P128 is a bacteriophage derived staphylococcal cell-wall degrading enzyme. This protein is under development in our laboratory for topical therapeutic use in humans. In this study, we tested the bactericidal activity of P128 protein on globally prevalent S. aureus clinical strains. We assessed the biological activity of P128 using various in vitro assays and under conditions designed to simulate physiological conditions.
P128 protein preparations used in this study were of > 95% purity. The protein expressed was in the soluble form in a standard E. coli
expression system and purified using a 2-step ion-exchange chromatography procedure [22
Susceptibility to P128 determined by MIC and MBC assay
Determination of MIC and MBC is a commonly used method to assess susceptibility to antimicrobial agents. We determined the MIC and MBC of P128 for a panel of 31 globally represented strains of S. aureus
using modified CLSI methods [23
]. Microtiter plate wells were pre-coated with BSA before adding P128 to minimize nonspecific adherence and loss of protein to the polypropylene surface. The MIC of P128 for the various strains of S. aureus
ranged from 1 to 64 μg/mL (Table ). The MIC at which 50% of the strains tested were inhibited (MIC50
) was 8 μg/mL. The MBC of P128 across S. aureus
strains tested also ranged from 1 to 64 μg/mL; and the MBC50
was found to be 16 μg/mL (Table ).
MIC and MBC of Vancomycin were determined using the same procedure that was used in case of P128. For the reference strain, S. aureus ATCC 25923 MIC and MBC of Vancomycin was found be 0.5 μg/mL and 2 μg/mL respectively. These values correlate with the reported MIC and MBC of Vancomycin for this strain, validating the assay used in this work. Vancomycin was also tested on a panel of S. aureus strains that represented the MIC range of P128 (1 to 64 μg/mL). MIC of Vancomycin for these strains ranged from 0.5 to 1 μg/mL and MBC ranged from 1 to 4 μg/mL (Table ).
MIC and MBC of Vancomycin against a panel of S. aureus isolates
Strains 1-6 were selected from a globally represented panel of distinct, typed clinical isolates (MSSA, strain 1; MRSA, strains 2-7) obtained from The Public Health Research Institute, New Jersey, USA; strain 7 is USA500/2, and 8 is S. aureus, ATCC 25923
Since MIC relates to growth inhibition activity of an antimicrobial agent, MBC may be a more appropriate measure of activity of P128 which is bactericidal in action.
Time-kill curve studies
Time-kill assays were performed in accordance with the CLSI guidelines, with a starting inoculum of 5 × 104 CFU/mL and, various multiples of the MICs. The objective of this assay was to evaluate concentration-dependent bactericidal activity. In order to find the optimal concentration required to achieve and maintain > 99.99% killing upto 24 h, sub-MIC levels were not considered. The detection limit of the time kill curve was 10 CFU/mL.
We determined the number of viable S. aureus cells remaining at different time intervals after adding P128 protein. Figure shows the time-kill curves of P128 for six representative strains of S. aureus, which included five MRSA strains and one MSSA strain. P128 showed rapid, dose-dependent bactericidal activity against the MSSA and MRSA strains tested, killing of 99.99% of cells in all six strains tested within 1 h at the respective MIC concentration. At the MIC, growth was inhibited up to 24 h for all five MRSA strains and up to 8 h for the MSSA strain (BK#9918). However, the cells of BK#9918 that grew after 8 h were susceptible to P128 (data not shown). Since a concentration 4× the MIC inhibited growth of this strain for up to 24 h, we surmised that higher concentrations of P128 or repeated treatments may be required in such cases.
Figure 2 Kill-kinetics of P128 on S. aureus strains. Time-kill curves of P128 at three different concentrations (MIC, MIC × 4, and MIC × 16) on five MRSA and one MSSA strains are shown. Cell control was maintained simultaneously for each strain. (more ...)
Efficacy of P128 gel formulation applied to S. aureus on agar surface
The efficacy of P128 hydrogel was tested on solid culture medium to simulate the conditions of topical nasal application.
The assay format was designed to check availability of the protein when applied as a gel formulation. The objective was also to test efficacy of P128 gel applied to a surface where low numbers of bacterial cells are present. We have used a range of 100-1 μg/mL of protein concentration in the gel formulation. P128 gel showed complete clearance at concentrations up to 1.56 μg/mL (Figure ).
Bactericidal activity of P128 against S. aureus COL in SNF
Functional efficiency and structural stability of enzymes can generally be influenced by pH, temperature, and the composition and concentrations of metal or inorganic ions in the reaction milieu. Our primary concern was that monovalent and divalent ions present in nasal fluid may have a deleterious effect on P128 activity. We therefore evaluated the activity of P128 in a composition that simulated the ionic content of normal human nasal fluid. We found that P128 reduced the staphylococcal viable count (CFU) by five orders of magnitude in SNF, comparable to the activity observed in case of P128 in physiological saline. Cells incubated in SNF that did not contain P128 were unaffected (Figure ). These results indicate that the protein would not be influenced by the ionic content of human nasal fluid.
P128 activity in simulated nasal fluid. Bactericidal activity of P128 against S. aureus strain COL was tested under conditions simulating the ionic composition of human nasal fluid.
Efficacy of P128 gel on nasal Staphylococci in their native physiological state
Secreted products and components such as exotoxins, exoenzymes, surface-associated adhesins, and capsular polysaccharide play a role modulating host responses to S. aureus
]. Production of capsular polysaccharide type 5 by Staphylococci has been reported in a study using a mouse model of S. aureus
nasal colonization [28
]. The same study also showed the inability of a capsule-defective mutant to persist in mouse nares, indicating that S. aureus
is encapsulated in the nares. The rate of methicillin resistance among CoNS isolates colonizing anterior nares of patients undergoing haemodialysis is reported to be higher than that of S. aureus
isolates; this is accompanied by the lack of susceptibility to other classes of antibiotics [7
]. Although S. epidermidis
is responsible for most CoNS infections, other CoNS species have been associated with a variety of human diseases [6
]. For example, S. haemolyticus
is the second most commonly encountered species in clinical infections, and S. lugdunensis
is a more recently described CoNS species [29
In this context, we evaluated the bactericidal activity of P128 on S. aureus and other staphylococcal species recovered from human nares. As the first step, we characterized the nasal commensal bacteria of 31 healthy people. Speciation was carried out using the HiStaph identification kit and the S. aureus carriage rate was also determined. Nasal Staphylococci of 71% of the healthy people sampled consisted of CoNS species, predominantly S. epidermidis and S. aureus was found in the remaining 29% of people. Other CoNS among nasal commensal bacteria included S. haemolyticus and S. lugdunensis (Table ). We examined nasal commensal populations in two randomly selected healthy people for comparability between the two nares with respect to bacterial load and staphylococcal species present and found both nares to be comparable (data not shown).
Speciation of nasal commensal Staphylococci of healthy people
Commensal bacteria recovered from nasal swabs of 31 healthy people were plated on blood agar, enumerated, and characterized by Gram stain, coagulase test, and speciation
We then evaluated the activity of P128 hydrogel on nasal Staphylococci of 31 healthy people.
In case of nasal swabs immersed in buffer-gel, colonies were numerous, ranging from 103 - 105 CFU; estimated based on results of a preliminary experiment, where S. aureus cells of known CFU counts (103, 104 and 105 CFU) were plated to vizualize the pattern of growth after overnight incubation of plates (data not shown). Of the swabs immersed in P128 hydrogel, 4/31 showed > 99.99% reduction in staphylococcal cell counts, 17/31 showed 99.9% reduction, 5/31 showed 99% reduction, and 5/31 showed 90% reduction (Table ). A few colonies that grew on the plate containing P128 were found to be sensitive to the protein when tested, and hence apparently escaped the protein action.
Efficacy of P128 gel on nasal Staphylococci in their native physiological state
This finding shows that P128 is bactericidal to nasal staphylococcal isolates. However, we did not evaluate the presence of capsular polysaccharides, which may be assessed in future studies in our laboratory. It is important to note that the cells were treated with P128 hydrogel immediately after isolation (i.e., without exposure to any other medium or subjection to any steps of cultivation). We conclude that both S. aureus and CoNS are susceptible to P128 in the physiological state relevant to nasal carriage. Considering the pathogenic potential and multidrug resistance of these species, it is significant that these species were fully sensitive to P128. Further studies are needed to determine the MIC and MBC of P128 on CoNS.
Reports point to the endogenous origin of most infective S. aureus
isolates and MRSA carriage poses an increased risk for invasive infections compared with MSSA carriage [30
]. The worldwide spread of MRSA strains, which are often multidrug-resistant [32
], combined with limited therapeutic options necessitates new approaches to combat this pathogen. Recent findings emphasize that commensal CoNS strains are also potential threats [33
]. Therefore an antibacterial agent, exemplified by P128, which can target antibiotic resistant S. aureus
as well as other clinically significant Staphylococci would meet the current medical need and warrants further development.