Exploring New Delhi Metallo Beta Lactamases in Klebsiella pneumoniae and Escherichia coli: genotypic vs. phenotypic insights.

Abstract

Background: Carbapenemase-producing Enterobacterales pose a serious clinical threat, particularly in high-burden settings of carbapenem-resistant Escherichia coli and Klebsiella pneumoniae (CREK), where rapid detection tools are essential to aid patient management. In this study, we focused on bla_NDM, the most frequently reported carbapenemase in the region, and evaluated a combined phenotypic (lateral flow) and genotypic (PCR and WGS) approach for its detection. This research underscores the utility of lateral flow assays as a practical alternative to resource-intensive genotypic methods, offering a scalable solution for settings with limited laboratory capacity.

Method: One hundred seventy-seven extensively drug-resistant strains were characterized using MALDI-TOF. Isolates were analyzed to detect Carbapenem-resistant Escherichia coli and Klebsiella pneumoniae (CREK) using disk diffusion, MIC test, and PCR targeting bla_NDM. Antibiotic susceptibility patterns were analyzed and visualized using single-linkage hierarchical clustering, with results displayed on a permuted heat map. Immunochromatographic assay, RESIST-5 O.K.N.V.I (Coris Bioconcept®) was used for CREK isolates [(n = 17), positive and negative)] and Oxford Nanopore Sequencing was conducted on subsets [(n = 5) bla_NDM-positive co-producers of bla_NDM and bla_OXA, and (n = 2) bla_NDM-negative bla_OXA producers) to evaluate the reliability of phenotypic and genotypic tests.

Result: Most of the XDR strains (90%) were CREK, with K. pneumoniae (71.2%) more prevalent than E. coli (28.7%) (p < 0.05). All CREK strains exhibited complete resistance (100%) to multiple antibiotics with 66% showing sensitivity to levofloxacin. Furthermore, K. pneumoniae (57.8%) had higher bla_NDM gene prevalence than E. coli (36.9%). Among bla_NDM-positive CREK, lateral flow assay revealed approximately half of each bacteria type co-produced bla_OXA (E.coli, 52.9%), and (K. pneumoniae, 47%). For bla_NDM-negative strains, bla_OXA was more prevalent in K. pneumoniae (82.35%) than E. coli (41%) (p < 0.05). Comparing phenotypic to genotypic assays, E. coli showed 100% (CI 80.49 - 100%) sensitivity and specificity with a high Kappa agreement coefficient (0.91) (CI 95% 0.661-1, p < 0.01), whereas K. pneumoniae assays had lower sensitivity and specificity (40%) (CI 5.27 - 85.34%), with a lower Kappa agreement coefficient (0.20) (CI 95% 0.104-0.298, p < 0.01).

Conclusion: This study demonstrates the value of the RESIST-5 O.K.N.V.I. lateral flow assay as a rapid and reliable diagnostic tool for detecting bla_NDM in Escherichia coli, with strong agreement to PCR and WGS. While performance for Klebsiella pneumoniae was lower, the assay offers a practical alternative in resource-limited settings, aiding antimicrobial stewardship and improving diagnostic capacities in high-burden regions.