Pretest

The application of BaKillus as a nasal spray implies the requirement of B. subtilis to survive the environment of the nasal mucosa. This does not go without saying, as B. subtilis naturally exists in the soil and not all microbes can co-exist. Additionally the bacteria have to cope with the host immune system. Naturally nasal-microbes have evolved to co-exist with each other and the immunesystem. So can B. subtilis, and in the future BaKillus, be introduced into this complex system and live long enough to accomplish its mission to hunt, disintegrate biofilms and destroy pathogens located within?

To test this we were intrigued by the idea of utilizing the B. subtilis wildtype, which holds the GRAS-status, and courageous team members in a quest to find answers. But first we wanted to clarify legal issues related to this.

Legal issues

LEGAL ISSUES

Main questions

The main questions are the following

• Is it possible to reliably detect B. subtilis in an assay to accomplish a convincing conclusion?
• Can B. subtilis co-exist with the nasal microbiome?
• Can B. subtilis live long enough in the nasal mucosa to be relevant for a medical treatment?

For this we conducted two tests:

• Co-cultivation of B. subtilis with nasal swabs
• Survival and detection of B. subtilis in the nasal mucosa

Co-cultivation of B. subtilis with nasal swabs

The first test targets the first two questions from above. Nasal swabs utilizing damped autoclaved Q-tips were taken from the team members and applied to an LB-plate and subsequently innoculated in a DSM-culture (to enable sporulation), which was split into two probes. To one probe B. subtilis was added. These were incubated for at least 24 hours, to enable sporulating bacteria to form spores. The spores are required for the detection, as only spores overlive a heat inactivation of 80 °C for over 20 minutes. One half of each probe was heat inactivated, while the other was not to distinguish all bacteria from spore formating bacteria. They were plated on LB-plates in different dilutions. The outcome is displayed in Figure 1.

Fig. 1: A representative selection of the outcome of this test. The heat inaktivation of the probes with W168 shows that B. subtilis can co-exist with the nasal microbiome, as colonies deriving presumably from B. subtilis spores grew, while only few are visible on the plates of the heat inactivation without W168.

Outcome:

• The nasal microbiome is quite different for most people
• It is possible to reliably detect B. subtilis in the nasal microbiome
• Mostly B. subtilis can co-exist with the nasal microbiome (with one exception not shown)
• With B. subtilis the nasal microbiome can change, probably due to the fact, that B. subtilis already produces cannabalism toxins and other antimicrobial substances outcompeting some of the other bacteria

Survival and detection of B. subtilis in the nasal mucosa

To answer the last question B. subtilis had to be introduced into the nasal cavaty. This was done by smearing a bit of a B. subtilis W168 overnight culture with Q-tips onto the nasal mucosa. After 7-9 hours a nasal swab was taken and applied onto an LB-plate as well as inocculated into a DSM-culture. The DSM-culture was incubated for at least 24 hours to enable spore formation. As above one half of the culture was heat inactivated and the other half was not. These were plated on LB-plates in different dilutions.

Fig. 2: A representative selection of the outcome of this test. The heat inaktivation of the probes with W168 shows that B. subtilis can survive in the nasal mucosa at least for some individuals, as colonies deriving presumably from B. subtilis spores grew, while only few are visible on the plates of the heat inactivation without W168.

Outcome:

• The application of B. subtilis might influence the quantity and composition of the nasal microbiome compared to the nasal swabs from the first test
• B. subtilis can survive the host immune system at least for some individuals for at least 7-9 hours, which is enough time for the application BaKillus
• No side-effect of the application of B. subtilis to the nasal mucosa occured, which is in line with the GRAS-status

Conclusion

This preliminary pretest demonstrates that it is feasible to utilize B. subtilis in the nasal mucosa as a BaKillus nasal spray, as B. subtilis can co-exist with the nasal microbiome, the host immune system and does not lead to obviuos side effects.