Why are we doing this?
The rise of antibiotic-resistant bacterial pathogens causing drug-resistant infections and its escalating health, social and economic consequences are now increasingly visible on a global scale.
Our team initially aim to focus on super resistant bacteria problems. During our early stage investigation in such an area, we gradually consider such problems have become one of the most rigorous threat to human society. While the overcommit of antibiotics increasing, the development of novel antibiotics has come to a deadlock. In 2018, only two novel antibiotic classes have been developed and approved by international drug agencies in the past 20 years (Luepke et al., 2017). Novartis have terminated the development of novel antibiotics. Previously, Sanofi, AstraZeneca and some other pharmaceutical companies have quit from developing antibiotics, since the high R&D cost and the bottleneck of developing. Such a dilemma may still last for a long time. We considered that the development of novel antibiotics should not be the exclusive strategy against antibiotics resistant bacteria and some other methods should have been tested by scientists (Table 1).
table 1（Trends in Microbiology, April 2019, Vol. 27, No. 4）
The emergence of SARS-Cov-2 and the heavy antibiotic use in these patients bring new urgency, because secondary bacterial infections is an indirect contributing factor to Covid-19’s mortality rate, the growth of bacteria causes the innate immune system to secrete additional inflammatory fluid to the lungs, inhibiting gas exchange. (Canton, Gijon, & Ruiz-Garbajosa, 2020; Shi & Gewirtz, 2018). In the last 9 months, SARS-CoV-2 has caused pandemic infection, over 10,000,000 people have been confirmed infected, over 500,000 people died of coronavirus infection and bacterial co-infection and other complications. In a study, 7% of 3834 COVID-19 patients had a bacterial co-infection, the commonest bacteria were Mycoplasma pneumonia, Pseudomonas aeruginosa (12% in all co-infected patients) and Haemophilus influenzae (Lansbury, Lim, Baskaran, & Lim, 2020). In clinical treatment, patients who have invasive catheters including endotracheal tube, arteriovenous catheter, results in increased susceptibility to co-infections of nosocomial multidrug-resistant pathogens including Pseudomonas aeruginosa which poses difficulties in antibiotic treatment (Zhang et al., 2020). Since SARS-CoV-2 remain globally spreading, it is urgent and significant to develop a therapy beyond antibiotics to against multiple drug resistant bacterial co-infections.
We continue our literature review and some fascinating aspect in such a dilemma was discovered. Phage therapy has been attempted and testing against bacterial infection hundred years ago, but such a field began maturing until the year 2000, coupled with the explosion of genomics and broad ecology-based phage research (Abedon, Kuhl, Blasdel, & Kutter, 2011). Some scientists have also urged to apply phage to combat pathogenic bacteria, especially in the developing countries (Nagel et al., 2016). It seems the phage therapy is renewing these years. These reviews and studies inspired us that phage therapy may be the next potential substitute when we are facing super resistant bacteria crisis. We starting to have interest in applying phage to combat resistant bacteria via synthetic biology method and technology.
Moreover，Pseudomonas aeruginosa，widely distributed in nature and normal human skin, intestinal tract and respiratory tract, is one of the more common clinical opportunistic pathogens（figure 1）.
Figure 1: infection caused by PA
Finally, we plan to engineer phage to give it better antibacterial effect and biosafety performance for phage therapy.
Briefly, we constructed two phage mutants. The first mutant T_S1 was obtained by inserting relE gene within the ORF73 of S1, a hypothetical protein. After editing by CRISPR-Cas9, all the plaques testes contained recombinant phages with desired mutation. For further biosafety consideration, we constructed a lysis-deficient phage (LyDT_S1) expressing the antibacterial toxin RelE following the DBTL (design→build→test→learn/redesign) engineering cycle. The mutant was obtained by replacing the gene of holing, a lysis-promoting phage toxin, with the relE gene. In lysis kinetics for characterization of bacteriostasis effect, high MOI (multiplicity of infection) group of mutants indicates that bacteria do not develop resistance to phages. It might help solve one of the main questions regarding phage therapy，the possible rapid emergence of phage - resistant bacterial variants, which could impede favourable treatment outcomes (Oechslin, 2018; Rohde et al., 2018).
After editing, the mutant phage LyDT_S1 can still inhibit bacterial growth through the expression of RelE but cannot induce cell lysis because the holin gene is eliminated. This is expected to reduce the amount of endotoxins released from the pathogen bacteria, with a corresponding reduction of systemic cytokine response and inflammation during bacterial infection. The dilution & purification and endotoxin release testing of LyDT_S1 are still under way.
This strategy of replacing the expression of holin with that of a toxin is a demonstrative example of the potential of this editing system based on CRISPR-Cas and λ-red plasmids, which we hope will serve as an efficient tool for editing the hard-to-engineer phages genomes, to better understand phage-host interactions and pave the way for the modular design of phages.
Abedon, S. T., Kuhl, S. J., Blasdel, B. G., & Kutter, E. M. (2011). Phage treatment of human infections. Bacteriophage, 1(2), 66-85. doi:10.4161/bact.1.2.15845
Canton, R., Gijon, D., & Ruiz-Garbajosa, P. (2020). Antimicrobial resistance in ICUs: an update in the light of the COVID-19 pandemic. Current opinion in critical care, 26(5), 433-441. doi:10.1097/mcc.0000000000000755
Lansbury, L., Lim, B., Baskaran, V., & Lim, W. S. (2020). Co-infections in people with COVID-19: a systematic review and meta-analysis. J Infect. doi:10.1016/j.jinf.2020.05.046
Luepke, K. H., Suda, K. J., Boucher, H., Russo, R. L., Bonney, M. W., Hunt, T. D., & Mohr, J. F., 3rd. (2017). Past, Present, and Future of Antibacterial Economics: Increasing Bacterial Resistance, Limited Antibiotic Pipeline, and Societal Implications. Pharmacotherapy, 37(1), 71-84. doi:10.1002/phar.1868
Nagel, T. E., Chan, B. K., De Vos, D., El-Shibiny, A., Kang'ethe, E. K., Makumi, A., & Pirnay, J.-P. (2016). The Developing World Urgently Needs Phages to Combat Pathogenic Bacteria. Frontiers in Microbiology, 7. doi:10.3389/fmicb.2016.00882
Oechslin, F. (2018). Resistance Development to Bacteriophages Occurring during Bacteriophage Therapy. Viruses-Basel, 10(7). doi:10.3390/v10070351
Rohde, C., Resch, G., Pirnay, J.-P., Blasdel, B. G., Debarbieux, L., Gelman, D., . . . Chanishvili, N. (2018). Expert Opinion on Three Phage Therapy Related Topics: Bacterial Phage Resistance, Phage Training and Prophages in Bacterial Production Strains. Viruses-Basel, 10(4). doi:10.3390/v10040178
Shi, Z., & Gewirtz, A. T. (2018). Together Forever: Bacterial-Viral Interactions in Infection and Immunity. Viruses-Basel, 10(3). doi:10.3390/v10030122
Zhang, G., Hu, C., Luo, L., Fang, F., Chen, Y., Li, J., . . . Pan, H. (2020). Clinical features and short-term outcomes of 221 patients with COVID-19 in Wuhan, China. J Clin Virol, 127, 104364. doi:10.1016/j.jcv.2020.104364