Emerging and re-emerging “superbug” infections are a global threat due to indiscriminate use of antibiotics. Methicillin-resistantStaphylococcus aureus(MRSA), one of the most lethal causative agents, has spread rapidly all over the world. To combat this global menace, the search for alternative antimicrobials is a pressing need, especially since most antimicrobials (glycopeptides) have exhibited decreased efficacy and susceptibilities against multidrug-resistant gram-positive infections. Antimicrobial peptides (AMPs) are gaining attention as a potential alternative to conventional antibiotics and small molecule drugs6-8. Earlier studies indicated that the cyclic molecules are among the most effective antimicrobials6,11. These studies have rendered AMPs as promising new classes of antibiotic candidates. A critical analysis of the altered ultrastructure of the cell membrane or the cell wall by electron microscopy (EM) enables us to understand the mechanisms of cell damage at sub-lethal to lethal concentrations of antimicrobial peptides. Earlier, we have reported the antibacterial spectrum and potency of ASP-1 against 29 bacterial isolates, including 12 MRSA isolates2,3. Our recent study revealed the narrow-spectrum of cyclic ASP-1 against methicillin/oxacillin resistantStaphylococcus(ORSA) and biofilm-forming bacteria had a lower chance of developing resistance3. The present study was aimed at determining the effect of reversed-phase high-performance liquid chromatography (RP-HPLC)-purified ASP-1 on the ultrastructure of MRSA-4, a clinical isolate along with a reference strainS. aureusATCC 29213 by scanning electron microscopy (SEM) and mode of action using a time-kill assay against a selected MRSA isolate.
The purification of ASP-1 was determined by a multistep process involving acid precipitation, solvent extraction, and adsorption with silica gel chromatography (230-400 mesh size) followed by semi-preparative and analytical reversed-phase high-performance liquid chromatography (RP-HPLC) as described earlier2,3. The purification process started with the preparation of cell-free supernatant (CFS) from the freshly cultured cells of the producer strainBacillussubtilisURID 12.1, which were grown at 37°C for 44h at 110rpm in modified tryptone soy broth (mTSB, pH 7.4±0.2), and the CFS was prepared by centrifugation at 12000rpm, 4°C for 20min. The bioactive fractions were pooled and further purified by RP-HPLC using a C18 column (4.6mm×250mm, particle size 5μm, Agilent, Santa Clara, CA, USA). The eluted active fraction with a retention time of 30.5min (data not shown) indicates its non-polar nature. The peptide purified at a semi-preparative scale was collected and tested for antistaphylococcal activity. These pooled active fractions, when subjected to re-chromatography on an analytical column, showed a single peak indicative of the pure fraction obtained.
In our previous study, we reported the antibacterial activity of ASP-1 against several MRSA isolates at a MIC of 0.3-16μg/ml and MBC of 2-128μg/ml, to be better than oxacillin2,3. In the present study, a total of 12 MRSA clinical isolates were used along with a reference strainS. aureusATCC 29213 to confirm the presence of two antibiotic resistance genes in MRSA isolates and one housekeeping gene specific toS. aureus. The bacterial cells were grown in Müeller Hinton Broth (MHB)/Agar (MHA, HiMedia, Mumbai, India). DNA was extracted from the standard test strain and test isolates using Power Lyzer® DNA isolation kit (MOBIO, HiMedia, Mumbai, India) following the manufacturer's protocol. Nucleic acid concentrations were estimated by NanoDrop™ spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). All indicator strains used in our previous study2,3were subjected to the detection ofmecA,femAandnucgenes by real-time (rt)-PCR as described by Li et al.13usingthefollowing primers:mecA1-5′-AAAATCGATGGTAAAGGTTGGC-3′,mecA2-5′-AGTTCTGCAGTACCGGATTTGC-3′14;nuc1-5′-GCGATTGATGGTGATACGGTT-3′,nuc2-5′-AGCCAAGCCTTGACGAACTAAAGC-3′10, andfemA1-5′-CAGTCACTAGCTGGGCCACTT-3′ andfemA2-5′-GTCAATTAGGTTATGTCGGAGGA-3′1. The amplification reaction was performed with Fast Start Universal SYBR Green Master (Roche Diagnostics, Indianapolis, IN, US) using 1μl of template and 40pmol each of forward and reverse primers. The gene targets were subjected to thermal cycling as follows: for 10min at 94°C and then 40 cycles of 30s at 94°C, 30s at 55°C, and 60s at 72°C for themecAgene primer set; 30 cycles of 45s at 94°C and 45s at 54°C, and 30s at 72°C forfemAgene primer set; 30 cycles of 30s at 94°C, 30s at 55°C, and 60s at 72°C fornucgene primer set. The reaction was performed by using a LightCycler® 96 Real-Time PCR system (Roche Diagnostics). The PCR products were then confirmed by 2% agarose gel electrophoresis.
To determine thein vitroantimicrobial activity of the test agent over time,i.e., the pharmacodynamics of the test agent (killing rate at different concentrations and time; or concentration- and time-dependent bactericidal or bacteriostatic activities), time-kill assays were conducted. MRSA-153(renamed as MRSA-5 in the present study) cells were subcultured in cation-adjusted Mueller-Hinton broth (CAMHB) (Hi-Media, India). Inoculum preparation, inoculation, and determination of the microbial count at respective MIC and MBC were performed as described previously3,15. MRSA-15 bacterial cells were washed using saline and adjusted to ∼5×104CFU/ml in CA-MHB and the test agent ASP-1 was added at final concentrations of 1× and 5× MIC and incubated at 37°C. In order to study the morphological and ultrastructural changes,S. aureusATCC 29213 and MRSA-4 cells were treated with ASP-1 at different concentrations and the changes were compared with the untreated cells of the two strains respectively using SEM. Bacterial cultures (108CFU/ml) were treated with ASP-1 at 1× MIC and 1× MBC for 0-12h and supernatant was drawn at hourly intervals. ASP-1-treated cells were then fixed on a clean grease-free coverslip using 2.5% glutaraldehyde in sterile phosphate-buffered saline (PBS, pH 7.3) at 4°C for 16h. Samples were washed thrice with sterile distilled water for 10min each, followed by treatment with osmium tetroxide as described elsewhere10. Subsequently, the samples were washed thrice with distilled water for 10min each; then treated with acetone gradient (30% to 100%) followed by critical point drying using CPD300 auto-program (Leica EM CPD300, Wetzlar, Germany). The dried specimens were mounted on stubs containing carbon adhesives and sputter-coated (Leica EM ACE600, Wetzlar, Germany) with gold. The images were captured by a Quanta 250 FEG (Thermo Fisher Scientific) at the SEM Facility, BITS Pilani, K. K. Birla Goa Campus, India.
Using the rt-PCR based identification approach, the presence ofnucandfemA(Supplementary Information Fig. S1A), andmecA, nucandfemAgenes (Supplementary Information Fig. S1B-D) was confirmed in all the 12 test isolates found to be resistant to oxacillin3. The amplification curves (Supplementary Information Fig. S1B-D) confirmed unequivocally the presence of themecA, nucandfemAgenes in all the MRSA isolates. The presence of the 300bp amplicon of the genefemA, and 270bp amplicon of the housekeepingnucgene determined by agarose gel electrophoresis confirmed the identity of all the test isolates and reference strain as beingS. aureus(Supplementary Information Fig. S2A and 2B). The presence of thefemAgene inS. aureusATCC29213 indicated that thefemAgene could also be present in methicillin-sensitive (mecA-negative)S.aureus16. ThemecAgene encodes for penicillin-binding protein-2 (PBP-2), and thefemAgene is responsible for penta-glycine inter-peptide bridge formation that is essential for cell wall peptidoglycan synthesis; these two activities together are responsible for a higher level of methicillin-resistance inmecA-positiveS.aureus13,16.
To determine the bacteriostatic or bactericidal nature of ASP-1 by a time-kill study we used MRSA-5 due to its sensitivity (MIC=16μg/ml) to test agent ASP-1 and resistance to oxacillin (MIC=1024μg/ml) revealed by the previous study3. The results in the form of a time-kill curve showed that our test agent ASP-1 was bacteriostatic at 1-5×MIC (Fig. 1), indicating that ASP-1 can possibly be used in combination with existing antibiotics for the treatment of highly resistant MRSA infections.
Previously, the time-kill curve of ASP-1 treatedS. aureusATCC 29213 cells showed reductions of 98.68% and 99.02% at 1× and 5× MICs respectively after 12h incubation3. Accordingly, the SEM images of ASP-treated ATCC29213 cells after 6 and 12h at 1× MIC and MBC (16μg/ml) showed extensive damages to the cell surface and distorted cell morphology, bringing forth interesting ultrastructure alterations (Fig. 2e, f, h and i). However, ASP-1 treated bacterial cells at 0h showed smooth cell surfaces with no discernible ultrastructural changes (Fig. 2b and c) and no ultrastructure alterations were observed in ASP-1 untreated cells at the three different time points (Fig. 2a, d and g).
For the present SEM study, the MRSA-4 isolate was selected for the ASP-1 treatment as this clinical isolate, which is resistant to oxacillin (32μg/ml), cefoxitin (>8μg/ml), vancomycin (>16μg/ml), mupirocin (>4μg/ml) and amoxicillin/clavulanate (>4/2μg/ml), was found to be susceptible to ASP-1 (MIC=8μg/ml)2,3. ASP-1 untreated bacterial cells were healthy with nearly spherical shape and smooth intact surface, and in the actively dividing stage showing the presence of septa at different time points (Fig. 3b, e and h). Scanning electron micrographs of ASP-1 treated cells (Fig. 3f and i) revealed surface deformities though cells at 0h showed normal morphology (Fig. 3c). In some fields, several cells showed minor depression on the cell surface, which might be due to vacuum artifacts during SEM processing; a similar observation was also previously noticed inS. aureuscells after SEM processing9.
The scanning electron micrographs (Fig. 2e and h) of ASP-1 (1×MIC=8μg/ml, 1×MBC=16μg/ml)-treated bacterial cells showed membrane distortions with surface depressions, and biconcave appearances with dents compared to ASP-1 untreated cells (Fig. 2a, d and g) at three different time points 0, 6 and 12h. The formation of holes on the cell surface and leakages of cellular contents were visible when the cells were incubated for 12h with ASP-1 at the same concentration (Fig. 2h). A similar ultrastructural change was observed at 16μg/ml (1×MBC) of ASP-1 treated cells after 12h of incubation (Fig. 2i) as compared to untreated cells. The majority of ASP-1 treated cells in many fields exhibited central depression or collapses with discernible holes on the cell surfaces9(Fig. 2e, f, h and i). Irregular cluster formation of deformedS. aureusATCC 29213 cells was observed when exposed to 1× MBC (Fig. 2f and h); this observation was consistent with the previous observation made by Greenwood and O’Grady (1972). When the effect of oxacillin (used as positive control) at 32μg/ml (1× MIC) on MRSA-4 cells was studied at different time points (Fig. 3a, d and g), the cell surface exhibited a dimpled and deformed appearance at 12h; the SEM image (Fig. 3g) also revealed a cell with a ring-like depression encircling a central perforation.
It is quite evident fromFigure 3f and i that exposure to ASP-1 at 1× MBC caused damages to different extents to the MRSA-4 cell surface as evidenced in dents and pit formations. The scanning electron micrograph (Supplementary information Fig. S3a and b) at higher magnification revealed the cell surface roughness, deep perforations or holes, along with complete lysis, collapse and shrinkage of several cells. When treated with oxacillin, the SEM images (Supplementary information Fig. S4a and 4b) exhibited dimpled and deformed MRSA-4 cells at 12h with ring-like depressions encircling a central perforation (Fig. S4a).
Membrane bleb formations were also observed in the antimicrobial peptide SMAP-29-treatedS. aureuscells with blebs at septum in actively dividing cells15. Some ASP-1 treated cells at 1× MBC displayed altered shapes and formation of membrane blebs as evident inFig. 2h, and i; similar observations were made in a separate study where membrane bleb formations were observed in human defensin 5-treatedEscherichiacoli5. The ultrastructure deformities at various concentrations of ASP-1 treated cells also corroborate the previous findings of the antimicrobial peptide DP-7 treated cells where membrane protuberances and plicated cell surfaces were recorded17. In a previous study, structural disorganization of phenothiazine drug methdilazine-treatedS. aureuscells was reported to be similar to ASP-1 mediated ultrastructural damage4.
These observations taken together indicate that ASP-1 may be a unique antibacterial peptide exerting its effect through membrane-targeted pore formation and membrane disruption, which rarely induce drug-resistance in bacteria12. This study suggests that since the test drug had strong anti-staphylococcal and anti-MRSA activity by probably acting on the cell surface, it could present a low risk of developing drug resistance, and therefore ASP-1 might be thought as an alternative to traditional antibiotics, especially those used for the treatment of skin and mucosal infections.
Conflict of interest
The authors declare that they have no conflicts of interest.
ACKNOWLEDGMENTS
The authors are thankful to the host University contingency. The laboratory assistance of Swetha Ramesh, a Department of Biotechnology (DBT)-sponsored Senior Research Fellow and Ms. Anviksha, a Masters in Engineering (M.E.) Biotechnology student are highly acknowledged.