Bacteriophages represent a rich and unique resource of anti-infectives to counter the global problem of antibiotic resis- tance. In this work, we assessed the bactericidal activity of two virulent staphylococcal phages, K and 44AHJD, against S. aureus isolates of clinical significance, and tested their efficacy in vivo. The phage cocktail lysed >85% of the clinical isolates tested. Both the phages were purified by ion-exchange column chromatography following propagation in bioreactors. The purity profiles of the ion-exchange purified phages were compared with those of phages purified using cesium chloride density gradient ultracentrifugation, and infectiousness of the purified phages was confirmed by plaque forming assay. The in vivo efficacy of a phage cocktail was evaluated in an experimental murine nasal colonization model, which showed that the phage cocktail was efficacious. To our knowledge, this is the first report of phage use in an in vivo model of nasal carriage.
During the last century, the human pathogen Staphylococcus aureus has become the main cause of nosocomial and community-acquired infections worldwide [
The emergence of MRSA in both hospital and community settings has prompted researchers to try to develop methods for the nasal decolonization of MRSA and methicillin-susceptible Staphylococcus aureus (MSSA) in specific patient groups. In the UK, it is recommended that MRSA carriers who are receiving prophylaxis for an operation should undergo nasal decolonization with mupirocin, the most commonly used antibiotic for Grampositive bacteria [
Squalamine, a water-soluble natural polyaminosterol isolated from the tissues of the dogfish shark (Squalus acanthias), has a 10,000-fold higher antimicrobial activity towards S. aureus than mupirocin [
The effectiveness of bacteriophages for phage therapy against pathogenic bacteria in both animals and humans is well documented [
It is generally accepted that virulent phages are more suitable candidates for therapeutic applications than temperate phages. The complications associated with temperate phages, such as super-infection immunity and possibility of integration into the host genome (lysogeny), and the possibility of transfer of genetic material, such as drug-resistance genes during infection, make temperate phages unsuitable for therapeutic purposes. It has been reported that free-living and virulent S. aureus phages in the environment are relatively low in numbers compared with phages infecting other bacterial species, although some virulent phages have been found in S. aureus [17- 19]. Therefore, we examined the potency of two of the broad host range lytic staphylococcal phages, namely K and 44AHJD, belonging to the families Myoviridae and Podoviridae, respectively. Complete nucleotide sequences for both of these phages have been reported previously [20,21].
Among 16 studied staphylococcal phages, 44AHJD is highly virulent because of the high translation efficiency of many of its genes [
In the present study, we purified phages K and 44AHJD by ammonium sulphate precipitation, followed by ionexchange chromatography. The levels of contaminating host proteins and endotoxins were determined and compared with phages purified by conventional cesium chloride (CsCl) density gradient centrifugation. Purified phages in the form of a cocktail were then evaluated for their in vivo efficacy in an experimental S. aureus nasal colonization mouse model.
Eighty-six S. aureus isolates, comprising 27 MRSA and 23 MSSA strains collected from hospitals in and around Bangalore, India, and 36 global strains (33 MRSA and 3 MSSA), were used to assess the bactericidal activity of the two virulent staphylococcal phages, K and 44AHJD (GenBank accession numbers AY176327 and AF513032 respectively). Thirty distinct, typed isolates of global representation were obtained from the Public Health Research Institute (PHRI), New Jersey, USA. Phage K (NC07814-02) was obtained from the Health Protection Agency Culture Collections, UK, and phage 44AHJD was a gift from Dr. Udo Blaesi, University of Vienna, Austria. All strains were cultured in Luria-Bertani (LB) broth at 37˚C on a rotary shaker (200 rpm), unless otherwise stated. S. aureus strain Newman was used in the in vivo experiments. Polyclonal antibody for S. aureus RN4220 was generated at Raj Biotech, Pune, India.
Phages K and 44AHJD were amplified in S. aureus strains RN4220 and KB600, as described previously [
Phage K crude lysate was precipitated using solid ammonium sulfate fractionation from 0% - 30% and 30% - 70% ammonium sulfate at room temperature, and then centrifuged at 12,860 × g for 45 min at 4˚C. The pellet obtained from the 30% - 70% fraction was dialyzed against 25 mM Tris-Cl pH 7.5 (buffer A) overnight. The dialyzed material was loaded onto a weak anion exchange DEAE cellulose (DE52) column (Whatman Inc., Florham Park, NJ, USA) using a Biologic Duoflo system (Bio-Rad, Hercules, CA, USA) equilibrated with buffer A at a flow rate of 5 mL/min. The column was washed with buffer A until the absorbance of the eluting fractions at 280 nm was zero. The bound phages were recovered by isocratic elution with 0.2 M NaCl in buffer A, dialyzed against buffer A, filter-sterilized through a 0.2 µm filter, and then analyzed by SDS-PAGE followed by silver staining. A similar protocol was followed for phage 44AHJD crude lysate.
Phages were enumerated from all of the chromatographic fractions, and percent phage recoveries were calculated taking the initial material as 100%.
One liter each of phage K and 44AHJD lysate was centrifuged at 25,000 × g for 2 h at 4˚C. The pellet, containing bacteriophage particles, was resuspended in approximately 1 mL of buffer A. CsCl density gradient ultracentrifugation of this phage concentrate was performed following standard methods [
Protein content at different stages of phage purification was determined according to the method of Lowry et al. (1951) [
The endotoxin content of the phage preparations was measured using an Endosafe Rapid LAL reagent kit (Charles River, Wilmington, MA, USA).
Healthy 6-week-old BALB/c mice (National Institute of Nutrition, Hyderabad, India) were used in all experiments. Animal experiments were performed at St. John’s Medical College and Hospital, Bangalore, India. The experiments were approved by the Institutional Animal Ethics Committee (IAEC) and the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). St. John’s Medical College is registered with CPCSEA (Registration No. 90/1999/CPCSEA dated 28/ 4/1999).
The commensal nasal flora of the mice was evaluated by nasal swabbing as described previously [
Mice were administered chloramphenicol sodium succinate at 0.5 mg/mL in drinking water, beginning at 24 h prior to inoculation of challenge strain, which continued until the end of the study.
Chloramphenicol-resistant S. aureus strain Newman was grown at 37˚C overnight on Columbia agar containing 2% NaCl to induce capsule formation [
Three groups of mice (n = 8) were used for the study. These were colonized with the challenge strain as described in 3.8. Daily doses of phage cocktail containing 1 × 1010 PFU of phage K and 4 × 1010 PFU of phage 44AHJD in 10 µL of 0.85% NaCl were administered intranasally to the test group on days 5, 6, and 7. The placebo control group was administered 10 µL of 0.85% NaCl. On day 8, the mice were euthanized and nasal tissue was taken for confirmation and enumeration of the test strain, as described above.
ELISA for detection of S. aureus derived proteins and S. aureus confirmation was performed using polyclonal antibodies generated for the S. aureus host RN4220 cell lysate. Dilutions of the RN4220 host lysate (from 1 ng to 1000 ng protein) served as the antigen, and were used for construction of a standard curve.
For western blot studies, all samples were run on a 12.5% SDS-PAGE gel and then transferred to a Biotrace nitrocellulose blotting membrane (Pall Corporation, Pensacola, FL, USA) and blocked with 3% BSA (in Tris-Cl, pH 8.0, buffered saline with 0.1% Tween 80:1 × TBST) overnight. Following washing with 1 × TBST, primary anti-RN4220 antibody (final concentration: 1:5000) was added, and the membrane washed again with 1 × TBST. Secondary goat anti-rabbit alkaline phosphatase (ALP) conjugate (final concentration: 1:500) was then added. The blot was developed using 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) substrate in conjunction with NBT (nitro blue tetrazolium).
For identification of isolates from the mouse nares, colonies taken from pure isolates from LB agar were suspended in 0.05 M carbonate-bicarbonate buffer pH 9.6, to a cell density of −1 × 109 CFU/mL. Two hundred microliters of this cell suspension were used to coat 96-well plates overnight at 4˚C. Wells were washed with TBST and blocked with 200 µL of 1% BSA in TBST for 1 h at 37˚C. After repeated washes with TBST, 100 µL of rabbit polyclonal anti-RN4220 antisera (1:20,000) was added and plates incubated for 1 h at 37˚C. Wells were washed again with TBST prior to addition of 100 µL of ALP-labeled goat anti-rabbit antibody (1:5000). Plates were incubated for 1 h at 37˚C. Following washing of the wells, 100 µL of substrate (PNPP) was added and plates were incubated for 40 min, after which absorbance was read at 405 nm.
While phage K was propagated using RN4220 as a propagating host, phage 44AHJD was propagated using KB600 host since we have earlier observed that 44AHJD requires endolysin supplementation for propagation in RN4220 [
A total of 86 isolates were tested for phage sensitivity, which included 30 distinct typed S. aureus isolates of global representation; six of Community acquired (CA)MRSA type strains and 50 clinical isolates from Indian hospitals (data not shown). Nearly 57% of isolates were susceptible to phage K and phage 44AHJD was lytic to 86% of the isolates tested. In case of both phages taken together, 74.4% of isolates were sensitive with plaque formation. In total, 88.3% isolates were susceptible to both phages in combination (
It has been shown earlier that a phage cocktail significantly reduces the frequency of mutation in bacteria in comparison to the use of a single phage preparation [
Due to its polyvalent nature, Staphylococcus phage K, has been studied in multiple applications, including in the treatment of subclinical bovine mastitis [
The recovery of phage K at various purification steps is summarized in
The total recovered phage titer following ammonium sulfate precipitation was 32%, with an almost eight-fold reduction in the total protein content.
Following anion exchange chromatography, the recovery was 31%, with enhanced specific activity (
Panel A: S. aureus from India-27 MRSA and 23 MSSA category; Panel B: Global S. aureus panel including PHRI strains and USA type strains-33 MRSA and 3 MSSA category; *Lysis seen due to lysis-from-without phenomenon due to phages at high MOI (>100).
precipitation/ion-exchange protocol (
Purified phage particles have two major uses: phage biology studies and therapeutic applications. To date, most phage preparations for therapeutic use have been purified by passing the lysate through filters to remove the host bacteria. While such purification reduces the risk of bacterial infections, it does not remove bacterial endotoxins, which can be harmful to patients. Moreover, costeffective phage purification methods would be beneficial for large-scale production. Because the available literature describes phage purification using a variety of methods, including cesium chloride gradient ultracentrifugation [
Sample 1: Crude phage lysate; Sample 2: 30% - 70% ammonium sulphate fraction; Sample 3: DEAE cellulose purified fraction.
Sample1: Crude phage lysate; Sample 2: 30% - 70% ammonium sulphate fraction; Sample 3: DEAE cellulose purified fraction.
columns [
Phages purified by anion exchange methods have been used successfully in a number of human studies [38,41]. Therefore, the systematic approach for phage purification used in the current study would benefit researchers in this field.
The results of our assessment of the purity of bacteriophages obtained by SDS-PAGE (
The cesium chloride phage preparations and the phage cocktail preparation showed host cell contamination of 10 - 100 ng/mL, as determined by ELISA with antiRN4220 antibodies. The western blot of samples from the CsCl and ion-exchange purified 44AHJD phages showed negligible signals for S. aureus host cell contaminants (
The endotoxin levels of the phage cocktail used in our study was in the range as reported by other researchers [
that bind to plastic and glass surfaces are efficiently removed by depyrogenation [
A recent report on purification of Staphylococcus phage VDX-10 showed that >90% of host proteins were removed, which is similar to our observations [
Only coagulase-negative staphylococci (S. gallinarum, S. arlettae, and S. equorum) were found in BALB/c mice used for experimentation. S. aureus was not detected in any of the animals (data not shown).
Of the 24 mice nasally inoculated with S. aureus strain Newman, 83.3% were colonized on day 7. On days 10 and 14, 25% (2/8) and 12.5% (1/8) of mice remained colonized, respectively. The carriage rate of S. aureus Newman in the currently employed BALB/c mice is similar to reported earlier [
The phage efficacy study involved evaluation of commensal bacterial flora from the mouse nares, then determination of the rate and extent of colonization of nasally inoculated S. aureus. Subsequently, efficacy of phage treatment was assessed in S. aureus-colonized mice. Phageeffected decolonization was evident in the animals treated with the phage cocktail. Daily doses of phage cocktail administered intranasally on days 5, 6, and 7 fully decolonized all eight animals inoculated with S. aureus strain Newman by day 8 while the colonization control group (seven of eight animals) and the group treated with placebo (six of eight animals) remained colonized (
S. aureus is not a normal commensal organism in mouse nares; therefore establishment of experimental colonization of S. aureus in these animals required optimization. We achieved sufficient maintenance of colonization to allow application of phage cocktail and test their efficacy. We found that 80% of mice remained colonized for 7 days in the model reported here. The mice were gradually decolonized of S. aureus naturally. Therefore, we chose a phage-treatment window within the 7- day period and ended the study on day 8. This afforded a good contrast between the treated and untreated groups. We observed decolonization of all animals in the treated group, while in the control group, 75% of animals remained colonized on day 8. We believe this study to be the first report of bacteriophage efficacy in a mouse nasal model of S. aureus carriage.
It is well established that S. aureus colonizes multiple sites in the human body, particularly the anterior nares [
in surgical patients and those on hemodialysis and continuous ambulatory peritoneal dialysis (CAPD) [
As there are no current guidelines on the bacteriophage titer that may be clinically effective against MRSA in the human nose, estimates can be made based on previous related studies of bacteriophage therapy. These include use of bacteriophage titers in respirable powders (108 - 109 PFU per 100 mg powder) [
Use of bacteriophages for treatment of various bacterial infections including S. aureus has been reviewed extensively [51,52]. Although temperate phages of S. aureus are more widely known [53,54], due to the potential problems of lysogeny and toxic gene transfer, its therapeutic use is limited [
The authors thank Dr. Barry Kreiswirth, PHRI, New Jersey, for the gift of clinical isolates, and Dr. Richard Novick for S. aureus strain RN4220. Thanks are also due to Dr. Udo Blaesi, University of Vienna, for the gift of phage 44AHJD, and Dr. Kenneth Bayles, Nebraska Medical Center, for S. aureus strain KB600. The authors thank Dr. Sudha Suresh, Pharmacology Division of St. John’s Medical College and Hospital, Bangalore, for assistance with animal experiments. The authors acknowledge Dr. Janakiraman Ramachandran, Chairman & CEO, Gangagen Inc, USA, for his support and encouragement, and Dr. M. Jayasheela, Head of Clinical Development and Regulatory Affairs, Gangagen Biotechnologies Pvt. Ltd., Bangalore, India. The authors acknowledge the upstream process development team for providing phage lysate for purification studies used in this work, and Mr. Naveen Kumar for formatting one of the figures in this paper.