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Bioeng Bugs. 2010 Nov-Dec; 1(6): 404–407.
PMCID: PMC3056090

The truncated phage lysin CHAPk eliminates Staphylococcus aureus in the nares of mice


The endolysin LysK derived from staphylococcal phage K has previously been shown to have two enzymatic domains, one of which is an N-acetylmuramoyl-L-alanine amidase and the other a cysteine/histidine-dependant amidohydrolase/peptidase designated CHAPk. The latter, when cloned as a single-domain truncated enzyme, is conveniently overexpressed in a highly-soluble form. This enzyme was shown to be highly active in vitro against live cell suspensions of S. aureus. In the current study, the IVIS imaging system was used to demonstrate the effective elimination of a lux labeled S. aureus from the nares of BALB/c mice.

Key words: Staphylococcus, decolonization, lysin, bacteriophage, nasal

Staphylococcus aureus (S. aureus) is responsible for a variety of infections ranging from skin conditions to fatal sepsis, endocarditis, septicemia, pneumonia and meningitis.1 Methicillin resistant S. aureus (MRSA) is frequently the cause of nosocomial infections globally, which can lead to prolonged hospital stays and increased mortality.2 It has been shown that a substantial proportion of these serious nosocomial infections originate from the patients own flora, nasal carriage of S. aureus in particular being a considerable risk factor.3,4 Currently, the topical application of mupirocin ointment is used to eliminate staphylococcal nasal carriage in clinical settings.5 Although successful in removing S. aureus from the nose when applied twice daily for up to 5 days, bacterial resistance to this agent is nonetheless increasing and so new antimicrobials are required.5

Lysins are phage-encoded peptidoglycan hydrolases, which, when applied exogenously as purified recombinant proteins to Gram-positive bacteria, bring about rapid lysis and death of the bacterial cell.6,7 Unlike antibiotics, phage lysins can be used to selectively target specific pathogenic bacteria without affecting surrounding commensal microflora. They are reported to have a narrow host range similar to that of their phage producers, rendering them generally either species8,9 or genus specific.10,11 The potential for these enzymes as novel therapeutics against a number of Gram-positive pathogens on mucosal surfaces and in systemic infections has previously been demonstrated.1221 In relation to bacterial resistance, no strains have yet been identified with resistance to phage lysins.6,8,19 It has been suggested that these enzymes have evolved to target specific molecules in the host peptidoglycan that are essential for cell viability, making resistance an unlikely event.22,23 In general, S. aureus phage lysins display a multi-domain modular structure comprising of a C-terminal cell wall binding domain and two N-terminal catalytic domains. Examples include LysK, MV-L, phi11 and LysH5.10,20,24,25

Native lysins of S. aureus, with the exception of MV-L, have typically shown poor expression, insolubility and low activity when generated as recombinant proteins in a heterlogous host.10,2628 However, their modular structure has enabled the construction of truncated (CHAPk),29 and chimeric versions of lysins (ClyS, P16–17)21,27 to help circumvent these problems. We previously showed that the activity of CHAPk (18.6 kDa) against live S. aureus including MRSA was two-fold higher than that of the native enzyme, LysK (54 kDa).29 To date, the multi-domain MV-L lysin from phage MR11 and the chimeric two-domain lysin, ClyS, are the only anti-staphylococcal lysins which have been evaluated in vivo.20,21 In this study we demonstrate the effectiveness of the highly-soluble, high-activity single-domain lysin, CHAPk to eliminate S. aureus from the nares of artificially infected BALB/c mice.

Bacterial Strain

The staphylococcal strain used in this study was S. aureus Xen29 (Caliper Lifesciences, UK). This strain is derived from the parental strain S. aureus ATCC 12600, a pleural fluid isolate which has been engineered with a stable copy of the modified Photorhabdus luminescens luxABCDE operon at a single integration site on the bacterial chromosome.21,30,32 When metabolically active this strain emits luminescence, which can then be rapidly detected and quantified for evaluating the efficacy of therapeutics, in animal infection models, using an In Vivo Imaging System (IVIS) (Xenogen, CA).3032 This enabled us to monitor the nasal colonization of individual mice in real-time and to directly compare the effect of the therapeutic agent with appropriate controls. S. aureus Xen29 was selectively grown at 37°C in tryptic soy broth (TSB) and tryptic soy agar (TSA) in the presence of 200 µg/ml kanamycin. CHAPk has been cloned into the pQE60 (Qiagen) expression system and heterlogously overexpressed in Escherichia coli XL1-Blue cells as previously described.29 Unless stated otherwise, all media was supplied by Sigma-Aldrich.

CHAPk In Vitro Activity

CHAPk was purified by ion-exchange chromatography to high purity and quantified with the BCA Protein Assay Kit (Pierce Scientific). CHAPk in vitro activity was assessed against live S. aureus Xen29 in a microtitreplate based assay. Briefly, S. aureus Xen29 was grown to early log phase at an optical density of 0.3 at 590 nm (OD590), centrifuged, and then resuspended to a final OD590 of 0.5 in 10 mM sodium acetate buffer (pH 7.4). 100 µl aliquots of CHAPk (at concentrations ranging from 1.0 µg/ml to 50 µg/ml) were mixed with 100 µl of the bacterial suspension and incubated at 37°C in a microtitreplate reader (Molecular Devices, Spectra Max 340) for between 5 to 15 min. Typically 5 µg/ml of CHAPk reduced the OD590 by 70% in 5 min (Fig. 1). To confirm that the loss in turbidity equated to a reduction in cell viability, aliquots of lysin treated cells were plated and CFUs enumerated on TSA plates containing 200 µg/ml kanamycin.

Figure 1
Activity of CHAPk against live S. aureus Xen29 in vitro. The decrease in optical density (OD) over time following the addition of CHAPk (5 µg/ml) at time zero (
An external file that holds a picture, illustration, etc.
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) in 10 mM sodium acetate buffer was monitored at 590 nm in a Molecular Devices SpectraMax

CHAPk In Vivo Nasal Decolonization

Experiments were undertaken with the approval of the University College Cork Animal Experimentation Ethics Committee. S. aureus Xen29 was grown overnight at 37°C with shaking at 170 rpm in TSB medium containing 200 µg/ml kanamycin. The culture was diluted 1:50 in fresh media and grown as above, to mid-log, centrifuged at 6,000 g, washed twice in PBS pH 7.4 and re-suspended in the same buffer to a predefined titer of 3.53 × 1010 CFU/ml. Inoculum titers were confirmed by serial dilution and plating on TSA containing 200 µg/ml kanamycin. Six to eight week old female BALB/c mice (Harlan, UK) were anesthetized using isofluorane gas (Abbott Laboratories, Kent, UK) to ensure accurate instillation of the inoculum. A total volume of 15 µl of 3.53 × 1010 CFU/ml of S. aureus Xen29 was intranasally instilled with a Gilson P20 pipette in a dropwise fashion, distributed equally in each nostril. One hour post-infection the mice were again anesthetized and imaged (Exposure: 5 minutes, Binning: medium, Color-scale: 70–500) using the IVIS system (Xenogen, CA). The luminescence from the nasal area of each animal [expressed as Relative Light Units (RLU)] was quantified using the Living Image software package (Xenogen, CA). Strong luminescence was detected in the nares of all mice, indicating successful colonization by S. aureus Xen29.

Mice were divided into two groups (n = 7) and administered either 60 µl (20 µl per nostril and 20 µl orally) of 925 µg/60 µl CHAPk in enzyme buffer (10 mM sodium acetate pH 7.4) or 60 µl of buffer alone. One hour post-treatment, mice were anesthetized by intraperitoneal injection with a mixture of ketamine (65 mg/kg) and xylazine (13 mg/kg) (Vetoquinol, Dublin, Ireland). This enabled the simultaneous visualization of an entire group at one time (n = 7) as opposed to the limit of 5 mice at a time using the gas method. Animals were imaged as described earlier. The RLU readings from the nasal area of each mouse in both groups were recorded. The mice were then euthanized, noses aseptically dissected, weighed, macerated in 500 µl of PBS and vigorously vortexed for 60 seconds. CFU counts for each nose were recorded by plating out dilutions of each suspension on TSA plates containing 200 µg/ml kanamycin.

One hour post-treatment, it was observed that luminescence was drastically reduced in the nares of the CHAPk-treated group compared to strong luminescence in the buffer-treated control group (Fig. 2). The mean RLU value of the CHAPk-treated group was 2,996 ± 254 while the mean RLU value of the buffer-treated control group was 23,366 ± 3,436. This represents approximately a 7-fold difference, which indicated a significant reduction in S. aureus Xen29 in the enzyme-treated group. Bacteriological analysis by plating confirmed that the single treatment with CHAPk brought about a 2-log reduction in S. aureus Xen29 numbers in the nares of the treated mice within one hour (p < 0.001). The mean log10 value of the CHAPk-treated group was 5.29 ± 0.26 CFU/g compared to a mean log10 value of 7.35 ± 0.10 CFU/g for the buffer-treated control group. The RLU and bacterial counts from both groups of animals were statistically analyzed using the Mann-Whitney rank sum test.

Figure 2
BALB/c mice following S. aureus Xen29 inoculation imaged using the IVIS system (Xenogen, CA). (A) Buffer-treated control group, imaged 1 hour post treatment with 10 mM Sodium acetate pH 7.4: the Mean RLU value of this buffer-treated control group was ...

In conclusion, a single treatment with CHAPk brought about a two-log reduction in S. aureus Xen29 cells in one hour from the nasal mucosa of mice. This single-domain truncated lysin demonstrates high solubility, rapid lytic activity and high specificity against staphylococci. These attributes, along with the low probability of bacterial resistance,6,8,19 strongly advocates the use of this enzyme as a potent alternative therapeutic option. In particular it should be efficacious for the elimination of multidrug-resistant S. aureus in both clinical and community settings such as hospitals and nursing homes. A single application of CHAPk administered intranasally as a spray has the potential to rapidly reduce the reservoir of pathogenic staphylococci in the nasal mucosa of humans and thus may be a valuable tool in the prevention and spread of life-threatening multi-drug resistant S. aureus. It would be particularly relevant in high-risk groups such as immunocompromised patients, chronic disease cases and patients requiring surgery, medical implants, hemodialysis etc.4,5 As CHAPk acts rapidly it could also be used to eradicate S. aureus from the nares of patients who require emergency surgery. This is a capacity that was previously unavailable as multiple applications of mupirocin are required for up to five days to effectively remove S. aureus from the nose.5 CHAPk also has potential to be used prophylactically to combat the increasing incidence of MRSA in close community settings such as military barracks and prisons.5


This work was financed by Science Foundation Ireland Ref 05/RF/BIM004.



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