Reprod Dom Anim 50 (Suppl. 2), 5–10 (2015); doi: 10.1111/rda.12553 ISSN 0936–6768
Review Article Challenges and Limits Using Antimicrobial Peptides in Boar Semen Preservation M Schulze1, M Grobbel2,3, K M€uller2, C Junkes4, M Dathe4, K R€ udiger1 and M Jung1 1 3
Institute for the Reproduction of Farm Animals Sch€ onow Inc., Bernau, Germany; 2Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany; Federal Institute for Risk Assessment, Berlin, Germany; 4Leibniz Institute of Molecular Pharmacology, Berlin, Germany
Contents Antibiotics are of great importance for the preservation of ejaculates for livestock breading. The use of antibiotics, however, is not an appropriate compensation for a lack of hygiene standards in artificial insemination (AI) centres. Sophisticated hygiene management and the proper identification of hygienic critical control points (HCCPs) at AI centres provide the basis for counteracting the development of antibiotic resistance in contaminant bacteria and their settlement in AI centres. In recent years, efforts have been made to use antimicrobial peptides (AMPs) in the preservation of boar semen. Investigations have included the testing of synthetic magainin derivatives and cyclic hexapeptides. One prerequisite for the application of AMPs is that they have a minor impact on eukaryotic cells. Bacterial selectivity, proteolytic stability, thermodynamic resistance, and mechanisms including synergistic interaction with conventional antibiotics have made cyclic hexapeptides highly promising candidates for potential application as peptide antibiotics for semen preservation.
Introduction Antimicrobial agents are of utmost importance to the liquid preservation of ejaculates from farm animals, both (i) to prevent the spread of epizootics such as brucellosis, chlamydiosis, or leptospirosis (Maes et al. 2008) and (ii) to prevent the growth of contaminant bacteria. To counteract increasing resistance to conventional antibiotics, novel antibiotic agents with different functional mechanisms must be developed. Increasing efforts have been made over the past few decades to design antimicrobial peptides (AMPs) as agents for clinical implementation; to date, the topical application of such agents has been the most successful approach (Hancock and Patrzykat 2002). For semen preservation, antibiotic additives must possess the following qualities: (i) broad-spectrum antimicrobial activity, (ii) absence of cytotoxicity, (iii) no interference with fertility, (iv) high stability, (v) ease of application, and (vi) low cost of production. Currently, more than 5500 natural and synthetic AMPs are known (Zhao et al. 2013). AMPs counteract multiple pathogens and display an impressive number of functions. In addition to their antimicrobial activity, AMPs participate in cell proliferation (Kamysz 2005), wound healing (Andreu and Rivas 1998), angiogenesis (Li et al. 2000), and reactions to acute inflammatory © 2015 Blackwell Verlag GmbH
responses (Levy 2000). AMPs are synthesized in mammalian organisms, particularly in the epithelial tissue of the skin (Gallo et al. 2002), the digestive tract (Zhang et al. 2000), the respiratory system (Boman 2003), and the reproductive tract (Frew and Stock 2011). In analogy to the defence mechanisms of lower organisms, higher organisms maintain an innate immune system, of which AMPs are important components. The variable biological activity of such peptides is based on their primary and secondary structure. However, cationic charge and amphipathicity are conserved characteristics that provide the basis for their selective effect on bacterial membranes that are rich in negatively charged lipids. Moreover, the outer membrane of gramnegative bacteria contains negatively charged lipopolysaccharides. Electrostatic and hydrophobic interactions between AMPs and the bacterial lipid matrix initiate a further sequence of bacteriotoxic effects (see below). The absence of anionic lipids and the high content of membrane cholesterol in eukaryotic membranes (which reduces their fluidity) prevent cytolytic and cytotoxic activity of AMPs against eukaryotic cells. However, the exposure of anionic sulfogalactosylglycerolipid on the sperm surface (Srakaew et al. 2014) and the comparatively low cholesterol content in the boar sperm membrane (Cross 1998) make sperm cells potentially susceptible to AMPs. Magainins, originally isolated from the African clawed frog Xenopus laevis, were even successfully applied as an intravaginal contraceptive (Reddy and Aranha 2000). The implementation of specifically designed AMPs as extender additives is a novel approach in artificial insemination (AI) management, but requires - in addition to analysing the AMPs’ antimicrobial activity - a careful examination of their compatibility with sperm.
Development of Antimicrobial Resistance After Routine Use of Antibiotics in Boar Semen Preservation National and international epizootic departments regulate the addition of antibiotics and their minimum concentrations (OIE 2013). In the European Union, regulations for international trade are outlined in Council Directive 90/429/EEC; however, some differences still
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M Schulze, M Grobbel, K M€ uller, C Junkes, M Dathe, K R€ udiger, and M Jung
exist among EU countries. In the last few years, there has been a tendency to use a cocktail of broad-spectrum, highly potent antibiotics in boar semen extenders (Morrell and Wallgren 2014). Still, 90% of Germany’s and Austria’s centres currently use only gentamicin as an extender additive (Schulze et al. 2015a). At the same time, between 2010 and 2011, in Germany and in Austria, gentamicin-resistant bacteria were isolated from extended semen in 26% (88/344) of AI doses and in 66.7% (18/24) of AI boar centres (Schulze et al. 2015a). A 2005 retrospective study in the USA found evidence of bacteriospermia in one-third of AI doses and, in 86% of these isolates, a further capacity for resistance to the antibiotics generally used in applied settings, such as amoxicillin, gentamicin, lincomycin, tylosin, and spectinomycin (Althouse and Lu 2005). Since the 1980s, penicillin–streptomycin combination antibiotics have had to be abandoned in diluents due to microbial resistance (Sone et al. 1982). Antibiotic resistance evolving in boar studs does not include all pathogenic agents and all substance groups uniformly (Althouse 2008), nor is the occurrence of resistant bacterial strains common across all studs (Schulze et al. 2015a). The majority of contaminants are gram-negative and attributable to the family Enterobacteriaceae (Ubeda et al. 2013). Species of the family Pseudomonadaceae also appear on a regular basis. Most of the latter are associated with the environment, but they are also opportunistic pathogens commonly related to nosocomial infections in humans and animals (Wallmann et al. 2003). According to Althouse et al. (2000), bacterial contaminants of boar semen are of both animal and non-animal origin. The majority of bacterial contamination occurs during the processing of ejaculates. During laboratory production, the hygienic critical control points (HCCPs) to be considered are the microbiological quality of the ultrapure water treatment plants, the inner surfaces of dilution tank lids, the manual operating elements, the laboratory surfaces, and the semen dyes used to label different boar breeds. The transportation of ejaculate and dumping of extenders and wasted ejaculates, respectively, are additional HCCPs (Schulze et al. 2015a). Extender fluids are perfect media for the growth of bacteria, especially when antibiotics lose their activity against various species of bacteria. Continued application of antibiotics in a specific medium facilitates unilateral selection pressure. Eliminating the susceptible bacterial flora from diluted ejaculates provides ‘growth niches’ for resistant bacteria. Factors of further potential influence on microbe content in diluted ejaculate include the composition of the extenders (Johnson et al. 2000), the duration of storage (Sone et al. 1992), the initial amount of bacteria in the native semen (Sone 1990), the rate of cooling (Althouse et al. 1998), and the temperature during preservation (Aurich and Spergser 2007). Rising temperatures during semen preservation lead to decreasing generational intervals for bacteria and bacterial counts (Appell and Evans 1978). The growth rate of
bacteria is affected by thermo-temporal dynamics and by temperature-dependent susceptibility to antibiotics (Althouse et al. 2008). In principle, initial proliferation should be precluded by immediate sperm processing; however, the relatively high storage temperature of 16–18°C favours the growth of mesophilic bacteria. Additional substances contained in extenders guarantee favourable growth conditions for bacteria through buffering oscillations of osmolarity and pH values. Toxic metabolic products of bacteria and spermatozoa are neutralized. The pH of diluted semen (6.8–7.2) supports the growth of most bacteria. Ultimately, resistant bacteria find perfect growth conditions in AI doses. Their participation in the regulation of bacterial density converts bacterial quorum-sensing components, which function as signal molecules, into bacterial messengers reporting the compensation of their division rate in relation to a limited or to an improved availability of nutritive substances (Gonzalez and Keshavan 2006). Particularly when using a ‘ready-to-use extender’, the practical effective concentration per AI dose differs greatly. This variation is caused by the variable dilution ratio, which results in a variable absolute quantity of antimicrobial substances. Therefore, future sperm production mandates the decoupling of antibiotic dosage from the dilution, resulting in a standardized final dosage (unpublished data). A standardized final dosage of antibiotics also has the advantage of problem-free waste disposal of the remaining extender; the extender would be free from antibiotics, and therefore these substances would not be discarded to the environment through the sewage system. Subinhibitory concentrations of antimicrobial substances, potentially achieved through suboptimal current dilution practices, are notorious for selecting for resistant bacteria and facilitating horizontal gene transfer, thereby contributing to the spread of resistance (Schulze et al. 2015a). Laboratory efflux systems thus form habitats for highly resistant biofilms of bacterial flora, spread via laboratory utensils. Several known causative agents of hospital-acquired infections, such as Burkholderia cepacia, Serratia marcescens, and Stenotrophomonas maltophilia, have repeatedly been isolated from extended boar semen (Althouse and Lu 2005; Schulze et al. 2015a). Problems with incompatibilities among extender components are also associated with the risk of breakdowns at critical HCCPs. Combinations of b-lactam and aminoglycoside antibiotics carry the potential for the deactivation of both components under specific conditions (Farchione 1981). Destabilizing agents such as ampicillin, for example, are susceptible to the high zinc concentrations (Beard et al. 1992) present in seminal plasma from boars (Massanyi et al. 2003). A reduction of the pH value to below 6.5 leads to strong decreases in gentamicin activity. Strong abatement in gentamicin activity also occurs in dextrose media (Graham et al. 1997). © 2015 Blackwell Verlag GmbH
Challenges and Limits Using Antimicrobial Peptides
Another criterion under investigation is the exact dosage of the extender. Currently, only 50% of AI centres work with precise doses of highly pure water to produce extenders (Schulze et al. 2015b). Finally, AI centres must maintain the proper storage of their extender powder. Suboptimal storage conditions, such as high temperatures, compromise the efficiency of extender components (unpublished data).
Antimicrobial Peptides - Characteristics of New Alternatives to Common Antibiotics AMPs share two characteristic properties. Most are highly cationic, and they are amphipathic, with their polar and hydrophobic amino acid residues located in separate structure surfaces. This spatial separation of cationic from hydrophobic surfaces is the essential prerequisite for the interaction of AMPs with bacterial membranes (Hancock and Rozek 2002). Coulomb interactions enhance binding of peptides to negatively charged head groups of lipids, such as phosphatidylglycerol or cardiolipin. Subsequently, hydrophobic interactions with the lipid acyl chains allow the peptides to penetrate into the hydrophobic region of lipid bilayers and to disturb the barrier function of the bacterial cell membrane. This membrane-permeabilizing mechanism is quite different to the mode of action of conventional antibiotics (Hancock and Chapple 1999). In addition, gram-negative bacteria have an outer membrane that is rich in strongly negatively charged lipopolysaccharides (LPSs). They consist of a lipophilic component, lipid A, and of a large hydrophilic region composed of polysaccharides (Junkes et al. 2008). Negatively charged phosphate groups are able to bind AMPs. AMPs self-capacitate by a so-called self-promoted uptake pathway necessary for crossing this LPS barrier and for penetrating the periplasmic space to reach the cytoplasmic membrane (Piers et al. 1994). The presence of anionic lipids and the lack of cholesterol are considered essential factors in AMP selectivity for prokaryotic membranes (Nikaido 2003). Permeabilization of the bacterial cytoplasmic membrane leads to destruction of the membrane potential, and exchange of extra- and intracellular compounds finally results in cell death (Wu et al. 1999). Several peptides are also able to translocate into the cytoplasm and to interfere with intracellular processes via binding to receptor molecules, including the induction of autolytic enzymes and the blocking of protein, DNA and cell wall synthesis. Other findings point to the reduction of membrane fluidity with consequences for the function of division proteins (Zhang and Falla 2004). In contrast to prokaryotic membranes, the negatively charged lipids of eukaryotic membranes are located mainly in the inner layer of the cell membrane, oriented towards the cytoplasm (Op den Kamp 1979). The outer layer of eukaryotic cell membranes, including sperm membranes, contains in particular neutral phospholipids, such as phosphatidylcholine, sphingomyelin, and © 2015 Blackwell Verlag GmbH
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phosphatidylethanolamine. Thus, electrostatic interactions with AMPs are minor.
The Use of Antimicrobial Peptides in Boar Semen Preservation In 1987, Zasloff isolated a group of AMPs from the mucous glands of the African clawed frog Xenopus laevis. These antimicrobial agents were defined as magainins. Magainins are linear a-helical peptides consisting of 23 to 27 amino acids. Even low micromolar concentrations exert high activity against a multitude of bacterial and fungal species (Zasloff 2002). The interaction of magainins with gram-negative bacterial membranes has been well investigated. The amphipathic helix of magainins binds with high affinity to LPS and the cytoplasmic membrane. Membrane depolarization and destabilization of the prokaryotic membrane by pore formation has been suggested to ultimately result in the lysis of the target cells (Dathe and Wieprecht 1999). For a long time, research aimed at increasing the antimicrobial effectiveness of peptides was focused on linear, potentially helix-forming peptides (Dathe et al. 2002). Small peptide sequences rich in specific amino acids, such as proline (P), phenylalanine (F), arginine (R), and tryptophan (W), occur as antimicrobial patterns in large natural proteins. R- and W-rich hexapeptides derived from a combinatorial library belong to this particular group of peptides (Blondelle et al. 1995). Cyclization of such hexapeptides led to the highly antimicrobial analogues c-RRWWWR (c-WWW) and c-RRWFWR (c-WFW) with three adjacent cationic and hydrophobic residues. Low enzymatic digestibility and modest size, limiting synthesis costs and a risk of immunogenicity are further characteristics of these AMPs (Wessolowski et al. 2004). Standardized synthesis procedures allow the rapid preparation of these AMPs with relatively high yield. The sequence of these synthetic peptides is relatively easy to modify, compared with those produced by recombinant techniques, and thus, their biological activity can be optimized (Chan et al. 2006). The antimicrobial effect is based upon the primary amphipathicity of the hexapeptides - three adjacent cationic and hydrophobic residues. These peptides strongly interact with the lipid matrix of bacterial cells. As shown on a model membrane level, the peptides induce lipid demixing (Arouri et al. 2009) and thus likely destroy the phospholipid organization of the bacterial membrane, which is related to local membrane destabilization (Junkes et al. 2011). Despite the urgent need to find alternatives to conventional antibiotics in boar semen extenders, reports on potential novel antimicrobials have been scarce. An investigation was performed on the impact of the two cyclic hexapeptides, c-WWW and c-WFW, and of the helical magainin II amide analogue MK5E on boar sperm quality during storage at 16°C for 4 days
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M Schulze, M Grobbel, K M€ uller, C Junkes, M Dathe, K R€ udiger, and M Jung
(Schulze et al. 2014). The study revealed peptide- and concentration-dependent negative effects on membrane integrity and motility in boar spermatozoa. These negative effects were far more pronounced for the magainin analogue MK5E than for the hexapeptides. The application of magainins did not provide the anticipated results. In boar semen, particular magainin derivatives led to irreversible membrane fluidization. In contrast, cyclic hexapeptides were partly able to stimulate linearly progressive sperm movement. In the presence of low concentrations of the cyclic hexapeptides, the sperm quality was comparable to that determined in standard extender over the course of preservation. C-WFW-supplemented boar semen resulted in normal fertility rates after AI. A second investigation addressed the suitability of the AMPs as an alternative to conventional antibiotics in liquid boar semen preservation (Speck et al. 2014). The antibacterial activity of c-WWW, c-WFW, and MK5E was studied in vitro with two gram-positive and eleven gram-negative bacteria. Isolates included ATCC reference strains, multiresistant E. coli, and bacteria cultured from boar semen. Using broth microdilution, the minimum inhibitory concentration (MIC) was determined for all AMPs. All AMPs revealed reactivity to the majority of bacteria, but not to Proteus spp. (all AMPs) or Staphylococcus aureus ATCC 29213 (MK5E), both of which are potential contaminants in raw boar semen. When tested in liquid-preserved boar semen in situ, hexapeptides effectively inhibited bacterial growth, especially in combination with a small amount of gentamicin. It has been shown that the presence of NaHCO3 significantly enhances the activity of AMPs against bacteria, and AMP activity depends on an ionic milieu comparable to that in mammalian body fluids (Dorschner et al. 2006). The standard BTS extenders contain NaHCO3 in addition to other components used to preserve sperm metabolic activity, such as EDTA (Johnson et al. 2000); the presence of NaHCO3 might explain the improved antimicrobial action of hexapeptides during sperm preservation. Our recent investigations revealed an additional protective ability of AMPs to neutralize LPS. It is well known that gram-negative bacteria release LPS, which acts as an endotoxin and exerts spermicidal activity (Hakimi et al. 2006). A recent study showed that LPS bound directly to the head region of porcine spermatozoa, decreased sperm motility, and induced sperm apoptosis (Okazaki et al. 2010). We found that LPS quantities were differentially neutralized by different peptides depending on the peptide concentration (Fig. 1). Adding 50 lM of c-WFW or c-WWW almost completely neutralized 50 EU per ml E. coli O55:B5 (Lonza, K€ oln, Germany). In contrast, gentamicin was unable to neutralize the pyrogenic activity of LPS at a concentration of 544 lM (250 lg/ml), which is the concentration typically used for boar semen preservation purposes. The linear hexapeptide lin-WWW (Dathe et al. 2004) is equally efficient to c-WWW in neutraliz-
Fig. 1. Potential for LPS neutralization (%) of different peptides and gentamicin (n = 4). Peptides were provided by Biosyntan GmbH, Berlin, Germany, and synthesized, purified, and characterized as described elsewhere (Dathe et al. 2002; Junkes et al. 2011). The peptides were dissolved in diluted HCL, lyophilized, and stored at 20°C. The day before use, stock solutions of different peptides were prepared in A. bidest. and stored at 4°C. For the experiments, 200 ll peptide samples (5 lM and 50 lM) were incubated with 50 EU/ml endotoxin (E. coli O55:B5, Lonza, K€ oln, Germany) at 23°C for 30 min. Following the incubation, a 100 ll sample volume was measured with the LAL-Kinetic-QCL test kit (Lonza) according to the manufacturer’s specifications.
ing LPS, although the antimicrobial effect was much weaker than that of the cyclic peptide. The a-helical peptides MK5E (Schulze et al. 2014) and KLA (Dathe et al. 2002), as well as the control peptide polymyxin PMX (Junkes et al. 2008), were also effective. The results suggest that AMPs, although not a complete alternative to traditional antibiotics, represent a promising solution for decreasing the use of conventional antibiotics and thereby limiting the selection of multiresistant strains (Speck et al. 2014). Testing the AMPs will require the implementation of validated standardized methods for in vitro and in situ studies. Research should aim to achieve a further reduction of sperm toxicity and a potential exploitation of AMPs’ synergistic effects with other conventional antibiotics. Moreover, peptide stability in different boar semen extenders and at different storage temperatures must be addressed. From a practical point of view, the tested AMPs are safe for laboratory personnel to handle in concentrated form. AMPs can be provided as a powder or in liquid form and are easily dissolved in water. Unfortunately, the production of these AMPs is currently too expensive for routine use.
Conclusions Antibiotics are mandatory components of semen extenders for the control of bacterial contamination and © 2015 Blackwell Verlag GmbH
Challenges and Limits Using Antimicrobial Peptides
growth. The increasing rate of worldwide resistance to conventional antibiotics in boar semen extenders requires the development of alternative processing strategies and alternatives to common antibiotics. Therefore, cationic antimicrobial peptides are of great interest as a novel class of antimicrobial additives in boar semen preservation.
The authors’ studies underlying this review were supported by IFN Sch€ onow e.V. (Germany), the Association for Biotechnology Research
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(FBF, Germany), and the AIF PRO INNO II: KF0140502MD6 (Germany). The authors thank American Journal Experts (AJE) for language editing.
Conflicts of interest None of the authors have any conflicts of interest to declare.
Author contributions
Acknowledgements
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Submitted: 4 Feb 2015; Accepted: 10 May 2015 Author’s address (for correspondence): M Schulze, Institute for Reproduction of Farm Animals Sch€ onow e. V., Bernauer Allee 10, D16321 Bernau, Germany. E-mail: m.schulze @ifn-schoenow.de
© 2015 Blackwell Verlag GmbH
Review Article Challenges and Limits Using Antimicrobial Peptides in Boar Semen Preservation M Schulze1, M Grobbel2,3, K M€uller2, C Junkes4, M Dathe4, K R€ udiger1 and M Jung1 1 3
Institute for the Reproduction of Farm Animals Sch€ onow Inc., Bernau, Germany; 2Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany; Federal Institute for Risk Assessment, Berlin, Germany; 4Leibniz Institute of Molecular Pharmacology, Berlin, Germany
Contents Antibiotics are of great importance for the preservation of ejaculates for livestock breading. The use of antibiotics, however, is not an appropriate compensation for a lack of hygiene standards in artificial insemination (AI) centres. Sophisticated hygiene management and the proper identification of hygienic critical control points (HCCPs) at AI centres provide the basis for counteracting the development of antibiotic resistance in contaminant bacteria and their settlement in AI centres. In recent years, efforts have been made to use antimicrobial peptides (AMPs) in the preservation of boar semen. Investigations have included the testing of synthetic magainin derivatives and cyclic hexapeptides. One prerequisite for the application of AMPs is that they have a minor impact on eukaryotic cells. Bacterial selectivity, proteolytic stability, thermodynamic resistance, and mechanisms including synergistic interaction with conventional antibiotics have made cyclic hexapeptides highly promising candidates for potential application as peptide antibiotics for semen preservation.
Introduction Antimicrobial agents are of utmost importance to the liquid preservation of ejaculates from farm animals, both (i) to prevent the spread of epizootics such as brucellosis, chlamydiosis, or leptospirosis (Maes et al. 2008) and (ii) to prevent the growth of contaminant bacteria. To counteract increasing resistance to conventional antibiotics, novel antibiotic agents with different functional mechanisms must be developed. Increasing efforts have been made over the past few decades to design antimicrobial peptides (AMPs) as agents for clinical implementation; to date, the topical application of such agents has been the most successful approach (Hancock and Patrzykat 2002). For semen preservation, antibiotic additives must possess the following qualities: (i) broad-spectrum antimicrobial activity, (ii) absence of cytotoxicity, (iii) no interference with fertility, (iv) high stability, (v) ease of application, and (vi) low cost of production. Currently, more than 5500 natural and synthetic AMPs are known (Zhao et al. 2013). AMPs counteract multiple pathogens and display an impressive number of functions. In addition to their antimicrobial activity, AMPs participate in cell proliferation (Kamysz 2005), wound healing (Andreu and Rivas 1998), angiogenesis (Li et al. 2000), and reactions to acute inflammatory © 2015 Blackwell Verlag GmbH
responses (Levy 2000). AMPs are synthesized in mammalian organisms, particularly in the epithelial tissue of the skin (Gallo et al. 2002), the digestive tract (Zhang et al. 2000), the respiratory system (Boman 2003), and the reproductive tract (Frew and Stock 2011). In analogy to the defence mechanisms of lower organisms, higher organisms maintain an innate immune system, of which AMPs are important components. The variable biological activity of such peptides is based on their primary and secondary structure. However, cationic charge and amphipathicity are conserved characteristics that provide the basis for their selective effect on bacterial membranes that are rich in negatively charged lipids. Moreover, the outer membrane of gramnegative bacteria contains negatively charged lipopolysaccharides. Electrostatic and hydrophobic interactions between AMPs and the bacterial lipid matrix initiate a further sequence of bacteriotoxic effects (see below). The absence of anionic lipids and the high content of membrane cholesterol in eukaryotic membranes (which reduces their fluidity) prevent cytolytic and cytotoxic activity of AMPs against eukaryotic cells. However, the exposure of anionic sulfogalactosylglycerolipid on the sperm surface (Srakaew et al. 2014) and the comparatively low cholesterol content in the boar sperm membrane (Cross 1998) make sperm cells potentially susceptible to AMPs. Magainins, originally isolated from the African clawed frog Xenopus laevis, were even successfully applied as an intravaginal contraceptive (Reddy and Aranha 2000). The implementation of specifically designed AMPs as extender additives is a novel approach in artificial insemination (AI) management, but requires - in addition to analysing the AMPs’ antimicrobial activity - a careful examination of their compatibility with sperm.
Development of Antimicrobial Resistance After Routine Use of Antibiotics in Boar Semen Preservation National and international epizootic departments regulate the addition of antibiotics and their minimum concentrations (OIE 2013). In the European Union, regulations for international trade are outlined in Council Directive 90/429/EEC; however, some differences still
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M Schulze, M Grobbel, K M€ uller, C Junkes, M Dathe, K R€ udiger, and M Jung
exist among EU countries. In the last few years, there has been a tendency to use a cocktail of broad-spectrum, highly potent antibiotics in boar semen extenders (Morrell and Wallgren 2014). Still, 90% of Germany’s and Austria’s centres currently use only gentamicin as an extender additive (Schulze et al. 2015a). At the same time, between 2010 and 2011, in Germany and in Austria, gentamicin-resistant bacteria were isolated from extended semen in 26% (88/344) of AI doses and in 66.7% (18/24) of AI boar centres (Schulze et al. 2015a). A 2005 retrospective study in the USA found evidence of bacteriospermia in one-third of AI doses and, in 86% of these isolates, a further capacity for resistance to the antibiotics generally used in applied settings, such as amoxicillin, gentamicin, lincomycin, tylosin, and spectinomycin (Althouse and Lu 2005). Since the 1980s, penicillin–streptomycin combination antibiotics have had to be abandoned in diluents due to microbial resistance (Sone et al. 1982). Antibiotic resistance evolving in boar studs does not include all pathogenic agents and all substance groups uniformly (Althouse 2008), nor is the occurrence of resistant bacterial strains common across all studs (Schulze et al. 2015a). The majority of contaminants are gram-negative and attributable to the family Enterobacteriaceae (Ubeda et al. 2013). Species of the family Pseudomonadaceae also appear on a regular basis. Most of the latter are associated with the environment, but they are also opportunistic pathogens commonly related to nosocomial infections in humans and animals (Wallmann et al. 2003). According to Althouse et al. (2000), bacterial contaminants of boar semen are of both animal and non-animal origin. The majority of bacterial contamination occurs during the processing of ejaculates. During laboratory production, the hygienic critical control points (HCCPs) to be considered are the microbiological quality of the ultrapure water treatment plants, the inner surfaces of dilution tank lids, the manual operating elements, the laboratory surfaces, and the semen dyes used to label different boar breeds. The transportation of ejaculate and dumping of extenders and wasted ejaculates, respectively, are additional HCCPs (Schulze et al. 2015a). Extender fluids are perfect media for the growth of bacteria, especially when antibiotics lose their activity against various species of bacteria. Continued application of antibiotics in a specific medium facilitates unilateral selection pressure. Eliminating the susceptible bacterial flora from diluted ejaculates provides ‘growth niches’ for resistant bacteria. Factors of further potential influence on microbe content in diluted ejaculate include the composition of the extenders (Johnson et al. 2000), the duration of storage (Sone et al. 1992), the initial amount of bacteria in the native semen (Sone 1990), the rate of cooling (Althouse et al. 1998), and the temperature during preservation (Aurich and Spergser 2007). Rising temperatures during semen preservation lead to decreasing generational intervals for bacteria and bacterial counts (Appell and Evans 1978). The growth rate of
bacteria is affected by thermo-temporal dynamics and by temperature-dependent susceptibility to antibiotics (Althouse et al. 2008). In principle, initial proliferation should be precluded by immediate sperm processing; however, the relatively high storage temperature of 16–18°C favours the growth of mesophilic bacteria. Additional substances contained in extenders guarantee favourable growth conditions for bacteria through buffering oscillations of osmolarity and pH values. Toxic metabolic products of bacteria and spermatozoa are neutralized. The pH of diluted semen (6.8–7.2) supports the growth of most bacteria. Ultimately, resistant bacteria find perfect growth conditions in AI doses. Their participation in the regulation of bacterial density converts bacterial quorum-sensing components, which function as signal molecules, into bacterial messengers reporting the compensation of their division rate in relation to a limited or to an improved availability of nutritive substances (Gonzalez and Keshavan 2006). Particularly when using a ‘ready-to-use extender’, the practical effective concentration per AI dose differs greatly. This variation is caused by the variable dilution ratio, which results in a variable absolute quantity of antimicrobial substances. Therefore, future sperm production mandates the decoupling of antibiotic dosage from the dilution, resulting in a standardized final dosage (unpublished data). A standardized final dosage of antibiotics also has the advantage of problem-free waste disposal of the remaining extender; the extender would be free from antibiotics, and therefore these substances would not be discarded to the environment through the sewage system. Subinhibitory concentrations of antimicrobial substances, potentially achieved through suboptimal current dilution practices, are notorious for selecting for resistant bacteria and facilitating horizontal gene transfer, thereby contributing to the spread of resistance (Schulze et al. 2015a). Laboratory efflux systems thus form habitats for highly resistant biofilms of bacterial flora, spread via laboratory utensils. Several known causative agents of hospital-acquired infections, such as Burkholderia cepacia, Serratia marcescens, and Stenotrophomonas maltophilia, have repeatedly been isolated from extended boar semen (Althouse and Lu 2005; Schulze et al. 2015a). Problems with incompatibilities among extender components are also associated with the risk of breakdowns at critical HCCPs. Combinations of b-lactam and aminoglycoside antibiotics carry the potential for the deactivation of both components under specific conditions (Farchione 1981). Destabilizing agents such as ampicillin, for example, are susceptible to the high zinc concentrations (Beard et al. 1992) present in seminal plasma from boars (Massanyi et al. 2003). A reduction of the pH value to below 6.5 leads to strong decreases in gentamicin activity. Strong abatement in gentamicin activity also occurs in dextrose media (Graham et al. 1997). © 2015 Blackwell Verlag GmbH
Challenges and Limits Using Antimicrobial Peptides
Another criterion under investigation is the exact dosage of the extender. Currently, only 50% of AI centres work with precise doses of highly pure water to produce extenders (Schulze et al. 2015b). Finally, AI centres must maintain the proper storage of their extender powder. Suboptimal storage conditions, such as high temperatures, compromise the efficiency of extender components (unpublished data).
Antimicrobial Peptides - Characteristics of New Alternatives to Common Antibiotics AMPs share two characteristic properties. Most are highly cationic, and they are amphipathic, with their polar and hydrophobic amino acid residues located in separate structure surfaces. This spatial separation of cationic from hydrophobic surfaces is the essential prerequisite for the interaction of AMPs with bacterial membranes (Hancock and Rozek 2002). Coulomb interactions enhance binding of peptides to negatively charged head groups of lipids, such as phosphatidylglycerol or cardiolipin. Subsequently, hydrophobic interactions with the lipid acyl chains allow the peptides to penetrate into the hydrophobic region of lipid bilayers and to disturb the barrier function of the bacterial cell membrane. This membrane-permeabilizing mechanism is quite different to the mode of action of conventional antibiotics (Hancock and Chapple 1999). In addition, gram-negative bacteria have an outer membrane that is rich in strongly negatively charged lipopolysaccharides (LPSs). They consist of a lipophilic component, lipid A, and of a large hydrophilic region composed of polysaccharides (Junkes et al. 2008). Negatively charged phosphate groups are able to bind AMPs. AMPs self-capacitate by a so-called self-promoted uptake pathway necessary for crossing this LPS barrier and for penetrating the periplasmic space to reach the cytoplasmic membrane (Piers et al. 1994). The presence of anionic lipids and the lack of cholesterol are considered essential factors in AMP selectivity for prokaryotic membranes (Nikaido 2003). Permeabilization of the bacterial cytoplasmic membrane leads to destruction of the membrane potential, and exchange of extra- and intracellular compounds finally results in cell death (Wu et al. 1999). Several peptides are also able to translocate into the cytoplasm and to interfere with intracellular processes via binding to receptor molecules, including the induction of autolytic enzymes and the blocking of protein, DNA and cell wall synthesis. Other findings point to the reduction of membrane fluidity with consequences for the function of division proteins (Zhang and Falla 2004). In contrast to prokaryotic membranes, the negatively charged lipids of eukaryotic membranes are located mainly in the inner layer of the cell membrane, oriented towards the cytoplasm (Op den Kamp 1979). The outer layer of eukaryotic cell membranes, including sperm membranes, contains in particular neutral phospholipids, such as phosphatidylcholine, sphingomyelin, and © 2015 Blackwell Verlag GmbH
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phosphatidylethanolamine. Thus, electrostatic interactions with AMPs are minor.
The Use of Antimicrobial Peptides in Boar Semen Preservation In 1987, Zasloff isolated a group of AMPs from the mucous glands of the African clawed frog Xenopus laevis. These antimicrobial agents were defined as magainins. Magainins are linear a-helical peptides consisting of 23 to 27 amino acids. Even low micromolar concentrations exert high activity against a multitude of bacterial and fungal species (Zasloff 2002). The interaction of magainins with gram-negative bacterial membranes has been well investigated. The amphipathic helix of magainins binds with high affinity to LPS and the cytoplasmic membrane. Membrane depolarization and destabilization of the prokaryotic membrane by pore formation has been suggested to ultimately result in the lysis of the target cells (Dathe and Wieprecht 1999). For a long time, research aimed at increasing the antimicrobial effectiveness of peptides was focused on linear, potentially helix-forming peptides (Dathe et al. 2002). Small peptide sequences rich in specific amino acids, such as proline (P), phenylalanine (F), arginine (R), and tryptophan (W), occur as antimicrobial patterns in large natural proteins. R- and W-rich hexapeptides derived from a combinatorial library belong to this particular group of peptides (Blondelle et al. 1995). Cyclization of such hexapeptides led to the highly antimicrobial analogues c-RRWWWR (c-WWW) and c-RRWFWR (c-WFW) with three adjacent cationic and hydrophobic residues. Low enzymatic digestibility and modest size, limiting synthesis costs and a risk of immunogenicity are further characteristics of these AMPs (Wessolowski et al. 2004). Standardized synthesis procedures allow the rapid preparation of these AMPs with relatively high yield. The sequence of these synthetic peptides is relatively easy to modify, compared with those produced by recombinant techniques, and thus, their biological activity can be optimized (Chan et al. 2006). The antimicrobial effect is based upon the primary amphipathicity of the hexapeptides - three adjacent cationic and hydrophobic residues. These peptides strongly interact with the lipid matrix of bacterial cells. As shown on a model membrane level, the peptides induce lipid demixing (Arouri et al. 2009) and thus likely destroy the phospholipid organization of the bacterial membrane, which is related to local membrane destabilization (Junkes et al. 2011). Despite the urgent need to find alternatives to conventional antibiotics in boar semen extenders, reports on potential novel antimicrobials have been scarce. An investigation was performed on the impact of the two cyclic hexapeptides, c-WWW and c-WFW, and of the helical magainin II amide analogue MK5E on boar sperm quality during storage at 16°C for 4 days
8
M Schulze, M Grobbel, K M€ uller, C Junkes, M Dathe, K R€ udiger, and M Jung
(Schulze et al. 2014). The study revealed peptide- and concentration-dependent negative effects on membrane integrity and motility in boar spermatozoa. These negative effects were far more pronounced for the magainin analogue MK5E than for the hexapeptides. The application of magainins did not provide the anticipated results. In boar semen, particular magainin derivatives led to irreversible membrane fluidization. In contrast, cyclic hexapeptides were partly able to stimulate linearly progressive sperm movement. In the presence of low concentrations of the cyclic hexapeptides, the sperm quality was comparable to that determined in standard extender over the course of preservation. C-WFW-supplemented boar semen resulted in normal fertility rates after AI. A second investigation addressed the suitability of the AMPs as an alternative to conventional antibiotics in liquid boar semen preservation (Speck et al. 2014). The antibacterial activity of c-WWW, c-WFW, and MK5E was studied in vitro with two gram-positive and eleven gram-negative bacteria. Isolates included ATCC reference strains, multiresistant E. coli, and bacteria cultured from boar semen. Using broth microdilution, the minimum inhibitory concentration (MIC) was determined for all AMPs. All AMPs revealed reactivity to the majority of bacteria, but not to Proteus spp. (all AMPs) or Staphylococcus aureus ATCC 29213 (MK5E), both of which are potential contaminants in raw boar semen. When tested in liquid-preserved boar semen in situ, hexapeptides effectively inhibited bacterial growth, especially in combination with a small amount of gentamicin. It has been shown that the presence of NaHCO3 significantly enhances the activity of AMPs against bacteria, and AMP activity depends on an ionic milieu comparable to that in mammalian body fluids (Dorschner et al. 2006). The standard BTS extenders contain NaHCO3 in addition to other components used to preserve sperm metabolic activity, such as EDTA (Johnson et al. 2000); the presence of NaHCO3 might explain the improved antimicrobial action of hexapeptides during sperm preservation. Our recent investigations revealed an additional protective ability of AMPs to neutralize LPS. It is well known that gram-negative bacteria release LPS, which acts as an endotoxin and exerts spermicidal activity (Hakimi et al. 2006). A recent study showed that LPS bound directly to the head region of porcine spermatozoa, decreased sperm motility, and induced sperm apoptosis (Okazaki et al. 2010). We found that LPS quantities were differentially neutralized by different peptides depending on the peptide concentration (Fig. 1). Adding 50 lM of c-WFW or c-WWW almost completely neutralized 50 EU per ml E. coli O55:B5 (Lonza, K€ oln, Germany). In contrast, gentamicin was unable to neutralize the pyrogenic activity of LPS at a concentration of 544 lM (250 lg/ml), which is the concentration typically used for boar semen preservation purposes. The linear hexapeptide lin-WWW (Dathe et al. 2004) is equally efficient to c-WWW in neutraliz-
Fig. 1. Potential for LPS neutralization (%) of different peptides and gentamicin (n = 4). Peptides were provided by Biosyntan GmbH, Berlin, Germany, and synthesized, purified, and characterized as described elsewhere (Dathe et al. 2002; Junkes et al. 2011). The peptides were dissolved in diluted HCL, lyophilized, and stored at 20°C. The day before use, stock solutions of different peptides were prepared in A. bidest. and stored at 4°C. For the experiments, 200 ll peptide samples (5 lM and 50 lM) were incubated with 50 EU/ml endotoxin (E. coli O55:B5, Lonza, K€ oln, Germany) at 23°C for 30 min. Following the incubation, a 100 ll sample volume was measured with the LAL-Kinetic-QCL test kit (Lonza) according to the manufacturer’s specifications.
ing LPS, although the antimicrobial effect was much weaker than that of the cyclic peptide. The a-helical peptides MK5E (Schulze et al. 2014) and KLA (Dathe et al. 2002), as well as the control peptide polymyxin PMX (Junkes et al. 2008), were also effective. The results suggest that AMPs, although not a complete alternative to traditional antibiotics, represent a promising solution for decreasing the use of conventional antibiotics and thereby limiting the selection of multiresistant strains (Speck et al. 2014). Testing the AMPs will require the implementation of validated standardized methods for in vitro and in situ studies. Research should aim to achieve a further reduction of sperm toxicity and a potential exploitation of AMPs’ synergistic effects with other conventional antibiotics. Moreover, peptide stability in different boar semen extenders and at different storage temperatures must be addressed. From a practical point of view, the tested AMPs are safe for laboratory personnel to handle in concentrated form. AMPs can be provided as a powder or in liquid form and are easily dissolved in water. Unfortunately, the production of these AMPs is currently too expensive for routine use.
Conclusions Antibiotics are mandatory components of semen extenders for the control of bacterial contamination and © 2015 Blackwell Verlag GmbH
Challenges and Limits Using Antimicrobial Peptides
growth. The increasing rate of worldwide resistance to conventional antibiotics in boar semen extenders requires the development of alternative processing strategies and alternatives to common antibiotics. Therefore, cationic antimicrobial peptides are of great interest as a novel class of antimicrobial additives in boar semen preservation.
The authors’ studies underlying this review were supported by IFN Sch€ onow e.V. (Germany), the Association for Biotechnology Research
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(FBF, Germany), and the AIF PRO INNO II: KF0140502MD6 (Germany). The authors thank American Journal Experts (AJE) for language editing.
Conflicts of interest None of the authors have any conflicts of interest to declare.
Author contributions
Acknowledgements
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Submitted: 4 Feb 2015; Accepted: 10 May 2015 Author’s address (for correspondence): M Schulze, Institute for Reproduction of Farm Animals Sch€ onow e. V., Bernauer Allee 10, D16321 Bernau, Germany. E-mail: m.schulze @ifn-schoenow.de
© 2015 Blackwell Verlag GmbH
