Multiplex high-resolution melting analysis as a diagnostic tool for detection of carbapenemes resistant and esbl genes in Klebsiella pneumoniae isolated from burn patients

Klebsiella pneumoniae is one of the important pathogens among burn inflammation patients, exhibiting high resistance to most antibiotics used for treatment, particularly carbapenems and cephalosporin antibiotics. In this study, 150 swab samples were collected from burn inflammation patients at Tikrit hospitals between the beginning of November 2022 and the end of November 2023. All swab samples were immediately inoculated with a sterile loop onto MacConkey agar and cultured on CHROMagar Orientation medium. To confirm the identification of the isolates, the VITEK2 system was used. A total of 80 K. pneumoniae isolates were obtained, with 30% of them coming from females. The prevalence of isolates was higher in females (66.6%) compared to males (33.4%). Antimicrobial susceptibility tests for all isolates were performed using the VITEK2 system method. The isolates were multidrug resistant. These results indicated that some isolates were multi-drug resistant (MDR), with the rate of sensitivity shown by Klebsiella pneumoniae being 100% to colistin and tigecycline, 91% to rifampicin, 90% to meropenem, 88% to imipenem, 87% to ertapenem, 85% to amikacin, 83% to ciprofloxacin, 82% to cefepime, 80% to ceftriaxone, 78% to gentamicin, 77% to amoxicillin-clavulanate, 59% to ceftazidime, 54% to cefotaxime, and 50% to cefixime. This study aimed to develop a multiplex RT-PCR with high-resolution melting (HRM) analysis for the rapid and accurate detection of six beta-lactamase genes, including ESBL and carbapenemase genes in Klebsiella pneumoniae isolates, as well as the housekeeping gene 16S rRNA. Six pairs of primers and RT-PCR conditions were designed using Beacon Designer software. The identification of the genes was conducted by analyzing the melting curves and temperatures with multiplex RT-PCR using a dedicated HRM reagent. This was the first assay to detect the main carbapenemase and ESBL genes in a two-tube assay, enabling the simultaneous detection of six key markers of resistance: three ESBL genes (TEM, CTX-M, and SHV), three carbapenemase genes (bla-KPC, bla-OXA-48, and bla-NDM), in addition to the 16S rRNA gene as an internal control. The optimal melting peaks for bla-OXA-48, bla-NDM, bla-KPC, 16S rRNA, SHV, CTX-M, and TEM were 94.1°C, 92.1°C, 92.6°C, 87.8°C, 83.7°C, 83.4°C, and 82.1°C, respectively, in one run lasting 2 hours. The presence of ESBL was observed in all 15 samples, indicating the presence of one or more of the genes shv, ctx, and tem. ESBL production is a common mechanism of antibiotic resistance in Klebsiella pneumoniae, allowing the bacteria to hydrolyze and inactivate β-lactam antibiotics. Regarding carbapenem resistance genes, five of these 15 samples contained carbapenem resistance genes, including OXA-48, KPC, and NDM. Carbapenem-resistant Klebsiella pneumoniae poses a significant clinical challenge due to limited treatment options and high mortality rates associated with infections caused by these strains. The frequency of occurrence of these genes in the samples was as follows: three isolates (20%) contained the OXA-48 gene, two isolates (13%) contained the KPC gene, and two isolates (13%) contained the NDM gene. The OXA-48 gene was found in 20% of the total isolates examined. OXA-48 is a class D carbapenemase that confers resistance to carbapenems, a class of broad-spectrum antibiotics. The relatively high frequency (20%) of OXA-48 detection suggests that this gene is fairly prevalent in the studied population or sample set.