Antibiotic resistance has been known to be a threat ever since the first antibiotic has been discovered. The mechanisms underlying the emergence of resistances were studied thoroughly but only this year, on Jan 4th 2023, an article came around, decifering the structure of a component of the two component system that is endogenous in Staphylococcus aureus and causes the downstream expression of a resistance gene: BlaR1. To us this felt like a milestone for the research on the mechanisms that underly resistances. What now?
Antibiotic resistance has emerged as one of the leading threats to global health and food safety in the 21st century and was classified by the World Health Organization as an emergent threat, needing immediate and coordinated combat worldwide (WHO, Newsroom, 31 July 2020). While new resistance mechanisms emerge and spread, the treatment of a growing list of infections become increasingly harder, with estimations proposing that by 2050 antimicrobial resistance (AMR) could take up to 10 million lives each year (Jim O'Neill 2016; Nguyen et al. 2021).
Figure: Deaths attributable to antimicrobial resistance (AMR) every year. Figure adapted from (Jim O'Neill 2016)
What are the methods so far? In the past 50 years Penicillin G, the compound discovered by Alexander Fleming, was altered. Other fungal species with other compounds were discovered. These were also altered. What mankind did was to invent and reinvent antibiotics. And they are effective - for a few months, sometimes years. Until the first resistance is documented. We can all agree that this is no long-term solution. It does not even cover the roots of the issue: Antibiotics pollution in our wastewater. In Germany and many other countries there is no step during the water treatment that covers filtration of antibiotics. In fact, it is not required by the german government. Some plants conduct tests for resistent strains and screen for resistance genes - when damage was already done.
This is where we saw room for change and improvement. An idea that might change our ways of living, shining light onto threats we will not even see coming. We have the structure - let's make some history!
Reasons for the rapid emergence of new multi-resistant pathogens are primarily the over- and misuse of antibiotics in healthcare and agriculture and the resulting environmental pollution. Our waterways suffer the most severe pollution, as they unite the wastewater of pharmaceutical companies, agriculture, and hospitals. This water combines in wastewater treatment plants (WWTPs), which now creates a selection pressure that favours the development of new antibiotic resistances, multiresistant strains and promote horizontal gene transfer, a mechanism by which antibiotic resistance genes are spread in bacteria (Grehs et al. 2021; Larsson and Flach 2022).
Simultaneously, inadequate treatment of the contaminated water promotes the release and spread of resistant pathogens into the environment. Therefore, WWTPs are a critical point for the acquisition and spread of antibiotics and the implementation of good detection and treatment technologies is essential. While there are some options to identify and monitor antibiotic resistant bacteria and antibiotic resistance genes in the wastewater, most of them are time-consuming and expensive requiring skilled professionals to perform the quantification (Langbehn et al. 2021).
What we wanted to achieve should be easy to use, affordable and applicable without laboratory equipment to adjust to requirements given by WWTPs. Most small plants do not have a laboratory and outsource their screening. Furthermore space is an issue. In our interviews with various treatment plants we were told, that space is rare as most of the plants were built during the 90s. The fourth stage of purification, the biological cleaning of wastewater, is yearning for space.
Our biosensor is based on β-lactam sensor proteins, which grant bacteria antibiotic resistance. They trigger the expression of β-lactamase enzymes, which inactivate β-lactams chemically. β-lactams such as penicillin and cephalosporins are the antibiotics most associated with resistance and contamination in healthcare settings. We settled on developing our biosensor around two two component system, VbrK/VbrR from Vibrio parahaemolyticus (Li et al. 2016) and BlaR1/Bla1 from Staphylococcus aureus (Alexander et al. 2023), and exchanging the resistance genes activated downstream of the cascade by reporter genes that are easily visible.
Another anchor of our mission was the versatility of our approach. Recognizing β-lactams is one thing. Differentiating among beta-lactams and even other antibiotics, another. The structure of BlaR1 (the receptor of the two component system BlaR1/BlaI) is well described and we can use it. We can alter the binding pocket in silico and find mutants that will detect specific antibiotics instead of the β-lactam family.
Now, how can you read out the result? Using β-galactosidases as a reporter gene with X-Gal as a substrate we can immobilize cells carrying the plasmid on paper strips. The only thing you need is a fridge and some time for the blue color to develop. This saves space and decreases costs for laboratory equipment in WWTPs.
You may ask yourself: Is this quantitative enough?
This method? No. That is why we have another approach with another reporter for reading out the
antibiotics concentration in your WWTP. Think of this: What if you could monitor antibiotics
pollution round-the-clock? To achieve this, we have established cells entrapped in alginate with
GFP
as a reporter for the detected antibiotics. By measuring the intensity of the fluorescence
emitted
by the fluorescent protein, you can monitor antibiotics pollution all day long.
Another question that may arise is how is all this possible with the current GMO regulation in the European Union? For the paper strip the amount of water should not exceed a few microliters. The cells never touch the source of where the water came from. Of course, responsible use is a must. When it comes to alginate capsules, the alginate is the trap itself. The cells are unable to escape while small molecules can enter uninterruptedly.
In his review on AMR, Jim O'Neill presents a compelling approach to tackle the growing problem of drug-resistant pathogens globally. Apart from the development of better surveillance mechanisms and tools for rapid detection, increasing public awareness for the emerging threat of AMR is a major part (Jim O'Neill 2016). The annual iGEM competition is a great opportunity to draw attention to one of the most dangerous developments of all time. We have presented our project to WWTPs, scientists and students. We were given the opportunity to actively participate in the global fight against the spread and development of muli-resistant pathogens. In our efforts we gained more supporters to put pressure on the government to act.
By developing our biosensor for reliable and affordable detection, we have joined the global initiative to combat this silent pandemic that, if left unaddressed, will take millions of lives.
As already mentioned we took advantage of already documented two component systems: BlaR1/BlaI and VbrK/VbrR. Two component systems work as follows: A molecular signal is recognized by the transmembrane receptor. The receptor forwards the signal into the cell by altering an effector which can now act as a transcription activator or inhibitor, initiating the expression of corresponding genes. In both cases, the receptors recognize β-lactams and the cascade induces the expression of resistance genes.
BlaR1, the receptor of the BlaR1/BlaI system, senses β-lactams through an acetylation of its sensor domain. The conformational change induced by the sensing leads to the activation of the cyctosolic metalloprotease domain of the receptor. The metalloprotease activity of the receptor enables it to cleave BlaI. BlaI in its uncleaved, dimeric state acts as an inhibitor of two resistance genes: blaZ (β-lactamase) and mecA (resistant cell wall transpeptidase). You can picture it as flexing a sling-shot and only releasing it when needed.
The VbrK/VbrR system is a more traditional two component system. The receptor VbrK is a histidine kinase that autophosphorylates upon β-lactam binding. The phophate group will then be transferred to VbrR which subsequently induces the expression of resistance factors like β-lactamase.
Figure: Design of our biosensor. This figure was created with Biorender.com
For the bacteria this creates an immense advantage when we try to kill them with β-lactams, like penicillin, ampicillin and all the other compounds from this familiy. Yet, we thought we could use this to our advantage. We exchanged the resistance genes with reporter genes: lacZ which encodes β-galactosidase and/or the GFP gene. The β-Galactosidase is known to cleave the snythetic substrate X-Gal, creating a blue color. GFP is a fluorescent protein that emitts light of a specific wavelenght upon excitation. It can be analyzed with a spectrometer or a fluorescence microscope. By replacing the resistance genes of named endogenous two component systems with our chosen reporter genes, we are able to detect and quantify β-lactam contaminations.
Out of breath? Wait for what comes next! We did not halt at β-lactams in general. Our team wanted to know which specific antibiotics circulate in our waste water. In 2023, a cryo-EM structure of BlaR1 was published by Alexander et al., with which it was possible to further elucidate the mechanism of beta-lactam sensing and resistance activation (Alexander et al. 2023). We want to use this structure as a basis for directed mutational studies inside the β-lactam binding pocket, with the aim of optimizing the specificity of BlaR1 for different β-lactam antibiotics. By using simulations of molecular dynamics and docking experiments in silico, we will be able to purify and characterize the protein variants best suited for the detection.