In Vitro Evaluation of Inhibitory Activity of Immunoglobulins Y on the Growth of a Multidrug-Resistant Strain of Klebsiella Pneumoniae

Abstract

Background: Multidrug-resistant (MDR) Klebsiella pneumoniae poses a significant global health threat, causing severe infections like pneumonia and bacteremia with high mortality rates, particularly in vulnerable populations. The rise of resistance to conventional antibiotics necessitates alternative antimicrobial strategies. Immunoglobulin Y (IgY) offers a promising, non-antibiotic approach due to its targeted action, high yield, and low risk of inducing resistance, as demonstrated in prior studies against other pathogens. This study aimed to evaluate the in vitro efficacy of IgY against an MDR K. pneumoniae strain to address gaps in data for resistant isolates.

Methods: An MDR K. pneumoniae strain, confirmed via API 20E and EUCAST 2022 antibiogram, was inactivated with 70% ethanol and used to immunize laying hens intramuscularly with Freund's adjuvant over six weeks. IgY was extracted from egg yolks and purified. Specificity and cross-reactivity were assessed by indirect ELISA against K. pneumoniae, Staphylococcus aureus, and Escherichia coli. Growth inhibition was evaluated in Muller Hinton Broth at IgY concentrations of 50, 100, and 150 µg/mL, measuring viable CFU counts at 0, 2, 4, and 8 hours.
Results: The MDR strain showed resistance to seven antibiotic classes but sensitivity to chloramphenicol and norfloxacin. Extracted IgY reached peak concentrations at week 7 (210.41 ± 80.72 mg/mL), with SDS-PAGE confirming purity (~186 kDa native, 65 and 27 kDa chains). ELISA revealed high specificity for K. pneumoniae with minimal cross-reactivity. Specific IgY exhibited dose- and time-dependent bacteriostatic effects, significantly reducing CFU (p<0.001) at 100 and 150 µg/mL, achieving up to 92.75% bacterial load reduction after 8 hours, while non-specific IgY had no effect.
Conclusion: These results highlight IgY's potential as a safe, scalable alternative for combating MDR K. pneumoniae, offering possible biotechnology development for antibody drugs to combat this bacterium.