensitivity against antifungal peptides (Thevissen et al., 2007).

Plants have been a promising alternative for eukaryotic systems and numerous strategies have been developed to optimize these organisms to attend commercial production (Desai et al., 2010). The most common plant hosts are *Nicotiana tabacum* and *Arabidopsis thaliana*, which have been reported in expression of AMPs derived from plants such as LjAMP1, SmAMP1, and SmAMP2, which have activities against 15 plant pathogens, including those of economic concern. Furthermore, considering AMPs in plants act like HDPs, such as defensins, the expression of these molecules, either in plants or in bacteria, has been successfully achieved. However, there is no report in the literature regarding expression of HDPs from animal sources expressed in plants. Diverse studies have demonstrated viable possibilities of using plants for production of biotechnological products, contributing to the development of new heterologous expression systems to reach large-scale production (Pogue et al., 2010). Nevertheless, it should be emphasized that there needs to be advances in technology before these organisms can be chosen over bacteria as an expression system for recombinant HDPs.

The heterologous expression technologies overviewed here are of paramount concern regarding the production of natural or synthetic peptides in large-scale for therapeutic research and development. The development of new technology and the widespread acceptance of protein therapy as listed above will very likely increase the percentage of HDPs and AMPs as therapeutic candidates for pharmaceutical and biotechnological industries.

#### **CONCLUDING REMARKS**

As focused in this report, promiscuous AMPs and HDPs are evolutionarily primordial defensive molecules widely distributed in different kingdoms suggesting that they have played an essential role in the development of multicellular organisms. This central function comes from the fact that these promiscuous peptides present the ability to bind to different targets. In addition to the direct effects against microorganisms, HDPs also show the ability to act in the host organism, thus monopolizing the control of infections at higher levels. This ability involves basic functions of acquired immunity such as cytokine signal transduction, TLR interaction, and primeval immune memory, thus acting as effective immune modulators in mammals. Unfortunately, even though research on HDPs has lead to a better understanding of their functionality, the number of outbreaks is continuously increasing while few alternatives are currently available to combat these infections.

In order to control this increasing problem, novel strategies for treating infectious illnesses will be necessary, requiring additional manipulation of immune responses in order to intensely diminish the infectious agents and also avoid tissue wounds due to inflammation. The interchange ability between immunogenicity, danger signaling, and immune modulation of HDPs could act in synergy with the immunity normally offered by humans. In this view, normal patients subjected to resistant bacteria would clearly benefit. Moreover, patients with a compromised immune system subjected to severe opportunistic infections could also have the opportunity to improve their quality of life, reducing multiple infectious symptoms, e.g., fever, skin abscesses, chest pain, and shortness of breath.

Nevertheless, many problems must be solved for efficient HDP selection and production. Regarding the selection issue, novel compounds must be screened from different and unusual sources exploring different biomes and environments. The production problems must be solved by using novel technologies of chemical synthesis, and also novel strategies of heterologous production. An additional problem is the low stability of some peptides. This can be improved by several methods including the production of mutants and also the design of novel particles that could protect the peptide against host–hostile environments, providing an efficient delivery system. One must ask whether peptides are capable of correctly modulating the host immune system. If not, the collateral effects will be worse than the proper infection. This does not seem to be the case, at least when considering the many trials using animal models. Nevertheless, several clinical trials using human beings must be performed in order to provide security for all patients. The use of the AMPs or HDPs is inevitable and extremely desirable, but more studies must be performed in order to really understand the mechanisms of action of such molecules. Indeed, researchers must use all the available technology possible including proteomics, genomics, transcriptomics, nanobiotechnology, and many others in order to reduce the detrimental effects on the population. The infectious storm is upon the human species and the best that we can do is be prepared for it by constructing and producing the best compounds possible.

#### **ACKNOWLEDGMENTS**

This work was supported by CAPES, CNPq, FAPDF, and UCB.

#### **REFERENCES**

J. M., Segal, D. M., Oppenheim, J. J., and Kwak, L. W. (2002). Toll-like receptor 4-dependent activation of dendritic cells by beta-defensin 2. *Science* 298, 1025–1029.

Efficacy of tachyplesin III, colistin, and imipenem against a multiresistant *Pseudomonas aeruginosa* strain. *Antimicrob. Agents Chemother.* 51, 2005–2010.

from *Momordica cochinchinensis*. *J. Biol. Chem.* 276, 22875–22882.

a model of inflammatory bowel disease, IL-10-deficient mice. *Inflamm. Bowel Dis.* 16, 1488–1495.

differentially affects transcript abundance of three antimicrobial peptides in hemocytes from the moth *Pseudoplusia includens*. *Insect Biochem. Mol. Biol.* 35, 1335–1346.

Raventos, D., Taboureau, O., Mygind, P. H., Nielsen, J. D., Sonksen, C. P., and Kristensen, H. H. (2005). Improving on nature's defenses: optimization & high throughput screening of antimicrobial peptides. *Comb. Chem. High Throughput Screen.* 8, 219–233.

infective cellulitis. *J. Infect. Dis.* 196, 1425–1430.

with multiple exploitable activities from shelf beans. *J. Pept. Sci.* 12, 341–346.

broad-spectrum antimicrobial peptides synthesized via ringopening polymerization of alphaamino acid-*N*-carboxyanhydrides. *Biomacromolecules* 11, 60–67.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships

that could be construed as a potential conflict of interest.

*Received: 16 September 2011; paper pending published: 28 September 2011; accepted: 01 November 2011; published online: 22 November 2011. Citation: Silva ON, Mulder KCL, Barbosa AEAD, Otero-Gonzalez AJ, Lopez-*

*Abarrategui C, Rezende TMB, Dias SC*

*and Franco OL (2011) Exploring the pharmacological potential of promiscuous host-defense peptides: from natural screenings to biotechnological applications. Front. Microbio. 2:232. doi: 10.3389/fmicb.2011.00232*

*This article was submitted to Frontiers in Antimicrobials, Resistance and Chemotherapy, a specialty of Frontiers in Microbiology.*

*Copyright © 2011 Silva, Mulder, Barbosa, Otero-Gonzalez, Lopez-Abarrategui, Rezende, Dias and Franco. This is an open-access article subject to a non-exclusive license between the authors and Frontiers Media SA, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and other Frontiers conditions are complied with.*

### Mechanism of honey bacteriostatic action against MRSA and VRE involves hydroxyl radicals generated from honey's hydrogen peroxide

#### *Katrina Brudzynski 1,2\* and Robert Lannigan3*

1 API-Medicals, Brock University, St. Catharines, ON, Canada

2 Department of Biological Sciences, Brock University, St. Catharines, ON, Canada

3 Department of Clinical Microbiology, London Health Sciences Centre, London, ON, Canada

#### *Edited by:*

Mirian A. F. Hayashi, Universidade Federal de São Paulo, Brazil

#### *Reviewed by:*

Atte Von Wright, University of Eastern Finland, Finland Charles Knapp, University of Strathclyde, UK

#### *\*Correspondence:*

Katrina Brudzynski, Department of Biological Sciences, Brock University, 500 Glenridge Avenue, St. Catharines, ON, Canada L2S 3A1. e-mail<EMAIL_ADDRESS>
It has been recently reported that honey hydrogen peroxide in conjunction with unknown honey components produced cytotoxic effects resulting in bacterial growth inhibition and DNA degradation. The objective of this study was twofold: (a) to investigate whether the coupling chemistry involving hydrogen peroxide is responsible for a generation of hydroxyl radicals and (b) whether •OH generation affects growth of multi-drug resistant clinical isolates. The susceptibility of five different strains of methicillin-resistant Staphylococcus aureus (MRSA) and four strains of vancomycin-resistant Enterococcus faecium (VRE) isolates from infected wounds to several honeys was evaluated using broth microdilution assay. Isolates were identified to genus and species and their susceptibility to antibiotics was confirmed using an automated system (Vitek®, Biomérieux®). The presence of the mec(A) gene, nuc gene and van(A) and (B) genes were confirmed by polymerase chain reaction. Results showed that no clinical isolate was resistant to selected active honeys. The median difference in honeys MICs against these strains ranged between 12.5 and 6.25% v/v and was not different from the MIC against standard Escherichia coli and Bacillus subtilis. Generation of •OH during bacteria incubation with honeys was analyzed using 3- -(p-aminophenyl) fluorescein (APF) as the •OH trap. The •OH participation in growth inhibition was monitored directly by including APF in broth microdilution assay. The growth of MRSA and VRE was inhibited by •OH generation in a dose-dependent manner. Exposure of MRSA and VRE to honeys supplemented with Cu(II) augmented production of •OH by 30-fold and increased honey bacteriostatic potency from MIC90 6.25 to MIC90< 0.78% v/v. Pretreatment of honeys with catalase prior to their supplementation with Cu ions fully restored bacterial growth indicating that hydroxyl radicals were produced from H2O2 via the Fenton-type reaction. In conclusion, we have demonstrated for the first time that bacteriostatic effect of honeys on MRSA and VRE was dose-dependently related to generation of •OH from honey H2O2.

**Keywords: hydroxyl radicals, H2O2, 3- -(***p***-aminophenyl) fluorescein, growth inhibition, MRSA, VRE, mechanism of honey antibacterial activity**

#### **INTRODUCTION**

Honey has well established function as an effective antibacterial agent with a broad spectrum of activity against Gram-positive and Gram-negative bacteria (for review, Lusby et al., 2002; Irish et al., 2011). Despite a progress in identification of compounds that are involved in growth inhibitory and bactericidal actions of honey, the mechanism underlying these activities remained unknown. A functional relationship between hydrogen peroxide produced in honey and antibacterial activity strongly pointed to H2O2 as the main contributor to this activity (White et al., 1963; Bang et al., 2003; Brudzynski, 2006). However, recent new findings question this view. Firstly, molecular H2O2 is relatively weak oxidant and it requires high concentrations to exert its cytotoxic effect (Rutala et al., 2008; Finnegan et al., 2010). Our data indicate that H2O2 content in honeys (0.4–2.6 mM), is much below its biocidal

levels. Yet, even at these low H2O2 concentrations, honeys effectively inhibited bacterial growth and caused DNA strand breaks (Brudzynski et al., 2011). Secondly, oxidative stress of endogenous H2O2 was clearly augmented by the action of unknown honey components (Brudzynski et al., 2011). Thirdly, in our accompanying paper, we provided the first evidence that honeys of high bacteriostatic activity (MIC90 12.5–6.25% v/v) possessed significantly higher levels of phenolic compounds of higher radical scavenging activities than honey of the average bacteriostatic activity (MIC90 25% v/v), thus suggesting the involvement of phenolics and H2O2 in bacterial growth inhibition (Brudzynski et al., 2012).

In our prior research, we have screened over 200 samples of Canadian honeys of different botanical origin for the antibacterial activity using broth microdilution assay (Brudzynski and Kim, 2011). Fifty percent of screened honeys showed MICs exceeding

that of sugar solution. Among the group of "active" honeys, MICs of honeys originating from buckwheat differed consistently by four to 16 doubling concentrations from the MIC of sugar solution. Canadian buckwheat honeys characteristically contained higher amounts of hydrogen peroxide than other honeys (Brudzynski, 2006) and possessed higher than average content of polyphenols of high antioxidant activity as measured by the oxygen radical absorbent capacity (ORAC) method (Brudzynski and Miotto, 2011; Brudzynski et al., 2012). These results have converged to recognize that the coupling chemistry between H2O2 and polyphenols, rather than hydrogen peroxide alone, may exert oxidative effect causing bacterial growth arrest and DNA degradation (Brudzynski et al., 2011, 2012). High ORAC values of polyphenols are usually indicative of their efficient peroxyl radical scavenging abilities (Cao et al., 1997; Price et al., 2006). However, the same polyphenols could become powerful pro-oxidants when oxidized in the presence of oxygen from air. Such situation occurs during honey harvesting from the comb and during storage. Oxidized polyphenols further generate hydrogen peroxide and in the presence of transition metals such as Cu(I) or Fe(II) they drive the generation of hydroxyl radicals from H2O2 via the Fenton reaction (Puppo, 1992;Hanasaki et al., 1994;Cao et al., 1997; Sakihama et al., 2002).

Together, these facts brought about a new hypothesis that the oxidizing action of honey on bacterial cells may result from the generation of hydroxyl radicals from H2O2. Such coupling reaction has never been shown to be associated with honey function in spite of the fact that honey possesses all necessary substrates for the Fenton reaction; H2O2, polyphenols as well as transition metal ions (Bogdanov et al., 2007).

In contrast to H2O2, •OH radicals are powerful oxidants that can oxidize molecules in all cellular compartments in a nonspecific manner. •OH cytotoxic effects have been shown for both prokaryotic and eukaryotic cells; bacteria, yeast, and human cells (Halliwell et al., 1985; Imlay and Linn, 1988; Perrone et al., 2008). For example, •OH production from H2O2 by neutrophil granules is a first line of defense against bacteria during acute inflammation. Similarly, the H2O2-induced injury in *Escherichia coli* appears to be mediated by •OH via Fenton reaction (Imlay and Linn, 1988; Imlay et al., 1988; Gutteridge et al., 1998). In the Fenton reaction *in vivo*, H2O2 is reduced to •OH radicals in the presence of ferrous (Fe II) or cuprous (Cu II) ions, according to the equation Me*n* +H2O2 = Me*n*+1 +•OH+OH−. Importantly however, the efficiency of this reaction increases dramatically in the presence of polyphenols (Cao et al., 1997; Sakihama et al., 2002).

Therefore, it appeared likely to us that hydroxyl radicals generated from hydrogen peroxide in the Fenton reaction rather than H2O2 are main cytotoxic agents that underlie honey antibacterial activity. However, there was no direct experimental evidence that (a) honey can generate hydroxyl radicals from H2O2 and (b) that H2O2-derived •OHs were responsible for bacterial growth and survival. In this study, we took advantage of 3- -(*p*aminophenyl) fluorescein (APF) as •OH trap to investigate generation of hydroxyl radicals during exposure of bacterial cultures to honey.

Since H2O2 is a common component in honeys, a byproduct of glucose oxidation by honeybee glucose oxidase, we also explored a possibility that generation of hydroxyl radicals from honey hydrogen peroxide represents a general mechanism by which honey affects bacterial growth. In support of such notion are the following facts: (a) honeys from different botanical and geographical origins have been shown to possess antibacterial activity and (b) at least in some honeys, this activity has been shown to be directed against both antibiotic-sensitive as well as multi-resistant bacteria. This putative mechanism against antibiotic-resistant bacteria may function in honeys derived from *Leptospermum* spp., (manuka), *Fagopyrum esculentum* (buckwheat),*Koompassia excelsa* (tualang), and honeydew honeys (Willix et al., 1992; Cooper et al., 2000, 2002a,b; French et al., 2005; Brudzynski and Lannigan, 2008; Blair et al., 2009; Tan et al., 2009; Majtan et al., 2010).

To provide a better insight into the involvement of hydroxyl radicals on bacterial growth, we have chosen several clinical isolates of methicillin-resistant *Staphylococcus aureus* (MRSA) and vancomycin-resistant *Enterococcus faecium* (VRE) and monitored simultaneously their growth (by absorbance) and •OH generation (by fluorescence) upon their incubation with active honeys. If the •OH generation proves to be implicated in the growth inhibition of standard bacteria (*E. coli* and *Bacillus subtilis*) as well as MRSA and VRE, then perhaps it could be concluded that honey action resembles that of antibiotics. In the latter case, the oxidative damage evoked by hydroxyl radicals has been suggested as a common mechanism for antibiotic-mediated cell death (Gutteridge et al., 1998; Kohanski et al., 2010).

#### **MATERIALS AND METHODS HONEYS**

Honeys were donated by beekeepers and included both commercial (pasteurized) and apiary (raw) samples. The list of honeys, their plant origin main physico-chemical characteristics is given in **Table 1**.

#### **BACTERIAL STRAINS**

Standard strains of *B. subtilis* (ATCC 6633) and *E. coli* (ATCC 14948) purchased from Thermo Fisher Scientific Remel Products (Lenexa, KS 66215, USA) were grown in Mueller–Hinton Broth (MHB; Difco Laboratories) overnight in a shaking water bath at 37˚C. Overnight cultures were diluted with broth to the equivalent of the 0.5 McFarland Standard.

Clinical isolates were obtained from the Clinical Microbiology Laboratory of the London Health Science Centre, London, ON, Canada, three strains of vancomycin-resistant *Enterococcus faecium* and four strains of MRSA. On receipt these were subcultured from swabs onto Mueller–Hinton II agar (Difco Laboratories). Originally the organisms were isolated and processed at the Department of Clinical Microbiology of the London Health Science Centres. An automated system (Vitek®, Biomérieux®) was used to identify bacterial isolates to genus and species and their susceptibility to antibiotics (**Table 2** and **3**).

#### **IDENTIFICATION OF CLINICAL ISOLATES AND CONFIRMATION OF ANTIBIOTIC RESISTANCE**

Isolates were identified to genus and species and their susceptibility to antibiotics was confirmed using an automated system (Vitek®, Biomérieux®). The presence of the *mec (A) gene*, *nuc gene*, and *van (A) and (B)* genes were determined by polymerase chain reaction.

#### **Table 1 | List of honeys.**

∗Hydrogen peroxide concentration was measured at honey dilution of 8× (25% v/v) and represents an average of three experimental trials, where each honey was tested in triplicate

#### **Table 2 | Susceptibility of clinical isolates to antibiotics.**

1, Beta-lactamase; 2, ampicilli; 3, benzylpenicillin; 4, ciprofloxacin; 5, clindamycin; 6, erythromycin; 7, gatifloxacin; 8, gentamicin; 9, gentamicin high level (synergy); 10, levofloxacin; 11, linezolid; 12, moxifloxacin; 13, nitrofurantoin; 14, oxacillin; 15, quinupristin/dalfopristin; 16, rifampicin; 17, streptomycin high level (synergy); 18, tetracycline; 19, trimethoprim/sulfamethoxazole; 20, vancomycin.

This work was conducted by the Clinical Microbiology Laboratory, London Health Sciences Centre, London, ON, Canada.

Reproducibility for each strain was determined at minimum two experiments and maximum three conducted in triplicate. MIC was determined by using a protocol as described in Methods according to the National Committee for Clinical Laboratory Standards (NCCLS, 1996).

The overnight cultures of each strain were diluted in MHB to obtain 108 cfu/ml using a 0.5 McFarland standard.

#### **POLYMERASE CHAIN REACTION**

The procedure for identification and confirmation for both MRSA and VRE were as follows: Nasal and perineal swabs were inoculated onto chromogenic media designed to identify possible MRSA and VRE isolates (Colorex VRE, Alere, Inc., London, ON, Canada; MRSASelectTM, Bio-Rad Redmond, WA, USA). Colonies suspected of being MRSA or VRE were confirmed using PCR. For MRSA confirmation the organism was assayed for the presence of the *mec (A)* gene and nuclease. Suspected VRE were assayed for

the presence of *van (A)* and *van (B)* genes. Sequences used were: MecA1: TGGCTATCGTGTCACAATCG (20 bases) MecA2: CTGGAACTTGTTGAGCAGAG (20 bases) Nuc1: GCGATTGATGGTGATACGGTT (21 bases) Nuc2: AGCCAAGCCTTGACGAACTAAAGC (24 bases) VanA1: GGGAAAACGACAATTGC (17 bases)

VanA2: GTACAATGCGGCCGTTA (17 bases)

VanB1: ATGGGAAGCCGATAGTC (17 bases)

VanB2: GATTTCGTTCCTCGACC (17 bases)

PCR was run according to a standard protocol that is used routinely in clinical microbiology laboratories.

#### **ANTIBACTERIAL ASSAY**

The antibacterial activity of honeys was performed using a broth microdilution assay in sterile,96-well microplates (Costar,Thermo Fisher Scientific, Canada) in compliance with requirements of NCCLS (1996). Wells in rows B to G contained 110μl of inoculated broth (106 cfu/ml final concentrations for each of microorganisms). Wells in row A were filled with 200μl of 50% solution of honey in sterile water and were inoculated with 20μl of 107 cfu/ml of bacterial culture. Serial twofold dilutions of honey were prepared by mixing and transferring 110μl of honey with 110μl of inoculated broth from row A to row H of a microplate. Row G contained only inoculum and served as a positive control and row H contained sterile MHB and served as a blank. In a single experiment, each honey was tested in triplicate.

After overnight incubation of plates at 37˚C in a shaking water bath, bacterial growth was measured at *A*595 nm using the Synergy HT multidetection microplate reader (Synergy HT, Bio-Tek Instruments, Winooski, VT, USA).

The contribution of color of honeys to the absorption was corrected by subtracting the absorbance of wells before (zero time) and after overnight incubation.

Statistical analysis and dose–response curves were obtained using K4 software provided by Synergy HT, Bio-Tek Instruments, Winooski, VT, USA.

Conversion of honey dilutions to honey concentrations (% v/v):

#### **DETERMINATION OF MICs**

The absorbance readings obtained from the dose–response curve were used to construct growth inhibition profiles (GIPs). The minimal inhibitory concentrations (MIC90) were determined from the GIPs curves and represented the lowest concentration of the honeys that inhibited the bacterial growth by 90% as measured by the absorbance at *A*595 nm.

QC/QA controls: estimation of the MICs was conducted only then when control bacterial strains in microdilution assay (row G) show sufficient growth. For each bacterial strain, 3–6 MICs were established in independent experiments. The obtained median MICs were the same or differed by± one doubling concentration.

#### **HYDROXY-RADICALS MEASUREMENTS**

3- -(*p*-Aminophenyl) fluorescein (APF; Invitrogen, Canada) was used for the detection of hydroxyl radicals produced by honeys. A broth microdilution assay in 96-well microplate format containing inoculum and twofold serially diluted honeys was adapted to assess both hydroxyl radicals generated and bacterial growth. To each well the APF solution was added to a final concentration of 10μM (in 50 mM potassium phosphate buffer, pH 7.4). The experimental wells (containing bacterial inoculum and honey dilutions) as well as the assay controls (bacterial inoculum) were supplemented with 10μM APF while the negative control consisted of experimental wells without APF.

Plates were incubated in a shaking water bath at 37˚C for 18 h. The plates were analyzed for both, bacterial growth using absorbance at *A*595 nm and hydroxyl radical generation using fluorescence excitation and emission wavelengths at 490 and 520 nm, respectively.

To measure hydroxyl radical generation in the presence of Cu ions, honeys were incubated with 400μM of CuCl2 dissolved in 50 mM potassium phosphate buffer, pH 7.4.

#### **HYDROGEN PEROXIDE ASSAY**

The hydrogen peroxide concentrations of the honeys were determined using the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit in 96-well microplate format according to the manufacturer manual (Molecular Probes, Invitrogen, Burlington, ON, Canada) and as described previously (Brudzynski et al., 2011). The Synergy HT multidetection microplate reader was used to measure the fluorescence formed during the reaction of honey's hydrogen peroxide with the Amplex Red reagent (10-acetyl-3,7 dihydroxyphenoxasine). The measurements were conducted at an emission wavelength of 590 nm, and an excitation wavelength of 530 nm. The standard curve constructed from the known concentration of H2O2 was used to calculate the hydrogen peroxide concentrations of the honeys. Each of the honey samples, and the standard curve, were tested in triplicate.

#### **CATALASE-TREATMENT OF HONEYS**

Honey were treated with catalase (13 800 U/mg solid; Sigma-Aldrich, Canada) at ratio of 1000 units per 1 ml of 50% honey solution in sterile water for 2 h at room temperature.

#### **RESULTS**

#### **BACTERIOSTATIC EFFECT OF HONEY AGAINST ANTIBIOTIC-RESISTANT CLINICAL ISOLATES**

From a large pool of over 200 honeys screened for their bacteriostatic activity against *E. coli* and *B. subtilis* (Brudzynski and Kim, 2011), we selected eleven honeys that showed MIC90 exceeding those of sugar solution (artificial honey). The list included six buckwheat honeys (H23, 58, 76, 77, 81, and 103), one clover honey (H11) and one blueberry honey (H15). The MIC90 of these honeys ranged from 6.25 to 12.5% (v/v), with exception of H15, with MIC90 of 25% (v/v). New Zealand Active Manuka honey (MIC90 6.25% v/v) was used as a reference (**Figure 1**).

These honeys were analyzed for their ability to inhibit growth of two vancomycin-resistant *Enterococcus faecium*,VRE 0 and VRE 2 and three strains of MRSA, MRSA 3, 4, and 5. **Figure 1** shows

that both VRE strains were susceptible to honeys. The median differences in honeys bacteriostatic activity varied only by twofold from the low MIC to the high MIC for each strains (MIC90 6.25 and 12.5% (v/v), respectively), with exception of honey H15. Both VRE strains displayed the same multi-drug resistant pattern (**Table 2**). There was no difference in MIC values of honeys against multi-drug resistant VRE strains and *E. coli* (**Figure 1**).

On the other hand, the susceptibility of the three strains of MRSA to honeys differed two- to four-fold ranging from MIC90 6.25–25% (v/v). MRSA 4 was the most resistant strain while MRSA 3 and 5 were more susceptible to different honeys. MRSA 4 was more resistant to honey action than *B. subtilis* (**Figure 2**).

The antibiograms performed on MRSA and VRE strains showed similar multi-resistance profiles to several classes of antibiotics (**Table 2**).

#### **CONFIRMATION OF THE PRESENCE OF RESISTANCE GENES IN CLINICAL ISOLATES**

These pathogens isolated from wounds were identified to the genus and species using the automated system Vitek II, Biomerieux. The presence or absence of antibiotic resistance genes [*van(A)*, *van(B)*, *mec(A)*, and *nuc*] was confirmed by the polymerase chain reaction. The presence of penicillin-binding protein (PBP2- ) in the *Staphylococcus aureus* strains were confirmed using an Oxoid agglutination kit.

Despite the multi-resistance pattern to antibiotics (**Table 2**), supported by a positive identification of the antibiotic-resistant genes (**Table 3**), all strains of MRSA and VRE were sensitive to honey bacteriostatic action (**Figures 1** and **2**). This observation together with the lack of significant differences in susceptibility between the antibiotic-resistant and standard bacteria suggested that bacteriostatic action of honey may proceeds via a common mechanism.

#### **DETECTION OF HYDROXYL RADICAL FORMATION FOLLOWING INCUBATION OF MRSA AND VRE WITH HONEYS**

In our most recent study, we have shown that hydrogen peroxide is implicated in bacterial growth inhibition but its inhibitory efficiency was modulated by unknown honey components (Brudzynski et al., 2011). We hypothesized that the formation of •OH from H2O2 rather than H2O2 itself may be directly implicated in bacterial growth inhibition.