Enterobactin-specific antibodies inhibit in vitro growth of different gram-negative bacterial pathogens

Enterobactin (Ent)-mediated high affinity iron acquisition is critically important for Gram-negative bacterial pathogens to survive and infect the host. Recently, we reported an efficient method to prepare novel Ent conjugate vaccines for inducing high level of Ent-specific antibodies, which displayed similar bacteriostatic feature as lipocalins, the host innate immune effectors with potent Ent-binding ability. The Ent-specific antibodies also showed a significant advantage over lipocalins by cross-reacting to various Ent derivatives including salmochelins, the glycosylated Ent that can help enteric pathogens evade the siderophore sequestration by host lipocalins. To demonstrate significant potential of the Ent conjugate vaccine for broader applications to prevent and control various Gram-negative infections in human and animal, in this study, we examined inhibitory effect of Ent-specific antibodies on the in vitro growth of three significant Gram-negative pathogens: Escherichia coli (n = 27), Salmonella enterica (n = 8), and Campylobacter spp. (n = 6). The tested strains were diverse with respect to hosts, geographical origins, serotypes, infection sites and siderophore productions. The Ent-specific antibodies significantly suppressed the growth of each tested strain under iron-restricted conditions. For example, the Ent-specific antibodies consistently exerted 2-5 log₁₀ units of growth reduction on most tested avian pathogenic E. coli (9 of 10 strains) isolated in five countries. Despite various dynamic growth responses observed, notably, the Ent-specific antibodies displayed significantly higher magnitude of growth reduction than lipocalin-2 (up to 5 log₁₀ units of difference) on majority of tested E. coli and S. enterica, which is likely due to sequestration of other siderophores (e.g., salmochelins) by the Ent-specific antibodies. Production of a variety of major siderophores by the tested E. coli and S. enterica strains was examined and confirmed by ultra high performance liquid chromatography-high resolution mass spectrometry analysis. Collectively, this study provides critical and compelling in vitro evidence supporting the feasibility of Ent-based immune interventions against several Gram-negative pathogens.