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 (). 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.
In conclusion, a single treatment with CHAP
k 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 CHAP
k 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 CHAP
k 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 CHAP
k 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
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