Antimicrobial resistance (AMR) has become a persistent global public health issue, with an estimated 10 million deaths annually worldwide by 2050 (1). According to the World Health Organisation, bacterial antimicrobial resistance was solely responsible for 1.27 million fatalities in 2019 alone, and it was also partially responsible for 4.95 million deaths (2). The widespread use of antimicrobial drugs has paved the way for selecting bacterial strains against these therapies, causing a tremendous rise in cases of anti-microbic resistance.
Recognizing the severity of this worldwide health concern, World Health Organisation WHO thus published a prioritized list contains antibiotic-resistant bacteria in 2017 (3). Enterobacteriaceae are classified as critically significant within this list, which includes key pathogens such as Escherichia coli and Klebsiella pneumoniae (4). These two infections put a heavy burden on healthcare resources and have a major effect on death rates.
This circumstance reflects a worrying trend where certain bacteria have evolved resistance to vitally needed drugs, such as carbapenems and third generation cephalosporins(5). The broad-spectrum action of carbapenems, a class of beta-lactam antibiotics, makes them essential for treating infections brought on by bacterial strains that are resistant to drugs. They are frequently regarded as antibiotics of last resort, saved for infections that are really severe or in situations when no other treatments have worked. Beta-lactam antibiotics, such as carbapenems, share structural similarities with penicillins and cephalosporins. Their mode of action is to prevent the production of bacterial cell walls(6). Since carbapenem drugs are frequently seen as the last line of defence against serious bacterial infections, bacteria generate enzymes called carbapenemases that impart resistance to these medicines. These enzymes cause carbapenems to hydrolyze, which makes them useless and adds to the worrying increase of bacterial strains that are resistant to the antibiotic. Because carbapenemases may induce resistance to a wide range of β-lactam antibiotics, they pose a substantial danger to the efficacy of antimicrobial treatment and should be taken seriously as a public health problem (7).
To comprehend and arrange carbapenemases in a methodical manner, the Ambler classification is frequently utilised. β-lactamases, which include carbapenemases, are classified into four different classes: A, B, C, and D. Classes A and B are especially important in the setting of carbapenemases. Serine carbapenemases, or class A carbapenemases, are characterised by the use of a serine residue in their active site for hydrolytic action. Notable examples of this kind of enzyme include Klebsiella pneumoniae carbapenemase (KPC). Conversely, class B carbapenemases are metallo-β-lactamases (MBLs) that need metal ions—typically zinc—in order to catalyse reactions. IMP (Imipenemase), VIM (Verona integron-encoded metallo-β-lactamase), and NDM (New Delhi Metallo-β-lactamase) are notable examples of Class B members(8, 9).New Delhi metallo-β-lactamase NDM and oxacillinases OXA is identified as leading indicators that affect Escherichia coli and Klebsiella pneumoniae (10).
The synthesis as well as the impact of NDM and OXA on multidrug resistance (MDR) highlight the significance of creating unique diagnostic approaches in addition to novel treatments derived from knowledge of clinical isolates of K pneumoniae and E. Coli. It has been customary to regularly screen these isolates for the NDM (8) and OXA (9) genes utilising molecular methods such as PCR tests. Nevertheless, research into other techniques has been spurred by the need for a speedier and more affordable diagnostic solution.
Loop-mediated isothermal amplification (LAMP) is an emerging diagnostic method for the detection of NDM and OXA enzymes in these pathogenic strains. It has several advantages over conventional PCR methods(11). The isothermal nature of the assay eliminates complex thermal cycling mechanisms thus being cost-effective and easy to use, especially in resource limited settings. As a result, it can be said that the efficiency of LAMP for fast amplification targeting DNA sequences allows detecting NDM and OXA genes in brief time space which is valuable from patients’ management point(12).
This study combines Real-Time Polymerase Chain Reaction (RTPCR) and melting curve analysis to enhance the diagnostics of LAMP. Real-time PCR offers quantitative measures of target gene presence; it gives an understanding about the prevalence and distribution of NDM and OXA genes in bacterial strains. This method allows for the distinction of particular DNA sequences according to their varying melting temperatures by examining the melting behaviour of amplified DNA. By using this capability, the diagnostic procedure may be further refined by identifying and differentiating between various NDM-1 and OXA variations. The study investigates the possibilities of combining melting curve analysis with loop-mediated isothermal amplification (LAMP) in real-time PCR (RT-PCR) devices, comparing the results with those obtained from conventional PCR and RTPCR. This study adds to the constantly developing toolkit against MDR bacteria and, in the process, aims to tilt the odds back in favour of humankind in the continuous fight against the threat of resistance.