Next Article in Journal
Photocatalytic Inactivation of Viruses and Prions: Multilevel Approach with Other Disinfectants
Next Article in Special Issue
Whole Genome Sequencing Evidences High Rates of Relapse in Clostridioides difficile Infection Caused by the Epidemic Ribotype 106
Previous Article in Journal
Surfactin Bacterial Antiviral Lipopeptide Blocks In Vitro Replication of SARS-CoV-2
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Applied Microbiology
Volume 2
Issue 4
10.3390/applmicrobiol2040053
applmicrobiol-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Selected Antimicrobial Peptides Inhibit In Vitro Growth of Campylobacter spp.

by
John Eric Line
1,*,
Bruce S. Seal
2 and
Johnna K. Garrish
1
1
United States Department of Agriculture, Agricultural Research Service, U.S. National Poultry Research Center, Athens, GA 30605, USA
2
Biology Program, Oregon State University Cascades, Bend, OR 97702, USA
*
Author to whom correspondence should be addressed.
Appl. Microbiol. 2022, 2(4), 688-700; https://doi.org/10.3390/applmicrobiol2040053
Submission received: 1 September 2022 / Revised: 15 September 2022 / Accepted: 18 September 2022 / Published: 21 September 2022
(This article belongs to the Special Issue Exclusive Papers Collection of Editorial Board Members and Invited Scholars in Applied Microbiology)
Browse Figure
Versions Notes

Abstract

:
Campylobacter is a major cause of acute human diarrheal illness. Broiler chickens constitute a primary reservoir for C. jejuni leading to human infection. Consequently, there is a need for developing novel intervention methods. Antimicrobial peptides (AMP) are small proteins which have evolved in most lifeforms to provide defense against microbial infections. To date, over 3000 AMP have been discovered; however, few of them have been analyzed specifically for ability to kill campylobacters. We selected and evaluated a set of 11 unique chemically synthesized AMP for ability to inhibit growth of C. jejuni. Six of the AMP we tested produced zones of inhibition on lawns of C. jejuni. These AMP included: NRC-13, RL-37, Temporin L, Cecropin–Magainin, Dermaseptin, and C12K-2β12. In addition, MIC were determined for Cecropin–Magainin, RL-37 and C12K-2β12 against 15 isolates of Campylobacter representing the three most common pathogenic strains. MIC for campylobacters were approximately 3.1 µg/mL for AMP RL-37 and C12K-2β12. MIC were slightly higher for the Cecropin–Magainin AMP in the range of 12.5 to 100 µg/mL. These AMP are attractive subjects for future study and potential in vivo delivery to poultry to reduce Campylobacter spp. populations.

1. Introduction

Antimicrobial peptides (AMP) are small proteins which have been found in almost every class of living organism where they have evolved as a host-defense mechanism against invading microorganisms [1]. For this reason, they are sometimes referred to as host-defense peptides and are considered key players in innate immunity [2]. AMP represent a unique and diverse group of molecules which can be divided into subgroups on the basis of their amino acid composition and structure [3]. They are generally short, 10–40 amino acids in length, cationic, amphipathic, and can be arbitrarily categorized according to their secondary structure as alpha-helices, beta-sheet, extended helices, and loops [4]. A growing number of AMP are being discovered and synthetic new ones are being designed thanks to tremendous research efforts in response to the dramatic and continued evolution of antibacterial-resistant strains of bacteria and the resulting international crisis in health care [1]. For example, a 2004 database contained 523 known peptides, whereas a 2013 database held 3904 natural and 1643 synthetic ones [5,6]. More recently, a 2021 database contains 22,259 entries which include 5891 general AMP, 16,110 patent AMP, and 77 AMP currently in preclinical or clinical stages of drug development [7]. While it is reasonable to expect that some of the AMP that have been discovered or designed de novo to kill a variety of Gram (−) bacteria might also inhibit G (−) Campylobacter spp., there have been very few reports in the literature describing effects of AMP on these specific pathogens, perhaps due to the relative difficulty of culturing campylobacters under microaerobic conditions necessary for its growth.
Campylobacter is one of the most important human pathogens worldwide, being a major cause of acute human diarrheal illness in the developed world [8,9,10,11,12]. Among foodborne bacterial infections, disease caused by Campylobacter jejuni is the most prevalent with approximately 1.5 million cases annually in the United States alone [13]. Commercial broiler chickens serve as a major reservoir for C. jejuni with colonization levels as high as 1010 CFU per gram of wet feces in the chicken [14,15,16]. Consequently, commercial broiler chickens constitute the primary reservoir for C. jejuni leading to human infections [15,17,18,19]. Common poultry-associated isolates are present in human clinical cases, providing evidence that poultry is a major contributor to human infection [20]. In addition, an increase in AMR among bacterial pathogens, due in part to the sub-therapeutic use of antibiotics in animal feed, has the potential to compromise public health therapies and remains a concern among scientists and the general public [21,22,23,24]. The current consensus of scientific and public opinion is that antibiotic use by humans and in food animals selects for the development of AMR among foodborne bacteria that can complicate public health therapies [25]. A major issue is that antibiotic resistance may not only occur among disease-causing organisms but may also become an issue for other organisms in the host and the environment [26]. Sub-therapeutic use of antibiotics as growth promoters has been discontinued in the European Union [27,28,29]. These regulations are justified due to the increase in antibiotic resistance among bacterial pathogens [21,30] including bacteria from healthy broilers [31]. Consequently, there is a need for developing novel intervention methods including narrow-spectrum antimicrobials and probiotics that selectively target pathogenic organisms while avoiding the killing of beneficial organisms [30].
Eliminating or dramatically reducing Campylobacter spp. contamination during poultry production will reduce foodborne infections [32,33,34]. Despite various intervention efforts against pathogens including campylobacters and salmonellae by poultry producers, processors, and regulatory agencies, the number of human foodborne diseases caused by these pathogens has not drastically declined [35,36]. There are currently no applicable, on-farm interventions for significantly reducing the colonization of poultry with C. jejuni. There is a need for effective interventions that may be practically applied in the poultry industry to reduce the colonization of poultry with C. jejuni and, subsequently, reduce consumer exposure to this pathogen [37]. A proposal for on-farm control measures for Campylobacter by the EU and implementation of in-plant performance standards for the pathogen by FSIS [38] highlight the urgent need for effective intervention methods. Hence, the objective of this study was to chemically synthesize representative AMP and screen them for ability to kill campylobacters in vitro with the long-term goal of potentially developing novel, practical intervention methods for reduction of these foodborne pathogens in poultry.

2. Materials and Methods

We selected and evaluated a set of 11 unique chemically synthesized and commercially available AMP (Table 1 and Table 2) for ability to inhibit growth of C. jejuni isolates 11,168 and 81–176 and seven additional pathogenic bacterial isolates including two Clostridium perfringens isolates, two Salmonella isolates, two Listeria monocytogenes isolates, and one E. coli O157:H7 in a spot-on-lawn assay. Peptides were synthesized using standard solid-phase (Fmoc) chemistry with a peptide synthesizer (CPC Scientific Inc., Sunnyvale, CA 94089, USA, C12K-2β12; AnaSpec, Fremont, CA 94555, all other AMP).
Target bacterial cultures, C. jejuni 11168 and 81–176 were propagated on Brucella agar with blood (BAB) overnight at 42 °C in a microaerobic gas atmosphere (5% O2, 10% CO2, 85% N2). Cells were aseptically harvested from the plates and suspensions were made in PBS equivalent to No. 1 McFarland standard. Approximately 200 µL of bacterial suspensions were applied to the surface of BAB plates containing 1.5% low electroendosmosis (EEO) agarose [50] (Sigma-A6013) using a swab dipped in the suspension once for the first half the plate, and again for the second half. The inoculated plates were air-dried for approximately 10 min and were then spotted with 10 µL of each antimicrobial peptide or sterile water (control). All AMP solutions were prepared as 1 mg/1 mL; therefore, a 10 µL spot equates to 10 µg. Plates with AMP spots were allowed to dry for approximately 5 min, then were incubated at 37 °C under microaerobic conditions. AMP efficacy was determined as visible zones of inhibition of growth at 24 h.
Six of the AMP we tested produced zones of inhibition on lawns of C. jejuni. Three AMP were chosen for further investigation on the basis of anti-Campylobacter activity, water solubility, and reported reduced cytotoxicity to mammalian cells. Cecropin-Magainin, RL-37, and C12K-2β12 were tested for ability to produce zones of inhibition in spot-on-lawn assays against 24 different bacteria including C. jejuni, C. coli, and C. lari isolates as well as two strains of Salmonella, Clostridium perfringens, Listeria monocytogenes, Lactobacillus, and E. coli O157:H7. The spot-on-lawn assay was conducted as described earlier with the addition of Brain Heart Infusion agar with 1.5% EEO agarose for salmonellas, E. coli, clostridia, and Listeria. Lactobacillus MRS agar with 1.5% EEO agarose was utilized for lactobacilli. Campylobacter and lactobacilli were incubated in a microaerobic atmosphere (10% CO2, 5% O2, 85% N2) at 37 °C, and ambient air cultivation at 37 °C was utilized for Salmonella, E. coli, and Listeria. An anaerobic environment (5% CO2, 5% H2, 90% N2) at 37 °C was used for incubation of C. perfringens.
In addition, a modification of the CLSI M26A [51] and Wu and Hancock [52] assays were utilized to determine minimum inhibitory concentrations (MIC) for the AMP Cecropin–Magainin, RL-37, and the oligo-acyl-lysyl (OAK) C12K-2β12 against 15 isolates of campylobacter in microtiter plates. Briefly, target campylobacter cultures were grown in Brucella broth for 18 h in 25 cm2 vented tissue culture flasks in a microaerobic gas environment (5% O2, 10% CO2, 85% N2) at 37 °C. Visual adjustment of suspensions equivalent to a 0.5 McFarland standard for 1.5 × 108 cfu/mL baseline were made using fresh Brucella broth. The standardized suspensions were further diluted in fresh Brucella broth 1:1000 for an inoculum of 1.5 × 105. Enumeration of inoculum levels was assessed on BAB. Cecropin-Magainin, RL-37, and C12K-2β12 antimicrobial peptides were serially diluted in polypropylene strip tubes using 0.2% bovine serum albumin and 0.1% acetic acid diluent. An amount of 10 µL of each peptide dilution was added to 90 µL of bacterial inoculum culture in Greiner 96-well V-bottom polystyrene tissue culture-treated microtiter plates in triplicate for targets. One well was inoculated for each target with 10 µL 0.2% bovine serum albumin and 0.1% acetic acid diluent with 90 µL of bacterial inoculum as positive control. One well was inoculated with 10 µL 0.2% bovine serum albumin and 0.1% acetic acid diluent with 90 µL of Brucella broth as negative control. Plates were placed in gallon-sized zip-lock freezer bags (SC Johnson, Racine WI 53403, USA), flush-filled with the microaerobic gas mixture, and incubated for 40 h with gas replacement at 24 h. MIC was defined as the minimum concentration that prevented growth based on visual observation using mirrored white light.

3. Results

A set of 11 unique AMP were chemically synthesized and evaluated for ability to lyse C. jejuni in a spot-on-lawn assay (Figure 1). Table 3 shows the initial screening results for the 11 AMP against two isolates of C. jejuni as well as additional human foodborne pathogens including isolates of Clostridium perfringens, Salmonella, Listeria, and enterotoxigenic E. coli. Six of the selected AMP produced obvious zones of inhibition against growth of C. jejuni isolates. These antimicrobial peptides included: NRC-13 Pleurocidin, RL-37, Temporin L, Cecropin–Magainin, Dermaseptin, and C12K-2β12. Interestingly, these same six AMP were also able to lyse most of the other pathogenic bacteria tested.
Cecropin–Magainin, RL-37, and C12K-2β12 were chosen for further investigation on the basis of anti-Campylobacter activity, water solubility, and limited cytotoxicity to mammalian cells and were tested for ability to produce zones of inhibition in spot-on-lawn assays against 24 different bacteria including 15 unique C. jejuni, C. coli, and C. lari isolates as well as two strains of Salmonella, Clostridium perfringens, Listeria monocytogenes, Lactobacillus, and E. coli O157:H7. These three active AMP produced obvious zones of inhibition on all 15 of the campylobacter isolates tested, regardless of the strain (Table 4). They also inhibited the other pathogens tested with the only exceptions being the lack of zone formation by the Cecropin–Magainin and RL-37 AMP on Lactobacillus helviticus and Clostridium perfringens 39.
MIC of C12K-2β12, Cecropin–Magainin, and RL-37 against 15 different C. jejuni, C. coli, and C. lari isolates are presented in Table 5. MIC for C12K-2β12 ranged from 1.6 to 3.1 µg/mL with no discernable differences between strains of campylobacter. MIC for RL-37 varied from 1.6 to 6.3 µg/mL also with no obvious differentiation between campylobacter strains. The MIC for the Cecropin–Magainin hybrid were significantly higher, ranging from 12.5 to 100 µg/mL. No significant differences between campylobacter stains were observed in response to the Cecropin–Magainin AMP.

4. Discussion

AMP display tremendous chemical diversity in nature, causing difficulty in their classification [53,54]. Amino acid sequence, net charge, secondary structural motif, and the abundance of specific amino acids may all differ between types of AMP [55]. However, AMP also maintain some common features, and classification of AMP may be based on source, activity, structural characteristics, and amino acid-rich species [54]. For example, most AMP are cationic in nature with net charges ranging from +2 to +9. This gives an electrostatic advantage to the AMP in binding to the negatively charged bacterial membranes [56]. Many AMP are membrane-acting peptides and have an amphipathic structure with hydrophobic and hydrophilic regions which play an integral part in interactions with target bacterial membranes, leading to cell lysis through a variety of proposed mechanisms including the toroidal pore or wormhole model, the barrel-stave model, or the carpet-like model [57,58]. Indeed, some AMP are non-membrane-acting and do not cause cell lysis, but rather pass through the intact bacterial lipid bilayer through endocytosis and, once in the cytoplasm, inhibit nucleic acid biosynthesis, protease activity, or DNA replication [51]. A number of peptide databases exist and are available for mining [5,59,60]. We initially chose 11 AMP for evaluation representing a variety of different sources, modes of action, and activities frequently discussed in the literature (Table 1). The selected AMP represent examples from insects, bacteria, amphibians, fish, mammals, and a case of de novo synthetic AMP. They also represent membrane-acting and non-membrane-acting peptides.
While very few reports exist in the literature specifically describing inhibition of campylobacters by AMP, C. jejuni has been reported to be highly susceptible to chicken host-defense peptides such as cathelicidin [61]. We therefore chose to evaluate RL-37, a 37-residue AMP of the cathelicidin family which is expressed in bone marrow of the rhesus monkey and utilizes an amphipathic α-helical structure to form pores and destabilize the membrane lipid bilayer of target bacteria. RL-37 is reported to inhibit Gram-negative organisms such as E. coli and Pseudomonas [48] and we found it to be quite efficacious against the Gram (−) campylobacters we tested, achieving MIC ranging from 1.6 to 6.3 µg/mL (Table 5). It was also reported to inhibit Gram (+) organisms such as Listeria monocytogenes and this was observed in our study as well (Table 4). We chose several additional α-helical AMP for evaluation including dermaseptin, NRC-Pleurocidin, Paracin I, and Temporin L. Dermaseptin, a 34-residue peptide isolated from frog skin (Phyllomedusa), demonstrates broad-spectrum lytic activity against Gram-positive and Gram-negative bacteria, yeast, and protozoa [43,62]. We found it to inhibit some, but not all, of our test isolates (Table 3). Since it did not inhibit both of our initial campylobacter isolates used for screening, we did not include it in further MIC evaluations. NRC-Pleurocidin, a 23-residue peptide isolated from the American plaice winter flounder has been reported to rapidly kill G (−) Pseudomonas aeruginosa and G (+) Methicillin-resistant S. aureus (MRSA) [45]. We found it capable of lysing all our test isolates except for C. perfringens 39. However, it was excluded from further analysis due to its moderate cytotoxicity. Paracin I is a 19-residue peptide isolated from catfish skin and has been reported to inhibit G (+) and G (−) organisms including E. coli and Salmonella [46] However, in the concentrations tested in our assay, Paracin I failed to inhibit any of our test isolates. Temporin L is a 13-residue peptide from European red frog skin and is among the shortest natural AMP known [49,63]. We found it to be efficacious against all our test organisms but because it is reported to be hemolytic and requires dimethylsulfoxide (DMSO) as a solvent rather than water, we did not think it practical for our purposes and did not include it in further analysis.
Some AMP contain a high content of certain amino acid residues such as proline, tryptophan, glycine, or histidine. These AMP are frequently extended with no regular structure [56]. Proline-rich AMP (Pr-AMPs) are cationic peptides which usually enter the cell through translocation of the inner membrane using a transporter-mediated uptake mechanism and do not lyse the cell. Pr-AMPs usually kill bacteria by targeting intracellular components. Pyrrhocoricin is a 20-amino acid residue Pr-AMP isolated from the European sap-sucking bug (Pyrrocoris apterus). Pyrrhocoricin interferes with protein biosynthesis in target bacteria by binding to the 70S ribosomal subunit and inhibiting heat-shock proteins such as DnaK, preventing protein folding [64]. In the concentrations tested in our assay, Pyrrhocoricin failed to inhibit any of our test isolates. Apidaecin 1B is 18-residue Pr-AMP isolated from honeybees. Apidaecin 1B is known to inhibit a large number of plant-associated bacteria and human pathogens via a bactericidal pathway without cell lysis [39]. Apidaecin 1B inhibited Salmonella enteritidis serovars Typhimurium and Heidelburg and E. coli O157:H7 in our assay, but did not kill Campylobacter jejuni, Clostridium perfringens, or Listeria monocytogenes isolates.
Bacteriocins are AMP produced by bacteria. Carnobacteriocin B2 is a Class II cationic bacteriocin which can form α-helices and lyse specific target bacteria [41]. Under the conditions of our spot-on-lawn assay, none of our target bacteria were affected by Carnobacteriocin B2. Dermcidin DCD is a 48-residue peptide isolated from human sweat glands. This anionic AMP forms oligomeric ion channels in lipid bilayers of specific target bacteria. This AMP was also unable to form any zones of inhibition against any of the bacteria utilized in our assay.
Despite the large number of natural AMP identified, isolated, and characterized, there are some inherent disadvantages associated with natural AMP. For example, the limited quantities of AMP isolated from natural sources, the uneconomic synthesis of longer AMP sequences, folding issues associated with natural AMP, short half-lives due to protease degradability, and difficulties in site-directed delivery [56]. This has led to a research emphasis on synthetic AMP in recent decades to overcome shortcomings of natural AMP. Several approaches have been employed for the design of improved synthetic AMP including template-based de novo synthesis based on natural AMP to improve activity and proteolytic stability (often through truncation), biophysical modeling in hydrophobic membrane environments, and virtual screening of peptide libraries to identify potential AMP sequences [56].
Various chemical modifications of AMP have been utilized to improve potency while decreasing cytotoxicity to mammalian cells, or improving peptide resistance to protease digestion, high temperatures, and low pH. Cecropin A (from the Cecropia moth) and Magainin 2 (from the African clawed frog, Xenopus Laevis) both individually exhibit potent antimicrobial effects against a broad spectrum of bacteria [65]. Shin and co-workers designed an engineered hybrid AMP composed of residues 1-8 of Cecropin A fused to residues 1–12 of Magainin 2 [66]. Cecropin–Magainin had greater antimicrobial activity against bacteria than either of the AMP individually while maintaining low toxicity. It has been suggested that the flexibility induced by Gly-Ile-Gly or Pro residues in the central part of Cecropin–Magainin may be important in the electrostatic and hydrophobic interactions of the N and C terminus with cell membrane surface and cell membrane, respectively [42]. We included Cecropin–Magainin in our spot-on-lawn assays and found it to inhibit all the bacterial species in our test, including the 15 Campylobacter sps. isolates, but not L. helviticus or C. perfringens 39 isolates.
Many naturally occurring AMP have rather long AA sequences which can limit their application as commercial drugs due to the high cost of producing them on an industrial scale [67]. Shorter synthetic AMP could potentially lower cost of synthesis and may be more resistant to proteolysis. Shorter synthetic AMP also could be less cumbersome to express in natural production vectors such as E. coli or Bacillus subtilis [68,69]. Oligomers of acylated lysines (OAKs) constitute a novel class of synthetic AMP made up of alternating amino acyl chains and cationic amino acids arranged in such a way as to create an optimal molecular charge and hydrophobicity for increased potency [70,71,72]. The synthetic OAK hexamer C12K-2β12 exhibits inhibitory activity in vitro against Gram-negative bacteria such as the gastric pathogen Helicobacter pylori [40]. Because H. pylori has some structural characteristics in common with campylobacters, we chose to evaluate C12K-2β12 in our spot-on-lawn assay. C12K-2β12 was very active and inhibited growth of all target bacterial isolates in our assay. The bactericidal effects of C12K-2β12 are exhibited by a dual mode of action. The OAK causes pore formation and cell lysis at higher concentrations while, at lower concentrations, it binds nucleic acids and proteins [55]. It demonstrated the lowest MIC of any of the AMP we tested against 15 isolates of Campylobacter representing jejuni, coli, and lari species, with a range of 1.6 to 3.1 ug/mL. C12K-2β12 is only 8 amino acid residues in length and is reported to be stable at low pH and after exposure to extreme temperatures [40]. These are all important considerations for an AMP to be potentially administered to poultry. This AMP also inhibited other pathogenic bacterial species associated with poultry (Salmonella, C. perfringens). These characteristics make C12K-2β12 a strong candidate for further investigation and potential development into a practical intervention for reduction of human foodborne pathogen populations associated with broiler chickens.
It is interesting that the AMP investigated in this study and found to be efficacious against Campylobacter spp. (NRC-13 Pleurocidin, RL-37, Temporin L, Cecropin–Magainin, Dermaseptin, and C12K-2β12) are all cationic AMP hypothesized to form amphipathic α-helical structures to initiate pore formation in target bacteria and subsequent cell lysis. It is also interesting that Pyrrhocoricin, which is also a cationic, amphipathic α-helical AMP, did not inhibit Campylobacter spp. There is evidence in the literature suggesting that the selectivity and potency of a specific AMP is determined primarily by the chemical composition of the target membrane [1]. It has likewise been hypothesized that differences in membrane composition between different strains of bacteria are responsible for the diversity in the potency and selectivity exhibited by a particular AMP against different strains of bacteria [73].
One of the most important factors in reducing the burden of foodborne disease has been identified as development of effective control measures [74]. By providing novel alternatives to antibiotic usage in poultry, the overall impact of our long-term research goals will be a reduction in bacterial pathogens associated with chickens and safer products for human consumption. Our working hypothesis is that AMP that inhibit the growth of Campylobacter spp. can be identified and subsequently utilized to reduce the Campylobacter load among commercially produced chickens. Identifying non-hemolytic AMP, such as C12K-2β12, RL-37, and Cecropin–Magainin, capable of killing campylobacters in vitro is a positive first step toward this goal. An obvious second step will be producing efficacious AMP in an economical fashion. Microbiological production of AMP through fermentation is becoming a feasible and economic method of production. Recent studies have described the development of a Bacillus subtilis culture expressing chicken NK-2 peptide [69]. Oral delivery of the B. subtilis culture can protect against Eimeria acervuline infection in broiler chickens. We are currently working to similarly express effective AMP in a microbial vector for in vitro analysis and subsequent oral delivery to chickens to protect against colonization by Campylobacter spp.

5. Conclusions

Selected AMP produced obvious zones of inhibition against growth of C. jejuni isolates. These AMP included: NRC-13 Pleurocidin, RL-37, Temporin L, Cecropin A–Magainin 2 hybrid, Dermaseptin, and C12K-2β12. MIC for C12K-2β12 ranged from 1.6 to 3.1 µg/mL with no discernable differences between strains of campylobacter. MIC for RL-37 varied from 1.6 to 6.3 µg/mL, also with no obvious differentiation between campylobacter strains. The MIC for Cecropin–Magainin were significantly higher, ranging from 12.5 to 100 µg/mL. No significant differences between campylobacter stains were observed in response to the Cecropin–Magainin AMP. The most efficacious of the AMP tested, the synthetic OAK C12K-2β12, is also heat- and acid-stable, making it an attractive subject for future study and potential in vivo delivery to poultry.

Author Contributions

Conceptualization, J.E.L., B.S.S., and J.K.G.; methodology, J.E.L., B.S.S., and J.K.G.; software, J.E.L.; validation, J.K.G.; formal analysis, J.E.L.; investigation, J.E.L. and J.K.G.; resources, J.E.L.; data curation, J.K.G.; writing—original draft preparation, J.E.L.; writing—review and editing, J.E.L., B.S.S., and J.K.G.; visualization, J.E.L.; supervision, J.E.L.; project administration, J.E.L.; funding acquisition, J.E.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by USDA, ARS Project Number 6040-32000-079-000D.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is contained within the article.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Venugopal, D.; Klapper, D.; Srouji, A.H.; Bhonsle, J.B.; Borschel, R.; Mueller, A.; Russell, A.L.; Williams, B.C.; Hicks, R.P. Novel antimicrobial peptides that exhibit activity against select agents and other drug resistant bacteria. Bioorg. Med. Chem. 2010, 18, 5137–5147. [Google Scholar] [CrossRef] [PubMed]
  2. Cuperus, T.; Coorens, M.; Van Dijk, A.; Haagsman, H.P. Avian host defense peptides. Dev. Comp. Immunol. 2013, 41, 352–369. [Google Scholar] [CrossRef] [PubMed]
  3. Yeaman, M.R.; Yount, N.Y. Mechanisms of antimicrobial peptide action and reisitance. Pharm. Rev. 2003, 55, 27–55. [Google Scholar] [CrossRef]
  4. Cao, Y.; Yu, R.Q.; Liu, Y.; Zhao, H.X.; Song, L.L.; Cao, Y.; Qiao, D.R. Design, recombinant expression, and antimicrobial activity of the cecropins-melittin hybrid antimicrobial peptides. Curr. Microbiol. 2010, 61, 169–175. [Google Scholar] [CrossRef]
  5. Aguilera-Mendoza, L.; Marrero-Ponce, Y.; Tellez-Ibarra, R.; Llorente-Quesada, M.T.; Salgado, J.; Barigye, S.J.; Liu, J. Overlap and diversity in antimicrobial peptide databases: Compiling a non-redundant set of sequences. Bioinformatics 2015, 31, 2553–2559. [Google Scholar] [CrossRef] [PubMed]
  6. Bahar, A.A.; Ren, D. Antimicrobial Peptides. Pharmaceuticals 2013, 6, 1543–1575. [Google Scholar] [CrossRef]
  7. Shi, G.; Kang, X.; Dong, F.; Liu, Y.; Zhu, N.; Hu, Y.; Xu, H.; Lao, X.; Zheng, H. DRAMP 3.0: An enhanced comprehensive data repository of antimicrobial peptides. Nucleic Acids Res. 2021, 50, D488–D496. [Google Scholar] [CrossRef]
  8. Ryan, K.J.; Ray, C.G.; Sherris, J.C. Sherris Medical Microbiology: An Introduction to Infection Diseases, 4th ed.; McGraw-Hill: New York, NY, USA, 2004. [Google Scholar]
  9. Ursing, J.B.; Lior, H.; Owen, R.J. Proposal of Minimal Standards for Describing New Species of the Family Campylobacteraceae. Int. J. Syst. Bacteriol. 1994, 44, 842–845. [Google Scholar] [CrossRef]
  10. Altekruse, S.F.; Stern, N.J.; Fields, P.I.; Swerdlow, D.L. Campylobacter jejuni—An Emerging Foodborne Pathogen. Emerg. Infect. Dis. 1999, 5, 28–35. [Google Scholar] [CrossRef]
  11. Janssen, R.; Krogfelt, K.A.; Cawthraw, S.A.; van Pelt, W.; Wagenaar, J.A.; Owen, R.J. Host-pathogen interactions in Campylobacter infections: The host perspective. Clin. Microbiol. Rev. 2008, 21, 505–518. [Google Scholar] [CrossRef] [Green Version]
  12. Kubota, K.; Kasuga, F.; Iwasaki, E.; Inagaki, S.; Sakurai, Y.; Komatsu, M.; Toyofuku, H.; Angulo, F.J.; Scallan, E.; Morikawa, K. Estimating the Burden of Acute Gastroenteritis and Foodborne Illness Caused by Campylobacter, Salmonella, and Vibrio parahaemolyticus by Using Population-Based Telephone Survey Data, Miyagi Prefecture, Japan, 2005 to 2006. J. Food Prot. 2011, 74, 1592–1598. [Google Scholar] [CrossRef] [PubMed]
  13. Centers for Disease Control and Prevention. Campylobacter (Campylobacteriosis), Questions & Answers. Available online: https://www.cdc.gov/campylobacter/technical.html (accessed on 31 August 2021).
  14. Sahin, O.; Morishita, T.Y.; Zhang, Q. Campylobacter colonization in poultry: Sources of infection and modes of transmission. Anim. Health Res. Rev. 2002, 3, 95–105. [Google Scholar] [CrossRef]
  15. Newell, D.G.; Fearnley, C. Sources of Campylobacter Colonization in Broiler Chickens. Appl. Environ. Microbiol. 2003, 69, 4343–4351. [Google Scholar] [CrossRef] [PubMed]
  16. Newell, D.G.; Koopmans, M.; Verhoef, L.; Duizer, E.; Aidara-Kane, A.; Sprong, H.; Opsteegh, M.; Langelaar, M.; Threfall, J.; Scheutz, F.; et al. Food-borne diseases—The challenges of 20years ago still persist while new ones continue to emerge. Int. J. Food Microbiol. 2010, 139, S3–S15. [Google Scholar] [CrossRef] [PubMed]
  17. Stern, N.J. Reservoirs for Campylobacter jejuni and approaches for intervention in poultry. In Campylobacter Jejuni: Current Status and Future Trends; Nachamkin, I., Blaser, M.J., Tompkins, L., Eds.; American Society for Microbiology: Washington, DC, USA, 1992; pp. 49–60. [Google Scholar]
  18. Mead, P.S.; Slutsker, L.; Dietz, V.; McCaig, L.F.; Bresee, J.S.; Shapiro, C.; Griffin, P.M.; Tauxe, R.V. Food-Related Illness and Death in the United States. Emerg. Infect. Dis. 1999, 5, 607–625. [Google Scholar] [CrossRef]
  19. Jore, S.; Viljugrein, H.; Brun, E.; Heier, B.; Borck, B.; Ethelberg, S.; Hakkinen, M.; Kuusi, M.; Reiersen, J.; Hansson, I.; et al. Trends in Campylobacter incidence in broilers and humans in six European countries, 1997–2007. Prev. Vet. Med. 2010, 93, 33–41. [Google Scholar] [CrossRef]
  20. Meunier, M.; Guyard-Nicodeme, M.; Dory, D.; Chemaly, M. Control strategies against Campylobacter at the poultry production level: Biosecurity measures, feed additives and vaccination. J. Appl. Microbiol. 2015, 120, 1139–1173. [Google Scholar] [CrossRef]
  21. Gyles, C.L. Antimicrobial resistance in selected bacteria from poultry. Anim. Health Res. Rev. 2008, 9, 149–158. [Google Scholar] [CrossRef]
  22. Aarestrup, F.M. The livestock reservoir for antimicrobial resistance: A personal view on changing patterns of risks, effects of interventions and the way forward. Philos. Trans. R. Soc. B Biol. Sci. 2015, 370, 20140085. [Google Scholar] [CrossRef]
  23. Doyle, M.E. Multidrug-Resistant Pathogens in the Food Supply. Foodborne Pathog. Dis. 2015, 12, 261–279. [Google Scholar] [CrossRef]
  24. Seal, B.S.; Lillehoj, H.S.; Donovan, D.M.; Gay, C.G. Alternatives to antibiotics: A symposium on the challenges and solutions for animal production. Anim. Health Res. Rev. 2013, 14, 78–87. [Google Scholar] [CrossRef] [PubMed]
  25. DuPont, H.L. The growing threat of food-borne bacterial enteropathogens of animal origin. Clin. Infect. Dis. 2007, 45, 1353–1361. [Google Scholar] [CrossRef] [PubMed]
  26. Yan, S.; Gilbert, J.M. Antimicrobial drug delivery in food animals and microbial food safety concerns: An overview of in vitro and in vivo factors potentially affecting the animal gut microflora. Adv. Drug Deliv. Rev. 2004, 56, 1497–1521. [Google Scholar] [CrossRef]
  27. Regulation EC. No. 1831/2003 of the European Parliament and the Council of 22 September 2003 on Additives for Use in Animal Nutrition. Available online: https://www.eur-lex.europa.eu/legal-content/EN/ALL/?uri=CELEX:32003R1831 (accessed on 20 September 2022).
  28. Casewell, M.; Friis, C.; Marco, E.; McMullin, P.; Phillips, I. The European ban on growth-promoting antibiotics and emerging consequences for human and animal health. J. Antimicrob. Chemother. 2003, 52, 159–161. [Google Scholar] [CrossRef]
  29. Castanon, J.I.R. History of the Use of Antibiotic as Growth Promoters in European Poultry Feeds. Poult. Sci. 2007, 86, 2466–2471. [Google Scholar] [CrossRef] [PubMed]
  30. National Research Council. Treating Infectious Diseases in a Microbial World: Report of Two Workshops on Novel Antimicrobial Therapeutics; The National Academies Press: Washington, DC, USA, 2006; ISBN 0-309-65490-4. Available online: http://www.nap.edu/catalog.php?record_id=11471 (accessed on 20 September 2022).
  31. Persoons, D.; Dewulf, J.; Smet, A.; Herman, L.; Heyndrickx, M.; Martel, A.; Catry, B.; Butaye, P.; Haesebrouck, F. Prevalence and Persistence of Antimicrobial Resistance in Broiler Indicator Bacteria. Microb. Drug Resist. 2010, 16, 67–74. [Google Scholar] [CrossRef]
  32. Stern, N.J.; Line, J.E. Campylobacter. In The Microbiological Safety and Quality of Food; Lund, B., Baird-Parker, T., Gould, G., Eds.; International Thomson Publishing: London, UK, 1885; pp. 1040–1056, 2000. [Google Scholar]
  33. Wagenaar, J.A.; Mevius, D.J.; Havelaar, A.H. Campylobacter in primary animal production and control strategies to reduce the burden of human campylobacteriosis. Rev. Sci. Tech.-Off. Int. Epizoot. 2006, 25, 581–594. [Google Scholar] [CrossRef]
  34. Nauta, M.; Hill, A.; Rosenquist, H.; Brynestad, S.; Fetsch, A.; van der Logt, P.; Fazil, A.; Christensen, B.; Katsma, E.; Borck, B.; et al. A comparison of risk assessments on Campylobacter in broiler meat. Int. J. Food Microbiol. 2008, 129, 107–123. [Google Scholar] [CrossRef]
  35. Crim, S.M.; Iwamoto, M.; Huang, J.Y.; Griffin, P.M.; Gilliss, D.; Cronquist, A.B.; Cartter, M.; Tobin-D’Angelo, M.; Blythe, D.; Smith, K.; et al. Centers for Disease Control and Prevention (CDC). Incidence and trends of infection with pathogens transmitted commonly through food—Foodborne Diseases Active Surveillance Network, 10 U.S. sites, 2006–2013. MMWR Morb. Mortal. Wkly. Rep. 2014, 63, 328–332. [Google Scholar]
  36. Crim, S.M.; Griffin, P.M.; Tauxe, R.; Marder, E.P.; Gilliss, D.; Cronquist, A.B.; Cartter, M.; Tobin-D’Angelo, M.; Blythe, D.; Smith, K.; et al. Preliminary incidence and trends of infection with pathogens transmitted commonly through food—Foodborne diseases active surveillance network, 10 u.s. Sites, 2006–2014. MMWR Morb. Mortal. Wkly. Rep. 2015, 64, 495–499. [Google Scholar]
  37. Line, J.; Hiett, K.; Conlan, A. Comparison of Challenge Models for Determining the Colonization Dose of Campylobacter jejuni in Broiler Chicks. Poult. Sci. 2008, 87, 1700–1706. [Google Scholar] [CrossRef]
  38. 81 FR 7285. Available online: https://www.govinfo.gov/content/pkg/FR-2016-02-11/pdf/2016-02586.pdf (accessed on 20 September 2022).
  39. Casteels, P.; Ampe, C.; Jacobs, F.; Vaeck, M.; Tempst, P. Apidaecins: Antibacterial peptides from honey bees. EMBO J. 1989, 8, 2387–2391. [Google Scholar] [CrossRef]
  40. Makobongo, M.O.; Kovachi, T.; Gancz, H.; Mor, A.; Merrell, D.S. In Vitro Antibacterial Activity of Acyl-Lysyl Oligomers against Helicobacter pylori. Antimicrob. Agents Chemother. 2009, 53, 4231–4239. [Google Scholar] [CrossRef] [PubMed]
  41. Quadri, L.; Sailer, M.; Roy, K.; Vederas, J.; Stiles, M. Chemical and genetic characterization of bacteriocins produced by Carnobacterium piscicola LV17B. J. Biol. Chem. 1994, 269, 12204–12211. [Google Scholar] [CrossRef]
  42. Shin, S.Y.; Kang, J.H.; Jang, S.Y.; Kim, Y.; Kim, K.L.; Hahm, K. Effects of the hinge region of cecropin A(1-8)-magainin 2(1-12), a synthetic antimicrobial peptide, on liposomes, bacterial and tumor cells. Biochem. Biophys. Acta 2000, 1463, 209–218. [Google Scholar] [CrossRef]
  43. Mor, A.; Van Huong, N.; Delfour, A.; Migliore-Samour, D.; Nicolas, P. Isolation, amino acid sequence and synthesis of dermaseptin, a novel antimicrobial peptide of amphibian skin. Biochemistry 1991, 30, 8824–8830. [Google Scholar] [CrossRef] [PubMed]
  44. Schittek, B.; Hipfel, R.; Sauer, B.; Bauer, J.; Kalbacher, H.; Stevanovic, S.; Schirle, M.; Schroeder, K.; Blin, N.; Meier, F.; et al. Dermcidin: A novel human antibiotic peptide secreted by sweat glands. Nat. Immunol. 2001, 2, 1133–1137. [Google Scholar] [CrossRef] [PubMed]
  45. Patrzykat, A.; Gallant, J.W.; Seo, J.-K.; Pytyck, J.; Douglas, S.E. Novel Antimicrobial Peptides Derived from Flatfish Genes. Antimicrob. Agents Chemother. 2003, 47, 2464–2470. [Google Scholar] [CrossRef]
  46. Park, I.Y.; Park, C.B.; Kim, M.S.; Kim, S.C. Parasin I, antimicrobial peptide derived from histone H2A in the catfish, Parasilurus asotus. FEBS Lett. 1998, 437, 258–262. [Google Scholar] [CrossRef]
  47. Bulet, P.; Hetru, C.; Dimarcq, J.-L.; Hoffmann, D. Antimicrobial peptides in insects; structure and function. Dev. Comp. Immunol. 1999, 23, 329–344. [Google Scholar] [CrossRef]
  48. Zhao, C.; Nguyen, T.; Boo, L.M.; Hong, T.; Espiritu, C.; Orlov, D.; Wang, W.; Waring, A.; Lehrer, R.I. RL-37, an Alpha-Helical Antimicrobial Peptide of the Rhesus Monkey. Antimicrob. Agents Chemother. 2001, 45, 2695–2702. [Google Scholar] [CrossRef] [PubMed]
  49. Simmaco, M.; Mignogna, G.; Canofeni, S.; Miele, R.; Mangoni, M.L.; Barra, D. Temporins, antimicrobial peptides from the European red frog Rana temporaria. Eur. J. Biochem. 1996, 242, 788–792. [Google Scholar] [CrossRef] [PubMed]
  50. Shafer, W.M. (Ed.) Antibacterial Peptide Protocols: Methods in Molecular Biology; Humana Press: Totowa, NJ, USA, 1997; Volume 78, p. 1173. [Google Scholar]
  51. Clinical and Laboratory Standards Institute. M26-A: Methods for Determining Bactericidal Activity of Antimicrobial Agents; Approved Guideline; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 1999; Volume 12. [Google Scholar]
  52. Wu, M.; Hancock, R.E.W. Interaction of the cyclic antimicrobial cationic peptide bactenecin with the outer and cytoplasmic membrane. J. Biol. Chem. 1999, 274, 29–35. [Google Scholar] [CrossRef] [PubMed]
  53. Browne, K.; Chakraborty, S.; Chen, R.; Willcox, M.D.; Black, D.S.; Walsh, W.R.; Kumar, N. A New Era of Antibiotics: The Clinical Potential of Antimicrobial Peptides. Int. J. Mol. Sci. 2020, 21, 7047. [Google Scholar] [CrossRef]
  54. Huan, Y.; Kong, Q.; Mou, H.; Yi, H. Antimicrobial Peptides: Classification, Design, Application and Research Progress in Multiple Fields. Front. Microbiol. 2020, 11, 582779. [Google Scholar] [CrossRef]
  55. Makobongo, M.O.; Gancz, H.; Carpenter, B.M.; McDaniel, D.P.; Merrell, D.S. The oligo-acyl lysyl antimicrobial peptide C12K-2β12 exhibits a dual mechanism of action and demonstrates strong in vivo efficacy against Helicobacter pylori. Antimicrob. Agents Chemother. 2012, 56, 378–390. [Google Scholar] [CrossRef]
  56. Sarkar, T.; Chetia, M.; Chatterjee, S. Antimicrobial Peptides and Proteins: From Nature’s Reservoir to the Laboratory and Beyond. Front. Chem. 2021, 9. [Google Scholar] [CrossRef]
  57. Moravej, H.; Moravej, Z.; Yazdanparast, M.; Heiat, M.; Mirhosseini, A.; Moghaddam, M.M.; Mirnejad, R. Antimicrobial Peptides: Features, Action, and Their Resistance Mechanisms in Bacteria. Microb. Drug Resist. 2018, 24, 747–767. [Google Scholar] [CrossRef]
  58. Kumar, P.; Kizhakkedathu, J.N.; Straus, S.K. Antimicrobial Peptides: Diversity, Mechanism of Action and Strategies to Improve the Activity and Biocompatibility In Vivo. Biomolecules 2018, 8, 4. [Google Scholar] [CrossRef]
  59. Wang, G. The antimicrobial peptide database provides a platform for decoding the design principles of naturally occurring antimicrobial peptides. Protein Sci. 2019, 29, 8–18. [Google Scholar] [CrossRef]
  60. Waghu, F.H.; Idicula-Thomas, S. Collection of antimicrobial peptides database and its derivitives: Applications and beyond. Protein Sci. 2019, 29, 36–42. [Google Scholar] [CrossRef] [PubMed]
  61. van Dijk, A.; Herrebout, M.; Tersteeg-Zijderveld, M.H.; Bokhoven, J.L.T.-V.; Bleumink-Pluym, N.; Jansman, A.J.; Veldhuizen, E.J.; Haagsman, H.P. Campylobacter jejuni is highly susceptible to killing by chicken host defense peptide cathelicidin-2 and suppresses intestinal cathelicidin-2 expression in young broilers. Vet. Microbiol. 2012, 160, 347–354. [Google Scholar] [CrossRef] [PubMed]
  62. Nicolas, P.; Amri, C. The dermaseptin superfamily: A gene-based combinatorial library of antimicrobial peptides. Biochem. Biophys. Acta 2009, 1788, 1537–1550. [Google Scholar] [CrossRef] [PubMed]
  63. Saviello, M.R.; Malfi, S.; Campiglia, P.; Cavalli, A.; Grieco, P.; Novellino, E.; Carotenuto, A. New Insight into the Mechanism of Action of the Temporin Antimicrobial Peptides. Biochemistry 2010, 49, 1477–1485. [Google Scholar] [CrossRef]
  64. Kragol, G.; Lovas, S.; Varadi, G.; Condie, B.A.; Hoffman, R.; Otvos, L., Jr. The antimicrobial peptide pyrrhocoricin inhibits the ATPase actions of DnaK and prevents chaperone-assisted protein folding. Biochemistry 2001, 40, 3016–3026. [Google Scholar] [CrossRef]
  65. Lee, D.G.; Hahm, K.-S.; Shin, S.Y. Structure and fungicidal activity of a synthetic antimicrobial peptide, P18, and its truncated peptides. Biotechnol. Lett. 2004, 26, 337–341. [Google Scholar] [CrossRef]
  66. Shin, S.Y.; Kang, J.H.; Hahm, K.-S. Structure-antibacterial, antitumor and hemolytic activity relationships of cecropin A-magainin 2 and cecropin A-melittin hybrid peptides. J. Pept. Res. 1999, 53, 82–90. [Google Scholar] [CrossRef]
  67. Cardoso, P.; Glossop, H.; Meikle, T.G.; Aburto-Medina, A.; Conn, C.E.; Sarojini, V.; Valery, C. Molecular engineering of antimicrobial peptides: Microbial targets, peptide motifs and translation opportunities. Biophys. Rev. 2021, 13, 325–369. [Google Scholar] [CrossRef]
  68. Daneshmand, A.; Kermanshahi, H.; Sekhavati, M.H.; Javadmanesh, A.; Ahmadian, M. Antimicrobial peptide, cLF36, affects performance and intestinal morphology, microflora, junctional proteins, and immune cells in broilers challenged with E. coli. Sci. Rep. 2019, 9, 14176. [Google Scholar] [CrossRef]
  69. Wickramasuriya, S.S.; Park, I.; Lee, Y.; Kim, W.H.; Przybyszewski, C.; Gay, C.G.; van Oosterwijk, J.G.; Lillehoj, H.S. Oral Delivery of Bacillus subtilis Expressing Chicken NK-2 Peptide Protects Against Eimeria acervulina Infection in Broiler Chickens. Front. Vet. Sci. 2021, 8, 684818. [Google Scholar] [CrossRef]
  70. Radzishevsky, I.S.; Rotem, S.; Bourdetsky, D.; Navon-Venezia, S.; Carmeli, Y.; Mor, A. Improved antimicrobial peptides based on acyl-lysine oligomers. Nat. Biotechnol. 2007, 25, 657–659. [Google Scholar] [CrossRef] [PubMed]
  71. Radzishevsky, I.S.; Kovachi, T.; Porat, Y.; Ziserman, L.; Zaknoon, F.; Danino, D.; Mor, A. Structure-Activity Relationships of Antibacterial Acyl-Lysine Oligomers. Chem. Biol. 2008, 15, 354–362. [Google Scholar] [CrossRef] [Green Version]
  72. Livne, L.; Kovachi, T.; Sarig, H.; Epand, R.F.; Zaknoon, F.; Epand, R.M.; Mor, A. Design and Characterization of a Broad -Spectrum Bactericidal Acyl-lysyl Oligomer. Chem. Biol. 2009, 16, 1250–1258. [Google Scholar] [CrossRef] [PubMed]
  73. Powers, J.-P.S.; Hancock, R.E. The relationship between peptide structure and antibacterial activity. Peptides 2003, 24, 1681–1691. [Google Scholar] [CrossRef]
  74. Quested, T.; Cook, P.; Gorris, L.; Cole, M. Trends in technology, trade and consumption likely to impact on microbial food safety. Int. J. Food Microbiol. 2010, 139 (Suppl. 1), S29–S42. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Applmicrobiol 02 00053 g001 550
Figure 1. “Spot-on-lawn” zone assay demonstrating inhibition of C. jejuni by selected antimicrobial peptides: (Counter-clockwise from top) RL-37, Cecropin A–Magainin 2 hybrid, the synthetic OAK C12K-2β12, ampicillin (positive control), and sterile water (negative control, no zone of inhibition).
Figure 1. “Spot-on-lawn” zone assay demonstrating inhibition of C. jejuni by selected antimicrobial peptides: (Counter-clockwise from top) RL-37, Cecropin A–Magainin 2 hybrid, the synthetic OAK C12K-2β12, ampicillin (positive control), and sterile water (negative control, no zone of inhibition).
Applmicrobiol 02 00053 g001
Table
Table 1. Sources and characteristics of antimicrobial peptides evaluated in this study.
Table 1. Sources and characteristics of antimicrobial peptides evaluated in this study.
AMPSourceAA #Structure and CharacteristicsReference
Apidaecin 1BHoneybee lymph18Cationic, no α-helix formation, high proline content, stable at high temp and low pH, small mol wt 2100[39]
C12K-2β12Synthetic oligo-acyl-lysyl (OAK) hexamer8Peptidomimetic, stable at high temp and low pH[40]
Carnobacteriocin B2Carnobacterium piscicola48Class II bacteriocin, cationic, single alpha-helices involved in coiled-coils or other helix–helix interfaces[41]
Cecropin A–Magainin 2 hybridCecropia moth/African clawed frog20Cationic, short helix–flexible–amphipathic helix, antibacterial as well as antitumor activity[42]
DermaseptinSkin of frog (Phyllomedusa)34Cationic, amphipathic α-helix[43]
Dermcidin DCDHuman sweat glands48Forms cation-stabilized oligomeric ion channels in lipid bilayers[44]
NRC-13 PleurocidinAmerican plaice-winter flounder23Amphipathic α-helix[45]
Parasin ISkin mucus of wounded catfish (Parasilurus asotus)19Amphipathic α-helix[46]
PyrrhocoricinEuropean sap-sucking bug (Pyrrhocoris apterus)20Cyclic, proline-rich peptide[47]
RL-37Bone marrow of Rhesus monkey (Macaca mulatta)37Cathelicidin, α-helix[48]
Temporin LEuropean red frog skin (Rana temporaria)13Stable α-helix, secondary amphipathicity, shortest natural AMP found to date[49]
Table
Table 2. Proposed modes of action, amino acid sequences, and hemolysis reactions of AMP employed in this study.
Table 2. Proposed modes of action, amino acid sequences, and hemolysis reactions of AMP employed in this study.
AMPAA SequenceNet ChargeProposed Modes of ActionHemolysis
Apidaecin 1BGNNRP VYIPQ PRPPH PRL3.1binding and irreversible combination with a periplasmic receptor/docking molecule, devoid of pore-forming activitynon-hemolytic
C12K-2β12C12K-KIK-KIK (C12 represents dodecanoic acid)4rapid membrane depolarization and cell permeabilizationnon-hemolytic at 1:64 (1.56 mcg/mL)
Carnobacteriocin B2VNYGN GVSCS KTKCS VNWGQ AFQER YTAGI NSFVS GVASG AGSIG RRP3.9cationic membrane-permeabilizing bacteriocin (Class II) not expected
Cecropin A–Magainin 2 hybridKWKLFKKIGIGKFLHSAKKF7.1trp2 insertion, Lys binding, alpha-helix membrane spanning due to flexible hingemoderate 1:2 (50 mcg/mL) to 1:32 (3.125 mcg/mL)
DermaseptinALWKT MLKKL GTMAL HAGKA ALGAA ADTIS QGTQ3.1forms amphipathic helices when integrated with membrane lipid bilayeryes
Dermcidin DCDSSLLE KGLDG AKKAV GGLGK LGKDA VEDLE SVGKG AVHDV KDVLD SVL−1.9transmembrane potential formed with nanopore formation upon insertionnot expected
NRC-13 PleurocidinGWRTLLKKAEVKTVGKLALKHYL5.1forms ion channels (probable toroidal pore) in planar lipid bilayers. Inhibits nucleic acid and protein synthesismoderate 1:2 (50 mcg/mL) to 1:256 (0.39 mcg/mL)
Parasin IKGRGK QGGKV RAKAK TRSS8binds to DnaK, inhibiting its major two functions: ATPase activity and misfolding proteins with inactivation by acting on internal targetsnon-hemolytic
PyrrhocoricinVDKGS YLPRP TPPRP IYNRN3binds to 70 kDa heat-shock protein DnaK, inhibiting protein foldingundetermined
RL-37RLGNFFRKVKEKIGGGLKKVGQKIKDFLGNLVPRTAS8amphipathic alpha-helical structure, pore formationno
Temporin LFVQWF SKFLG RIL2allows Temporin A and B to bypass LPS and access the cytoplasmic membrane by preventing their oligomerization to LPSyes
Table
Table 3. Formation of zones of inhibition by selected AMP against bacterial isolates.
Table 3. Formation of zones of inhibition by selected AMP against bacterial isolates.
AMPCj1Cj2Cp1Cp2S1S2Lm1Lm2Ec
Apidaecin 1B----++--+
C12K-2β12+++++++++
Carnobacteriocin B2---------
Cecropin A–Magainin 2 hybrid++-++++++
Dermaseptin+-++-++++
Dermcidin DCD-------±-
NRC-13 Pleurocidin++-+±++++
Parasin I---------
Pyrrhocoricin---------
RL-37++++-++++++
Temporin L++++++++++
Ampicillin (control)--+++++++++++
Acetic acid (control)--+++++++
Key to bacterial target isolates: Cj1 = Campylobacter jejuni 11168; Cj2 = Campylobacter jejuni 81–176; Cp1 = Clostridium perfringens CP39; Cp2 = Clostridium perfringens CP509; S1 = Salmonella enteritidis serovar Typhimurium 14028; S2 = Salmonella enteritidis serovar Heidelburg 130NR; Lm1 = Listeria monocytogenes A49594 (4b); Lm2 = Listeria monocytogenes 311-WT; Ec = Escherichia coli O157:H7 (233_RC1-WT). Key to table symbols: - no inhibition; ±presence of satellite colonies within zone of inhibition; + 5 mm mean zone diameter; ++ 13.5 mm mean zone diameter.
Table
Table 4. Formation of zones of inhibition by selected AMP against various target bacteria.
Table 4. Formation of zones of inhibition by selected AMP against various target bacteria.
Target BacteriaC12K-2β12Cecropin A–Magainin 2RL-37
Campylobacter jejuni 14118++++++
Campylobacter jejuni 81-116+++++
Campylobacter jejuni 81-176 a+++++
Campylobacter jejuni 11168 ^a+++++
Campylobacter jejuni RM1221 a+++++++
Campylobacter jejuni A74C++++++
Campylobacter jejuni A49943 *+++++
Campylobacter jejuni A33250 *+++++
Campylobacter jejuni A29428 *+++++
Campylobacter coli Epi 33-WT+++++++
Campylobacter coli A49941 *+++++++
Campylobacter coli A33559 *++++++
Campylobacter lari RM2100+++++++
Campylobacter lari A35221 *+++++
Campylobacter lari “slaughter beach”+++++
Salmonella enterica serovar Typhimurium Epi 3++++
Salmonella enterica serovar Heidelberg Epi 42++++
Lactobacillus acidophilus-WT++++
Lactobacillus helveticus-WT++--
Clostridium perfringens 39+--
Clostridium perfringens 509+++
Listeria monocytogenes A49594 (4b)+++
Listeria monocytogenes 311 WT+++
Escherichia coli O157:H7+++
a Alternate ATCC designations; ^ National Collection of Type Cultures (NCTC) isolate; * American Type Culture Collection (ATCC) isolate. Key to table symbols: - no zone of inhibition; + 5 mm mean zone diameter; ++ 13.5 mm mean zone diameter; +++ 18 mm mean zone diameter.
Table
Table 5. Minimum inhibitory concentrations (MIC) of selected AMP against Campylobacter jejuni, coli, and lari isolates.
Table 5. Minimum inhibitory concentrations (MIC) of selected AMP against Campylobacter jejuni, coli, and lari isolates.
Target Campylobacter spp. IsolateC12K-2β12
MIC (µg/mL)		Cecropin A–Magainin 2
MIC (µg/mL)		RL-37
MIC (µg/mL)
Campylobacter jejuni 141183.1501.6
Campylobacter jejuni 81-1161.612.53.1
Campylobacter jejuni 81-176 a3.1503.1
Campylobacter jejuni 11168 ^a3.1253.1
Campylobacter jejuni RM1221 a1.6501.6
Campylobacter jejuni A74C1.6503.1
Campylobacter jejuni A49943 *3.1253.1
Campylobacter jejuni A33250 *3.11003.1
Campylobacter jejuni A29428 *3.11006.3
Campylobacter coli Epi 33-WT1.6501.6
Campylobacter coli A49941 *1.61001.6
Campylobacter coli A33559 *3.11001.6
Campylobacter lari RM21001.6251.6
Campylobacter lari A35221 *3.11003.1
Campylobacter lari “slaughter beach”3.11003.1
a Alternate ATCC designations; ^ National Collection of Type Cultures (NCTC) isolate; * American Type Culture Collection (ATCC) isolate.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Line, J.E.; Seal, B.S.; Garrish, J.K. Selected Antimicrobial Peptides Inhibit In Vitro Growth of Campylobacter spp. Appl. Microbiol. 2022, 2, 688-700. https://doi.org/10.3390/applmicrobiol2040053

AMA Style

Line JE, Seal BS, Garrish JK. Selected Antimicrobial Peptides Inhibit In Vitro Growth of Campylobacter spp. Applied Microbiology. 2022; 2(4):688-700. https://doi.org/10.3390/applmicrobiol2040053

Chicago/Turabian Style

Line, John Eric, Bruce S. Seal, and Johnna K. Garrish. 2022. "Selected Antimicrobial Peptides Inhibit In Vitro Growth of Campylobacter spp." Applied Microbiology 2, no. 4: 688-700. https://doi.org/10.3390/applmicrobiol2040053

Article Metrics

Back to TopTop