Article pubs.acs.org/jmc
Synthesis and Biological Evaluation of Novel Peptide BF2 as an Antibacterial Agent against Clinical Isolates of Vancomycin-Resistant Enterococci Kusum Singh,† Suresh Kumar,† Shashank Shekhar,† Benu Dhawan,‡ and Sharmistha Dey*,† †
Department of Biophysics and ‡Microbiology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110 029, India S Supporting Information *
ABSTRACT: Enterococci are the leading cause of nosocomial infections worldwide and acquired resistance to a variety of antibiotics. Antimicrobial peptides represent a promising molecule against the antibiotic resistance in bacteria and an indispensable component of the innate immune system. The aim of the study was to develop an antimicrobial peptide against vancomycin-resistant enterococci (VRE). We have designed a series of peptides based on Sapecin B as template. An in vitro antibacterial study of synthetic peptide BF2 against the clinical isolates of vancomycin-resistant and control strains of enterococci showed rapid killing effect on enterococci by killing 99.9% of bacterial cells in 60 min and susceptibility at minimum inhibitory concentration (MIC) range of 6.25−12.5 μg/mL. Synergy of BF2 was observed in combination with vancomycin and teicoplanin. The peptide was bactericidal and nontoxic to mammalian cells. An in vivo study also revealed the antibacterial activity against enterococci-infected Wistar albino rats. BF2 may be used synergistically with antibiotics.
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INTRODUCTION Enterococci are Gram-positive bacteria that are important pathogens responsible for serious infections but are also part of the normal intestinal flora of humans and animals. Although many Enterococcus species have been identified, only two are responsible for the majority of human infections, i.e., E. faecalis and E. faecium. The major reason for survival of these organisms is the development of resistance to currently available antibiotics either by mutation or by receipt of foreign genetic material through the transfer of plasmids and transposons1 with increasing antibiotic resistance; enterococci are recognized as feared nosocomial pathogens that can be a therapeutic challenge for alternative antibiotics. Vancomycin is the ultimate drug for the multidrug-resistant Gram-positive bacteria like enterococci. Because of the emergence of vancomycin-resistant enterococci (VRE) as nosocomial pathogens, there is a severe crisis in hospitals. A high percentage of hospital-acquired infections are caused by highly resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and VRE. E. faecalis and E. faecium, which cause many serious infectious diseases, are clinically isolated and represent the most prominent VRE.2−4 A very essential need in recent years is an effort to develop a new alternative for vancomycin. Antimicrobial peptides (AMPs) are one of the key elements of the innate immunity against bacteria.5 The knowledge acquired in the past two decades of a new group of AMPs, having mainly cationic group, makes them the basic element of a novel generation of drugs for the treatment of © XXXX American Chemical Society
bacterial infection that are ideal for the fast and efficient defense against microbes.6−8 In the present study, we have designed a peptide based on the sequence of Sapecin B, an antibacterial protein of Sarcophaga peregrina (flesh fly), effective against Gram-positive bacteria9,10 and assessed for in vitro and in vivo antibacterial activity against E. faecalis and E. faecium and clinical isolates of the same.
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RESULTS Synthesis, Purification, and Characterization of Peptides. In our previous paper, we reported one designed antimicrobial peptide based on Sapecin B as a template, which was active against MRSA and methicillin-sensitive Staphylococcus aureus (MSSA) and not against VRE.11 In this study, another series of peptides were designed by modifying the same template. Nine peptides were synthesized by replacing the terminal residues with cationic and hydrophobic amino acids. The peptides were purified by reverse-phase high-performance liquid chromatography (RP-HPLC), and the purity of BF2 was found to be ≥95% and K′ was 0.38 in solvent 1 (acetonitrile) and 0.48 in solvent 2 (methanol). The purity and K′ for both the solvents of other peptides are shown in Supporting Information, Table S1. The mass of BF2 Received: June 25, 2014
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dx.doi.org/10.1021/jm500960s | J. Med. Chem. XXXX, XXX, XXX−XXX
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comparable to the positive control as linezolid decreased 93.8% bacterial cell counts. The quantitative analysis of bacterial load in the blood of all three groups of rats infected by enterococci and treated with BF2 showed a decrease in the number of bacterial counts significantly (P < 0.0001) in a dose-dependent manner as compared with untreated rats (Figure 4).
determined by electrospray ionization and quadrupole time-offlight (ESIQ-TOF) was 1361.87 Da (Supporting Information, Figure S1). Minimum Inhibitory Concentration and Minimum Bactericidal Concentration Determination. All the peptides were tested for antibacterial activity against VRE (Table 1). BF2 (RWRLLLLKKH) peptide showed the best MIC among all the peptides. Hence, all other in vitro and in vivo studies were performed with BF2 peptide.
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DISCUSSION AND CONCLUSION A high percentage of hospital-acquired infections are caused by highly resistant bacteria such as MRSA and VRE. Due to the development of resistance against vancomycin, it is an urgent need to find a new alternative to solve this crisis. In this study we have focused on the development of AMPs against VRE. AMPs have a lot of advantages over conventional therapy, as key components of the innate immune system, like broad spectrum, rapid killing, and neutralizing endotoxin.13 Because of the wide range of biological functions of AMPs studied in recent years, they are used as a blueprint for the development of alternative therapy. In our previous study, the peptide RWRLLLLLRΔL(SD-8) was found to be antibacterial against MRSA and MSSA.11In this study, we have designed another series of peptides with the same template by interchanging the terminal amino acids with cationic and hydrophobic residues and reducing the internal leucine chain. In this series of 9 peptides, only 5 peptides were active against E. faecalis and E. faecium and not against MRSA and MSSA. The MIC of BF2 was the lowest among all the active peptides against VRE. These two enterococci, E. faecalis and E. faecium, acquired resistance due to the acquistion of different genetic information and are involved in alternation of the terminal amino acid residue of normal-condition D-alanyl-D-alanine, to which vancomycin binds. Some antimicrobial peptides are reported to have a killing activity aginst VRE12,14 with very low killing time. Time kill studies showed a rapid bactericidal effect of BF2 against the enterococci. In the present study, in vitro experiments with BF2 were performed to determine their bactericidal activities and to determine whether synergism, antagonism, or indifference would be the predominant response when these peptides were tested in combination with other clinically used antibiotics against enterococci. Peptides are synergistic with conventional antibiotics and do not easily select resistant mutants. The present AMP, BF2, was found to be synergistic in combination with clinically used glycopeptides antibiotics. BF2 was nontoxic to both human cells (RBCs and HEK 293) at a high concentration, even at 5-fold MIC, despite being highly active at its antibacterial concentration against the enterococci. The main focus of this study was to check the antimicrobial activity of BF2, so for the in vivo study, just after 1 h of infection, the blood was collected to perform the experiment. All the animals survived after infection and were sacrificed after 2 days. Hence, the in vivo study also confirmed the antibacterial effect of BF2 on rats infected with enterococci. It can be concluded that BF2 peptide may be considered as a potential molecule for the future design of antimicrobial drugs against enterococci.
Table 1. Primary Screening of Synthesized Peptides against VRE (ATCC 29212 and Clinical Isolates) peptide a
SD-8 BF2 BF3 BF4 BF5 BF6 BF7 BF8 BF9 BF10
sequence
MIC (μg/mL) range
RWRLLLLLRΔL RWRLLLLKKH RWRLLLLKKR RWRLLLLKKF HWRLLLLKKH RWRLLLLKRH RWRLLLLKWH RWRLLLLLRWR RWRLLGLLKRH RWRLLLLLKRH
N/A 6.25−12.5 N/A 25−50 25−50 12.5−25 N/A N/A N/A 25−50
a
SD-8, a novel therapeutic agent active against multidrug-resistant Gram-positive cocci.11
BF2 inhibited the growth of ATCC 29212 and ATCC 51299 at 25 μg/mL. In contrast, the clinical isolates of resistant enterococci (E. faecalis and E. faecium) showed more susceptibility with BF2 in the MIC range of 6.25−12.5 μg/ mL (Table 2). The minimum bactericidal concentration (MBC) of BF2 was in the range of 25−50 μg/mL. Time Course Killing Assay. BF2, at a concentration of two times MIC, showed a rapid bactericidal effect for both ATCC 29212 and the clinical isolate at 60−120 min exposure period with a decrease of 3 log10 CFU/mL (Figure 1). Cytotoxicity Assay. At 5-fold MIC of BF2, the hemolysis was 0.5−4, no interaction; and >4.0, antagonism.20 In Vivo Studies. In vivo antibacterial activity of BF2 was performed by dose-response manner on Wistar albino female rats of 40−45 days old in 5 groups (3 rats per group) of 125−150 g per rat. Enterococci was grown in the MH broth after reaching the log phase of growth. The suspension was centrifuged at 1000g for 15 min. The supernatant was discarded, and the bacteria were diluted in PBS to achieve a concentration of 4 × 106 CFU/mL of PBS. All rats were inoculated intravenously with 4 × 106 CFU/mL of enterococci cells. After 1 h of inoculation, rats were treated in group I with 12.5 mg/kg (MIC) BF2, in group II with 25 mg/kg (2MIC), and in group III with 37.5 mg/kg (3MIC) intravenously (i.v.) . Rats in group IV were injected with 0.1 mL of PBS i.v., which served as the vehicle (negative control). Rats in group V were treated with 25 mg/ kg of linezolid i.v. (positive control). To perform the quantitative evaluation of the bacteria in the blood of all groups, the blood samples were taken from the tail vein of rats after 1 h of BF2 treatment and plated on blood agar plate. The plates were incubated at 37 °C in ambient air overnight. The number of enterococci cells was counted the following day. The main focus of this study was to check the antimicrobial activity of BF2; hence, after 1 h infection the blood was collected to perform the experiment. All the animals survived in healthy condition after treatment and were sacrificed after 2 days. Statistical Snalysis. Statistical analysis was carried out by one-way ANOVA test (P < 0.001). The values were represented as mean ± SD.
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ACKNOWLEDGMENTS The authors acknowledge financial support from the Council of Scientific and Industrial Research and AIIMS for the fellowship of K.S. and S.K., respectively.
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ABBREVIATIONS USED AMPs, Antimicrobial peptides; VRE, Vancomycin-resistant enterococci; DMF, Dimethylformamide; HBTU, 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; MIC, Minimum inhibitory concentration; MBC, Minimum bactericidal concentration; MRSA, Methicillin-resistant Staphylococcus aureus; MSSA, Methicillin-sensitive Staphylococcus aureus; PBS, Phosphate-buffered saline; hRBCs, Human red blood cells; FIC, Fractional inhibitory concentration; MH, Mueller−Hinton; RP-HPLC, Reverse-phase high-performance liquid chromatography; ESI-QTOFMS, electrospray ionization and quadrupole time-of-flight mass spectrometry
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