The world states us facts.
Antimicrobial resistance (AMR) has emerged as a major threat to public health estimated to cause 10 million deaths annually by 2050.
Collaboration with team VIT_Vellore made us be aware of serious phenomena in India. India carries one of the largest burdens of drug-resistant pathogens worldwide. It is no surprise that emergence of enzyme New Delhi metallo-beta-lactamase (NDM-1), named after the national capital of India, in 2008 rapidly spread to other countries. And various mutants of NDM-type MBLs have emerged, including our enzyme of interest, NDM-23.
Also, in China, to find new compounds via synthetic way have been listed in national strategy.
To find the arrow hitting Achilles’ Heel of metallo-beta-lactamase, what should the arrow be? How can we catch the arrow, redesign or search in libraries? Are we on the proper track towards therapeutics?
There are voices we collected below.
Recognition from Professors
Weihui Wu, Nankai University
At the early stage of entire project, we decide to listen to related experts’ advice first. Dr. Weihui Wu researches on bacterial molecular biology and infectious diseases, including drug resistance mechanism of Pseudomonas aeruginosa and pathogenic gene expression regulation. Since this is a project to solve antibiotic resistance problem and our main gene is originated from P. aeruginosa., we consulted with professor Wu.
After hearing the brief introduction of our project, he thinks our program is completely feasible . We can use beta lactamase for biological testing, or study the role of inhibitors on bacteria, as well as intracellular detection. He pointed out that the reactants must not only have enzymatic activity, but more importantly, it should be able to act on bacteria.
At that time, some team members came up with an idea which is to overlap different beta-lactamases in one vector to express them together, then explored their impacts on each other. However, professor thinks that multiple genes are carried out in one operon. For example, simultaneous detection of Class B and C beta-lactamases does not involve mutual regulation at the level of gene expression.
To test for drug resistance at the bacterial level, the professor believes that there are two criteria: (1) the degree of degradation of the substrate, expressed by measuring enzymatic activity. (2) The degree of change in antibacterial concentration of antibiotics. The relationship of expression level of the enzyme and antibiotic resistance are not linear, and the clinical effect is only concerned with bactericidal or bacteriostatic effects.
Cen Shi, Deputy Director of Shanxi Food and Drug Inspection Institute
Drug inspection institute is charged with the responsibility of controlling the quality of drugs, before they come into the market. Or if there are some cases caused by poisonous food and drug, they can check them. So staff there are quite familiar with approved drug. And we went to communicate with them to discuss the possibility of our project aiming at drug development.
At the beginning, she affirmed our idea of screening effective beta-lactamase inhibitors. And in the institution, they have evaluated some similar drugs, for example, sulbactam and ampicillin.
Then we introduced our methods to her. Since in the institution they use different facilities to check whether the valid component in drugs meet the national standard. She thought UV-vis method is a simple and direct method to test the absorbance peak of our target substances. However, its value was greatly influenced with others in the system. She suggested using HPLC.
On the theme of approved drugs, we asked how far it is between screening out the inhibitors and combine them with antibiotics for therapeutics. She introduced the procedure of drug development, a new drug can be eventually used in clinic after experiments on animal and human beings for several stages. What we do is just the first step towards approved drug, however, it does play a role on promoting a new kind drugs for therapeutics and brings hope to solve the illness issue related to these metallo-beta-lactamases.
Communications and Contacts
5th Synthetic Biology Young Scholar Forum
On August 16 and 17, we presented 5th Synthetic Biology Young Scholar Forum held in Tianjin Binhai. We attended lectures given by several professors in the field of synthetic biology and did a short project introduction presentation in iGEM session. And we had communications with other 4 2019 iGEM teams, Tianjin, OUC-China, SJTU- BioX-Shanghai, and Tsinghua-A.
In presentation session, professors gave us two important comments. One is about our innovativeness and the other is related to safety. In poster session, we presented some figures of our results, which was approved by some attendees. And one asked us detailed fluorescent mechanism of using fluorescent probe and hoped we could show its structure in poster directly. We reconsidered these valuable advice and highlighted in description and safety.
Safety consultation with management teacher
Buckets of waste bacterial liquid were produced during this summer. Following the safety standard rules, we added high concentration sodium hydroxide to prevent gene transfer. We consulted with Yong Li, a management teacher in our experimental center, to study laboratory waste treatment.
He told us our college's waste liquid is generally divided into four types of waste liquid: organic waste liquid, inorganic waste liquid, acid waste liquid, alkaline waste liquid.
As for our treatment to liquid waste, it’s no problem with high concentration of NaOH. The other way is adding “84” disinfectant.
When discussing about the antibiotic pollution, whether engineering bacteria transferred with antibiotic-resistant genes have impacts on it is regarded unclear for both of us. But as the possibility exists, we cannot ignore it’s potential risks. Teacher recognized our attention of safety, and we would pay more attention to supervision of our waste bacteria and other polluted materials.
Heng Zheng, China Pharmaceutical University
Dr. Zheng’s lab works on microbial drug development design based on computer programs. In other words, they are experienced in virtual screening.
Initially, a direct idea to solve antibiotic resistance problem is to develop new kind of antibiotics. We considered designing more typical structure of antibiotics and achieved the build via genetic tools. And computer is regarded indispensable in this process. So we contacted with Dr. Zheng via email.
But he gave us a new angle towards it. The market of antibiotics is close to full, and few people would like to develop it. Why don’t we try to search for new inhibitors and combine it with existing antibiotics as double compounds (beta-lactam/beta-lactamases inhibitor) ? Computational design on drug structure have more wide spaces to explore.
Chunling Xiao, Peking Union Medical College
Focusing on drug screening of Tuberculosis bacillus and Pseudomonas aeruginosa, Dr. Xiao and her group members are experts in high-throughput screening. We considered to do the screening in FDA approved drug libraries, because it is a rapid way to find flexible molecules comparing with synthesis of new small molecules. And it is a new field we decided to stepped into. Thus we got contact with Dr. Xiao.
Having the experience of virtual screening, they affirmed that new compound design is not easy considering of synthetic methods and screening in approved drug libraries shorten this process. There are many ways of HTS including absorbance detection, fluorescent intensity measurement and colorful changing and so on.
Also, they kindly introduced some principles in HTS. For example, when we established the system for HTS, the condition we try to explore are NaCl concentrations, Zn2+ concentrations and pH. But as we all know, temperature, type of buffer are also important factors influencing the enzyme activities. They told us their usual way to execute temperature changing experiment is by water bath since the machine cannot control it automatically. But our microplate reader is not on the same floor with water bath so it’s difficult to guarantee its accuracy. So they advised us to ensure the measurement procedure on the average of room temperature.
Winding Roads in Execution
Tao Wang, Tianjin University
Dr. Wang researches on immune and vaccine development. Although he frankly said that he cannot give us specific suggestion on antibiotics, from general experience he helped us optimize measurement procedure. We should make fluorescent data quantitative in order to compare each inhibitors’ effect, so that our result would be more convincible.
Since there’s a lot of differences between facility and conditions, we finally decide to test typical inhibitors’ inhibition kinetic constants such as IC50, Ki and analyze their inhibition mechanisms.
Kewu Yang, Northwest University
The key step of our project is to evaluate new inhibitors’ effect on living bacteria. Via reading articles, we found that most of them choose to test minimum inhibitory concentration(MIC). However, real condition blocks us to cultivate pathogenic bacteria such as P. aeruginosa in our laboratory because we have to guarantee both experimenters and circumstance’s safety first. So it is vital to look for proper method to test on engineered E.coli.
Dr. Yang’s article gave us inspiration. His group researching targets are closely related to biochemical drugs and the proteins and metallo-enzymes. UV-vis is not a fresh way and it is widely used in researches. When applied to living bacterial cells together, it is more meaningful to monitor the level of beta-lactams rather than beta-lactamase inhibitors. At that time, UV-vis is a simple way to detect the degree of antibiotic hydrolyzation.
 Dixit A, Kumar N, Kumar S, Trigun V. Antimicrobial resistance: Progress in the decade since emergence of New Delhi metallo-beta-lactamase in India. Indian J Community Med 2019;44:4-8
 Nordmann P, Naas T, Poirel L. Global spread of carbapenemase-producing Enterobacteriaceae. Emerg Infect Dis 2011;17:1791-8.
 Yonghong Xiao, Lanjuan Li,China's national plan to combat antimicrobial resistance, The Lancet Infectious Diseases, Volume 16, Issue 11, 2016, 1216-1218.