Bovine mastitis is the most important production disease in dairy farming, causing redness, pain, a warm and swollen udder, and abnormal milk that causes mammary gland inflammation.1,2 It reduces product yield and causes mortality, resulting in an economic loss of $US131 per cow per year.3 The etiological agents can be contagious (eg, Staphylococcus aureus, Streptococcus agalactiae, Mycoplasma spp) or environmental (eg, Escherichia coli, Enterococcus spp, Streptococcus uberis) and include a range of gram-positive and gram-negative bacteria.4 Mastitis infections can be classified as either subclinical mastitis (SCM) or clinical mastitis (CM).1 Subclinical mastitis is defined as an infection without local inflammation and hence no visible indications.2 Conversely, CM is an inflammatory reaction that results in clearly abnormal milk.1,2,4 Antibiotic therapy is still an important part of mastitis control regimens today. However, antimicrobial resistance (AMR) to antibiotic treatments is becoming more common in human and animal infections, posing a global threat. AMR develops when bacteria can overcome the effects of previously effective antibiotics, necessitating the search for new treatment options.5 Antimicrobial peptides (AMPs) generated from synthetic and natural sources are excellent candidates for overcoming AMR. They display broad-spectrum antimicrobial activity with high specificity and minimal toxicity.6 Many researchers have used AMPs from organisms and their derived peptides to treat a range of ailments over the last half-century.7,8 The antimicrobial peptide database was updated on April 25, 2022, with a total of 3,324 AMPs (https://aps.unmc.edu/). The majority of AMPs are cationic and amphipathic, with an average of 5 to 40 amino acid residues.9 They are regarded as amphipathic because they have both hydrophobic and hydrophilic units, which are thought to contribute to their broad spectrum of antibacterial activity. Toxicity, lack of stability, and high production costs are a few challenges in applying AMPs as clinical candidates. Natural AMPs or synthetic AMPs based on natural templates are bioactive proteins that have low potential and do not promptly conduce to resistance when compared with conventional antibiotic, because they have an electrostatic interaction that results in rapid bacterial cell death, a multimechanism for bacterial attacking. Moreover, they are innate immune modulators that act through the immune system resulting in less possibility of resistance.10,11 Developing shorter and more readily available AMPs, also known as short antimicrobial peptides, can help solve this problem.12 Short peptides comprising only Leu and Lys units have shown to be effective against gram-negative and gram-positive bacteria.13
Pm11 is a nonribosomally derived peptide that is produced from pleurocidin, an alpha-helix cationic family found in Pleuronecies americanus.14,15 Since finding the active center, scientists have been deleting and replacing amino acids in the natural polypeptide pleurocidin to create a new antibacterial polypeptide with bactericidal properties.15 Zhang and coworkers15 reported that pleurocidin congeners, including Pm11, were found to have activity against Streptococcus mutans, Streptococcus sanguinis, and Streptococcus sobrinus, which are common cariogenic microorganisms that operate directly by transferring their genetic material into the target and altering normal cell processes or passively through inflammation or immunological suppression.15 In the current study, we further investigated the antimicrobial activity of Pm11 in terms of minimum bactericidal concentration (MBC), half-maximum inhibitory concentration (IC50), and time-kill kinetics against bovine mastitis pathogens such as Streptococcus agalactiae SCM1084 and Streptococcus uberis SCM1310. The bacteria were screened at the Large Animal Hospital and Students Training Center (Nakornpathom), Faculty of Veterinary Science, Chulalongkorn University, Thailand.
Materials and Methods
Microorganisms
Bovine mastitis pathogens were obtained from clinical (CM) and subclinical mastitis (SCM) cows including Escherichia coli (SCM1249), Klebsiella spp (SCM1282), Staphylococcus aureus (CM967), Streptococcus agalactiae (SCM1084), and Streptococcus uberis (SCM1310). Mastitis bacterial strains were determined as described by the National Mastitis Council.16 The bacteria stored in glycerol stock (stored at −20 °C in brain heart infusion [BHI] broth with 25% glycerol) were grown on fresh BHI broth at 37 °C and then subcultured on blood agar.
Preparation of Pm11 peptide
The Pm11 peptide was commercially synthesized (GeneScript with HPLC purify ≥ 95.0%). It was a short polypeptide chain consisting of 13 amino acids (WFKFFKKFFKKWK) with a theoretical molecular weight of 1895.35, and solubility in phosphate-buffered saline (PBS) was < 2 mg/mL. Pm11 stock solution was prepared by dissolving 1 mg/mL of Pm11 powder in PBS, and the concentrations were adjusted to 160, 80, 40, 20, 10, 5, 2.5, 1.2, and 0.6 μM by a 2-fold serial dilution.
Bactericidal analysis
The MBC was performed to determine antimicrobial activity.17 Serially diluted Pm11 peptide and approximately 105 bacterial colony-forming units (CFU)/mL of mastitis pathogens were added to the wells of a 96-well plate. The mixtures were incubated for 3 hours at 37 °C with slow shaking at 100 rpm. After incubation, 10-fold serial dilution of the mixtures was obtained in PBS, and 100 μL of each dilution was spread on tryptic soy agar (TSA) (Merck) plates. The plates were incubated overnight at 37 °C to observe colonies of viable cells for calculating CFU per milliliters. For drop plate assay, 3 μL of each dilution was dropped on TSA plates to demonstrate the inhibition activity of the Pm11 peptide at various concentrations in the same plate. In this assay, tryptic soy broth (Merck) medium was applied as the negative control, and bacterial culture without peptide was used as untreated control. IC50 is the amount of an inhibitory agent required to stop a biological process in half. GraphPad prism5 was used to determine the IC50.
Time-kill assay
The in vitro bactericidal properties of Pm11 against bovine mastitis pathogens were evaluated at various time points using a time-kill assay.18 The bovine mastitis pathogens at a concentration of 105 CFU/mL were added with Pm11 antimicrobial peptide at the MBC. The mixture was cultured at 37 °C with slow shaking for 12 h. During incubation, 100 μL of the sample was taken at different time intervals such as 0, 1, 2, 4, 8, and 12 h and serially diluted in sterile PBS. Each dilution was plated onto TSA plates, and the plates were incubated overnight at 37 °C to calculate the viable colonies in CFU per milliliters.
Minimum hemolytic concentration
Pm11 cytotoxicity was measured using the minimum hemolytic concentration method on sheep red blood cells (RBCs) supplied from Clinag Co, Ltd. The sheep RBCs were centrifuged to remove the buffy coat for 10 min at 3,000 rpm at 25 °C and then washed multiple times with PBS until the bright color was achieved. Then, sheep RBCs were finally suspended in PBS at a concentration of 4% (vol/vol). Furthermore, in a sterile 96-well plate, 100 μL of suspended sheep RBCs was mixed with 100 μL of Pm11 peptide at final concentrations of 160, 80, 40, 20, 10, 5, 2.5, 1.2, 0.6, and 0.3 M. The mixtures were incubated at 37 °C for 1 h. After incubation, the mixtures were centrifuged at 25 °C for 5 min at 1,000 X g. Optical density of the supernatant was measured at 414 nm in a UV spectrophotometer. The percentage of hemolysis was calculated as described previously.19
Scanning electron microscopy
The visual attack of the Pm11 antimicrobial peptide on gram-positive pathogen S aureus CM967 and gram-negative pathogen E coli SCM1249 was observed via scanning electron microscopy (SEM). The pathogens were incubated with 1X MBC of Pm11 for 1.5 hours. After that, the cells were collected and fixed using 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2), kept at 4 °C for at least 4 h, and then filtrated using a microfilter of 0.2 μm. The filtrated paper was dehydrated by a series of different ethanol concentrations: 25%, 50%, 75%, 95%, and 100% ethanol. A critical point drying machine was used to dry the sample. After that, the samples were mounted on stub with glue and gold-coated by a sputter coater machine. The samples were observed under a scanning electron microscope and energy dispersive x-ray spectrometer (JEOL InTouchScope series SEMs, the JSM-IT500HR).
Results
Antimicrobial activity of Pm11 peptide to bovine mastitis pathogens was conducted after determining the MBC of Pm11 peptide to bacterial lab strains such as E coli DH5α and Bacillus subtilis.20 Pm11 peptide had the most potent activity against E coli SCM1249 with an MBC of 2.5 µM and IC50 of 0.32 µM. It was followed by MBC 5 µM to gram-positive pathogens such as S agalactiae SCM1084, S uberis SCM1310, and S aureus CM967, with an IC50 of 0.87, 0.97, and 1.33 µM, respectively. Moreover, the pathogen Klebsiella spp SCM1282 was inhibited by Pm11 peptide with an MBC of 10.0 µM and IC50 of 2.07 µM (Table 1). The drop plate assay showed that the antimicrobial activity of Pm11 at MBC concentration inhibited the growth of E coli SCM1249, S agalactiae SCM1084, S uberis SCM1310, S aureus CM967, and Klebsiella spp SCM1282 (Figure 1). The results showed that Pm11 peptide at concentrations of 0.1 to 10 µM significantly reduced bacterial growth and eliminated pathogen colonies at MBC values as compared with those of untreated controls.
Antimicrobial activity of Pm11 against bovine mastitis pathogens.
| Pathogenic microorganisms | MBC (μM) | IC50 (μM) |
|---|---|---|
| E coli SCM1249 | 2.5 | 0.32 ± 0.03 |
| S agalactiae SCM1084 | 5.0 | 0.87 ± 0.18 |
| S uberis SCM1310 | 5.0 | 0.97 ± 0.15 |
| S aureus CM967 | 5.0 | 1.33 ± 0.19 |
| Klebsiella spp SCM1282 | 10.0 | 2.07 ± 0.47 |
Drop plate assay of bovine mastitis pathogens, Escherichia coli SCM1249 (A), Streptococcus agalactiae SCM1084 (B), Streptococcus uberis SCM1310 (C), Staphylococcus aureus CM967 (D), and Klebsiella spp SCM1282 (E). Pathogens (105 CFU/mL) were treated with Pm11 peptide at various concentrations for 3 h. The 3 μL of 10-fold serial dilution of the treated bacteria were dropped on tryptic soy agar plates and incubated at 37 °C for overnight.
Citation: American Journal of Veterinary Research 84, 1; 10.2460/ajvr.22.06.0096
Time-kill kinetics of the Pm11 peptide on bovine mastitis pathogens was examined after determining the same MBC. The Pm11 peptide at different MBCs was used to treat E coli SCM1249 (MBC = 2.5 µM), Klebsiella spp SCM1282 (MBC = 10 µM), S uberis SCM1310 (MBC = 5 µM), S aureus CM967 (MBC = 5 µM), and S agalactiae SCM1084 (MBC = 5 µM), for 0, 1, 2, 4, 8, and 12 h. The Pm11 peptide time-kill kinetics to pathogens were graphed in log10 bacterial colony count over time (Figure 2). The results revealed that the Pm11 peptide caused the complete cell death of S uberis SCM1310 and S agalactiae SCM1084 from 105 to 0 CFU/mL within 1 h. Similarly, E coli SCM1249 and S aureus CM967 were also completely killed by the Pm11 peptide within 4 h. However, the Pm11 peptide could reduce the cell density of Klebsiella spp SCM1282 from 105 to 103 CFU/mL within 4 h and after which Klebsiella spp SCM1282 could grow again.
Time-kill kinetics of Pm11 antimicrobial peptide on bovine mastitis pathogens show in various incubation times. Each value shown is the mean ± standard error of the mean from three experiments.
Citation: American Journal of Veterinary Research 84, 1; 10.2460/ajvr.22.06.0096
The impact of the Pm11 peptide targeting S aureus CM967 and E coli SCM1249, the representative of gram-positive and gram-negative bovine mastitis pathogens, was observed using SEM compared with the untreated control (Figure 3). The results showed that the untreated control cells were unaffected and appeared smooth without cell lysis. Meanwhile, the surface of the cells treated with Pm11 peptide induced dramatic morphological changes such as blebbing, roughening, nicks, shape loss, and formation and accumulation of cell debris.
Morphological observations of E coli SCM1249 (A–D) and S aureus CM967 (E–H) treated with Pm11 antimicrobial peptide (A, B, E, and F, untreated control; C, D, G, and H treated with Pm11 at minimum bacterial concentrations for one and half hours). The blue arrows indicate cell roughening, nicks, and blebbing. The green arrows indicate shape loss, cell debris, and accumulation of cell debris. (A, C, and E–H: bar = 1 μm; B and D: bar = 500 nm).
Citation: American Journal of Veterinary Research 84, 1; 10.2460/ajvr.22.06.0096
The capability of the Pm11 peptide to lyse the erythrocytes was measured after incubation of different peptide concentrations varying from 0 to 160 μM with the sheep red blood cells. The results showed (Table 2) that the Pm11 peptide at 0.3 to 80 μM was exceptionally low hemoglobin release. The hemolysis was just 10.19% even at the maximum concentration of 160 μM.
Hemolytic activity of Pm11 antimicrobial peptide on sheep red blood cells.
| Final concentration of peptide (μM) | Average percent hemolysis |
|---|---|
| 160.0 | 10.19 ± 2.29 |
| 80.0 | 1.96 ± 0.13 |
| 40.0 | 0.44 ± 0.44 |
| 20.0 | 0.15 ± 0.23 |
| 10.0 | 0.65 ± 0.22 |
| 5.0 | 0.34 ± 0.31 |
| 2.5 | 0.06 ± 0.08 |
| 1.2 | 0.22 ± 0.20 |
| 0.6 | 0.13 ± 0.23 |
| 0.3 | 0.09 ± 0.16 |
| 0.0 | 0.00 ± 0.00 |
Discussion
Mastitis is one of the most dangerous production diseases in dairy farming. CM affects 20% to 40% of all lactating cows annually.3 Furthermore, subclinical (invisible to the naked eye) mastitis affects 10% to 30% of all lactating cows on a farm at any given time. Antibiotics are generally used in dairy cattle to cure or prevent diseases and boost milk output and feed efficiency. Although antibiotics remain the main treatment strategy, their effectiveness is limited, and the emergence of antibiotic-resistant bacterial strains has become a major concern in antibiotic treatment.21,22 As a result, hundreds of peptide antibiotics have been developed in the last half-century, demonstrating the therapeutic potential of peptides generated from natural peptides.23 Pm11 peptide, which is generated from the natural antibiotic pleurocidin, has an alpha-helix structure with +6 net charged lysine as cationic and phenylalanine and tryptophan as hydrophobic amino acids and is an amphipathic antimicrobial peptide.15 In a study, Zhang et al15 reported that significant antibacterial activity of Pm11 peptide inhibited S mutans within 30 min at the 33.8-μM concentration, which is consistent with the current study, in which Pm11 peptide inhibited S agalactiae SCM1084 and S uberis SCM1310 within 1 h at the 5.0-μM concentration. In the current study, the Pm11 peptide was shown to have microbicidal activity against both gram-positive and gram-negative bovine mastitis bacteria, demonstrating broad-spectrum antibacterial activity similar to that of its parent peptide, pleurocidin.14 However, in this investigation, the Pm11 peptide failed to kill Klebsiella spp. The findings were consistent with a prior study that reported the penetration inhibition of AMPs by the extracellular polysaccharide capsule of Klebsiella pneumoniae, resulting in reduced effectiveness.24 K pneumoniae is also known as hypermucoviscous, due to the presence of a mucoviscous exopolysaccharide bacterial coating capsule that is tough than other gram-negative capsules. They aid infection by concealing the pathogen from immune detection, and they are particularly effective against peptide-based antimicrobials.24,25 When a peptide interacts with a bacterial capsule, antimicrobial peptide structural changes occur, causing sequestration and preventing the peptide from reaching its pathogen membrane target.26,27 As can be seen in the SEM micrographs, the appearance of bleb morphology, including blebs at the division plane, cell pole, and multiple blebs found on E coli SCM1249 with Pm11 peptide treated, indicated the membrane damage and periplasmic leakage. This outcome was similar to bleb development, cellular elongation, and clumping in E coli treated with human defensin 5.28
Furthermore, the self-assembly of AMPs with the diphenylalanine motif has been linked to antimicrobial activity.29 Similarly, the linkage of Pm11 peptide (WFKFFKKFFKKFK) with diphenylalanine amino acid may induce amyloid oligomer interaction with the polar head group on the bacterial membrane, known as lipid coaggregation. Diphenylalanine interacts with gram-negative membranes, causing outer membrane permeation and inner membrane depolarization and significant alterations in membrane shape, such as the development of nicks and rips.30 The hemolytic activity of peptides that affected hemoglobin release in mammalian RBCs was reported to be associated with charge and hydrophobicity.31 The ability to form amphipathic molecules of peptides has been linked to improved antimicrobial activity and increased hemolytic activity once a threshold of hydrophobicity has been reached.32,33 Trp, Lys, and Arg are the amino acids that strongly affect hemolytic activity through hydrophobicity or charge. Through the indole moiety, tryptophan residues are implicated in the binding of peptides to cholesterol found in biological membranes.34 Hemolysis is the most applied initial toxicity assessment. New medicines with potential for systemic use must have low toxicity to erythrocytes.19 The in vitro hemolysis guidance for the pharmaceutical scientist reported that the hemolytic cutoff for the various species is represented in percent. Any hemolysis value below 10% is considered nonhemolytic, whereas values above 25% are at risk for hemolysis.35 Although the Pm11 amino acid sequence contains more than 50% hydrophobic residues and has a strong basic charge, the results suggest that the Pm11 peptide has a low hemolytic activity to lyse sheep RBCs and is emerging as an attractive therapeutic agent with a high clinical potential.
Acknowledgments
This research was supported by Agricultural Research Development Agency, Thailand (grant No. CRP6305032110).
The authors declare that there were no conflicts of interest.
We thank Enago for English language editing and Dr. Sirin Saranyuthanon for kindly proofreading the revised edition.
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