The World Health Organization has named antimicrobial resistance (AMR) as one of the most pressing threats to global health. Over 1.27 million deaths worldwide have been attributed to bacterial AMR in 2019 alone. As many as 10 million people could die from AMR infections each year by 2050 if no action is taken. 

Current AMR testing results need 2 to 3 days to be produced. This represents a critical time delay that increases treatment costs and elevates the risk of death. Such a delay also makes AMR monitoring in the environment harder. The biotech industry has made efforts to develop new antibiotics. Nonetheless, a holistic approach to combating AMR also needs a means to track its spread.

Having tools that can quickly identify antimicrobial-resistant microbes is essential for addressing the AMR crisis. That’s where the polymerase chain reaction (PCR) comes in. For one, PCR can detect AMR genes in the environment. PCR can also help inform clinicians of the antibiotics that a microbe is resistant to. Most importantly, PCR assays can be done within a single day, cutting the time needed to begin antibiotic treatment.

But how does PCR produce these results? To better understand PCR’s role in fighting the AMR crisis, we first need to discuss how AMR arises in microbes. We’ll then discuss how PCR works and how it can detect resistant microbes in the environment. We will then end by highlighting a series of companies developing innovative PCR kits tailored for detecting AMR. 

How Does Antimicrobial Resistance Work?

AMR arises when infecting microbes continue to survive even after doctors administer antibiotics. Microbes can gain resistance through several mechanisms.

• Intrinsic Resistance: The way a bacterium is structured may confer on it an innate ability to survive antibiotic treatment. Here, the cells don’t have to be changed to survive after antibiotic exposure. Cells can be naturally resistant to an antibiotic in many ways. For example, the cells of some microbial species have thick cell walls that prevent antibiotics from entering them. 
• Acquired resistance - mutations: Microbes can also acquire resistance through genetic mutations. Mutations alter a kind of biomolecule called proteins that affect how cells behave. Genes whose mutations make a microbe more resistant to an antibiotic are called AMR genes. Below are some examples of proteins where gene mutations can affect antibiotic resistance:

• Efflux pumps: These are proteins that force toxins such as antibiotics out of the cell. Any antibiotic that must enter the cell will not work as well when the efflux pumps are working. Mutations can increase their activity, contributing to AMR.
• Beta-lactamases: These proteins break open what’s called a beta-lactam ring. These rings provide the foundation of beta-lactam antibiotics like penicillin. Beta-lactam antibiotics target proteins that help bacteria form the cell wall. When this ring is broken, beta-lactam antibiotics lose their activity. 
• Proteins involved with DNA replication: Some antibiotics bind to enzymes that mediate DNA replication, such as DNA gyrase. DNA replication allows genomes, an organism’s genetic material, to be copied to the next generation. Stopping this process with antibiotics leads to death. However, mutations to the genes encoding these enzymes can prevent antibiotics from binding.

• Acquired resistance - taking in foreign DNA: Microbes can also take genetic material from other bacteria or free-floating DNA in the environment. This occurs through horizontal gene transfer (HGT), where genetic information is moved between organisms from divergent evolutionary lineages. This information can be picked up from the environment or through mobile genetic elements (MGEs) that can jump around within a genome. MGEs can also be transferred to another organism’s genome through HGT.

Doctors must be careful not to administer antibiotics that microbes are naturally resistant to. Nevertheless, knowing whether a microbe acquired resistance is a much harder problem to solve.  That’s why techniques that help people know when and how AMR arises and spreads will help curb the AMR crisis.

What Is PCR?

PCR is one such technique used to monitor AMR spread. It is a foundational technique that allows DNA sequences to be found and amplified. Every PCR assay requires the following reagents and components for it to work:

• Nucleotides: The building blocks of DNA. Every nucleotide is composed of a phosphate-sugar backbone with a nitrogen-containing base attached to it. Four types of nucleotides in equal abundances are required to replicate DNA and run the PCR assay: adenine, guanine, thymine, and cytosine.  
• Template DNA: This is the DNA extracted from a clinical or environmental sample where AMR is being surveyed. Care must be taken to ensure that the target DNA sequences are present for amplification.
• DNA polymerase: This is the enzyme that drives DNA replication in the PCR assay. Without this enzyme, no DNA amplification can take place. The polymerase must also have high fidelity to ensure that the replicated DNA contains the correct sequence.
• Primers: These short, synthesized DNA sequences, or oligonucleotidesprovide the boundaries for DNA polymerase to replicate DNA. These sequences are typically 18-30 bases and anneal to the ends of the DNA target. Primers can cover DNA sequences as short as 100 base pairs (bp) and as long as thousands of bp in length. Primer design tools such as Primer3plus are necessary to make sure the PCR assay amplifies only the target sequence efficiently.
• PCR buffers: These solutions contain the molecules and ions necessary for the DNA polymerase to operate optimally. These include molecules that stabilize the reaction’s pH, ions that activate DNA polymerase, and other ions that allow primers to anneal to the target sequence. By definition, buffers also prevent changes to acidity, or pH, that impacts how well the reactions work.
• Thermal cyclers: These machines carry out all PCR assays. They contain metal blocks that allow tight temperature control while running the PCRs. These machines also have software that allows users to input the thermal cycling conditions. 

Once these reagents are present, the PCR assay proceeds in a series of up to 35 cycles. Each cycle contains a series of three steps that enable DNA targets to be doubled:

• Denaturation: DNA starts as two strands linked together in a helix. In this step, the strands are separated by heating at 94^oC. 
• Annealing: In this step, the single-stranded DNA molecules are cooled to the annealing temperature (~50-65^oC). At this temperature, the primers can bind to the regions flanking the DNA target. 
• Extension: This step features the DNA polymerase amplifying the DNA from one end of the primer (the 3’ end) to the other (the 5’ end).

PCR has many extensions and modifications that have been used for a wide range of applications. Two of these include:

• qPCR: In a quantitative PCR (qPCR), assay, fluorescent dyes or DNA probes are added to a typical PCR assay. The binding of the dye or probe sequence to the DNA target allows not only the gene target to be amplified, but its abundance to be measured as well. This can help researchers assess how much and how quickly antimicrobial resistance could be spreading in diverse environments such as wastewater, urban aquatic environments, and soil.
• Multiplex PCR: Multiplex PCR introduces multiple primer sets in a single PCR to search for multiple gene targets at the same time. This allows health monitors and scientists to identify MDR bacteria and produce a comprehensive AMR survey for clinical treatment. 

Irrespective of the PCR extension employed, PCR assays proceed with a series of common ingredients that must be prepared in the right amounts. Additionally, it’s the primers that allow researchers to detect AMR genes in the clinic and environment. 

Biotech Companies Using PCR to Combat Antimicrobial Resistance

PCR assays can produce AMR data more quickly than conventional assays. Multiple biotech companies have sought to develop kits for detecting AMR. Many of these kits can test AMR spread or help doctors diagnose resistant infections. 

• Resistomap and Takara Bio: These companies have teamed up to prepare an integrated qPCR workflow to monitor AMR over time. The pipeline begins with Takara Bio’s Smartchip Real-time PCR system performing the qPCRs. The qPCRs feature two sets of primers designed to target up to 384 AMR genes at the same time. Then, Resistomap’s ResistApp digitally analyzes the generated qPCR data. The app determines the extent of AMR spread by measuring the amount of an AMR gene present relative to a bacterial control gene called 16S rRNA. Absolute quantification of an AMR gene is also possible provided standards containing known amounts of DNA are also prepared.
• Cepheid: The fight against AMR also depends on knowing which microbes are causing the disease. The Xpert Xpress Strep A test provides a way to screen for a specific type of microbe called Group A Streptococcus (GAS). These bacteria cause a wide range of diseases such as Strep throat, rheumatic fever, and necrotizing fasciitis. The assay allows researchers to diagnose GAS more easily without needing to wait for days by growing the infecting microbes in the lab. 
• Streck: The Nebraska-based company has developed a series of qPCR kits specifically designed to detect different sets of AMR genes. Their specialty lies in detecting different kinds of ampC genes, each of which encodes a type of b-lactamase such as OXA. The kits can also help find multiple variants of the same ampC gene.
• Hologic: The Novodiag System is an innovative platform that uses multiplex qPCR assays to detect AMR microbes in clinical settings. With this system, patients can obtain results within an hour after sample processing. The Novodiag CarbaR+ panel assay also searches for resistance to two types of antibiotics: carbapenems (eg. Imipenem) and Colistin.

Need a PCR Machine for Your Research?

Diagnostic companies are taking the AMR fight to the environment and the clinic. You too can join the fight. Maybe you’re ready to do the PCR assays, but don’t know what thermal cycler to use. 

At Excedr, we have the full complement of PCR thermocyclers available for you to lease. By leasing with us, you can acquire the thermal cycler best suited to your needs and keep your workflows cost-effective. Help the world fight antimicrobial resistance today and ask us how we can help. 

Interested in applying to lease a PCR machine today? Let us know!