While the COVID-19 pandemic rages on, the silent pandemic of antimicrobial resistance (AMR) continues to fester. Approximately 700,000 people die from drug-resistant infections every year, making AMR one of the top 10 public health threats facing humanity. As COVID-19 lockdowns lift, there has been an uptick in respiratory diseases caused by other viruses and bacteria. Accurate diagnosis is the first step toward effective treatment of these illnesses. An understanding of the mechanisms underlying AMR will refocus efforts to develop novel diagnostic techniques to combat AMR.
Mechanisms of antimicrobial resistance
“Approximately 700,000 people die from drug-resistant infections every year, making AMR one of the top 10 public health threats facing humanity.”
There are four key drivers of AMR:
Reduced drug accumulation: Microorganisms develop gene mutations that limit drug uptake or increase drug efflux. For example, mutations in lysosomal transporter genes promote drug efflux and enable the Plasmodium falciparum parasite to confer resistance to the antimalarial drug chloroquine.
Drug inactivation: The action of drugs can be affected by a microorganism’s metabolic processes, reducing the amount of active drug available to bind its target. For example, resistance of Mycobacterium tuberculosis to isoniazid, an antituberculosis prodrug, can result from mutations in the gene encoding a bacterial catalase-peroxidase enzyme, preventing its conversion to its active form.
Modification of drug targets: Antimicrobial agents target proteins that are crucial for microbial survival and/or replication. The human immunodeficiency virus, or HIV, mediates resistance to antiretroviral drugs by introducing mutations in its reverse transcriptase enzyme.
Evolution of new cellular processes: Microbes become resistant by developing new pathways that bypass a drug’s intended target. For instance, group A streptococcus bacteria acquire mutations that bypass the folate biosynthetic pathway and allow folate uptake directly from the host.
Acquired AMR genes may be transferred across bacterial species leading to the formation of intractable superbugs.
Identifying antimicrobial resistance
Overuse and misuse of antimicrobial agents is the leading cause of AMR. Diagnostic tests that rapidly and accurately identify pathogens and determine antimicrobial susceptibility to available drugs are the cornerstone in tackling AMR.
Here are the pros and cons of current methods for identifying AMR:
Culture-based diagnostics: Culture-based methods such as disk diffusion or broth dilution are the current standard of care since they allow phenotypic resistance detection. Microbial growth is assessed in the presence of specific drugs at various concentrations and compared to growth in the absence of the drug. Their main drawback is a long turnaround time of 16 to 48 hours. Culture-based diagnostics are ideal for low resource settings since they do not require expensive equipment or reagents, but they cannot be used as point-of-care tests. Automated commercial platforms are also available.
Mass spectrometry: Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) is used in clinical laboratories to identify and assess pathogens for drug susceptibility. MALDI-TOF measures several metabolites that generate a unique biochemical fingerprint of the pathogen. With frequent sampling, the growth rate of the microorganisms can be quantified and used to determine drug susceptibility. This method provides results in a few hours with high sensitivity and specificity but needs specialized instruments and trained personnel.
Molecular diagnostics: Nucleic acid amplification tests, such as polymerase chain reaction (PCR) and isothermal amplification, can be performed directly on small volumes of biological samples and provide rapid results. The tests are highly sensitive and can identify multiple pathogens in parallel. Isothermal amplification offers the added advantage of not requiring specialized equipment like a thermal cycler. However, both assays probe only a limited number of known resistance genes without providing phenotypic information on drug susceptibility.
Immunodiagnostics: Lateral flow assays (LFA) based on antibody–antigen conjugates provide rapid, inexpensive identification of pathogens from clinical samples at the point of patient care but lack antimicrobial susceptibility testing.
Innovations in detecting antimicrobial resistance
Whole genome sequencing: The detailed genetic information on point mutations, insertions, and deletions provided by whole genome sequencing (WGS) is used to predict the emergence of AMR in various microorganisms. DNA purified from cultured samples is sheared into fragments, amplified, and sequenced. The sequenced fragments are compared to an organism’s reference genome to identify alterations that give rise to resistance genes. WGS has been used to investigate the mechanisms of drug resistance and identify novel resistance genes in Staphylococcus aureus, Escherichia coli, and Mycobacterium tuberculosis.
“Acquired AMR genes may be transferred across bacterial species leading to the formation of intractable superbugs.”
CRISPR-based diagnostics: Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins, originally identified as bacterial defense mechanisms, are now being evaluated as potential diagnostic tools to identify pathogens and AMR genes. Coupled to guide RNA, the CRISPR-Cas molecular scissors cleave targeted nucleotide sequences within a pathogen’s genome. By introducing DNA and RNA reporters that fluoresce when cleaved, the CRISPR-Cas system can function as a diagnostic tool. Combining CRISPR with nucleic acid amplification has been used to detect Mycobacterium tuberculosis and dengue and Zika virus from patient samples. Efforts are underway to develop a lateral flow assay for point-of-care testing in low resource settings. CRISPR-based technology can also be combined with transcriptomic analysis to quickly screen bacterial genomes for resistance-related mutations and changes in gene expression in response to antibiotics.
Optical diagnostics: Advances in optical technologies can pave the way for rapid point-of-care diagnostics. Fourier transform infrared spectroscopy (FTIR) quantifies the spectra generated by infrared light absorption of various microbial components, such as lipids, proteins, lipopolysaccharides, and nucleic acids. Changes in the bacterial cell wall and associated polysaccharides of Escherichia coli give rise to different molecular spectra that have been used to measure resistance to several antibiotics.
Similarly, atomic force microscopy-infrared spectroscopy (AFM-IR) has been used to identify changes in phospholipids and carbohydrates associated with vancomycin and daptomycin resistance in Staphylococcus aureus.
The high cost and technical requirements of FTIR may limit its wide acceptance in clinical microbiology laboratories. Though recently, an affordable portable bench top spectrometer that uses machine learning to predict antibiotic resistance in Escherichia coli with 89 percent sensitivity and 66 percent specificity may present a viable alternative.
Additionally, integrating conventional laboratory techniques with advances in automation, microfluidics, and nanotechnology can help develop lab-on-a-chip devices that overcome the limitations of current diagnostics. However, these are prototypes that must undergo rigorous testing before they being implemented in clinical practice.
Overcoming the limitations of current AMR diagnostics
Although several commercial and laboratory-developed tests are available for identifying pathogens, those that can effectively predict drug susceptibility are limited. Advances in nucleic acid sequencing, CRISPR-Cas diagnostics, and optical technology provide innovative alternatives to overcome the limitations of current AMR diagnostics.
Due to falling costs of nucleic acid sequencing, WGS is also gaining acceptance as a clinical diagnostic tool. However, WGS may provide false positive results since the presence of resistance genes does not always translate into phenotypic resistance. CRISPR-Cas diagnostics and infrared spectroscopy are promising tools that remain to be optimized for clinical use.