Short Ligand, Cysteine-Modified Warnericin RK Antimicrobial Peptides Favor Highly Sensitive Detection of Legionella pneumophila

Abstract

Culture-based methods for the detection of Legionella pneumophila are prohibitively slow and frequently inadequate. The problem has been addressed with biosensing technology that employs a variety of ligands for the specific capture of bacteria. However, the limited success of the application of mammalian antibodies, aptamers, and nucleic acid-based probes for sensitive biosensing has generated growing interest in exploring alternative biosensing architectures, such as those based on antimicrobial peptides (AMP) that are known for their attractive therapeutic applications. We report on the successful employment of cysteinemodified warnericin RK AMP for the operation of a highly sensitive biosensor of L. pneumophila based on digital photocorrosion of GaAs/AlGaAs nanoheterostructures. The replacement of the relatively cumbersome procedure commonly applied for the attachment of antibodies to COOH-terminated mercaptohexadecanoic acid self-assembled monolayers has allowed for a significant reduction in the distance at which bacteria are immobilized above the biosensor surface. An important consequence of this approach is the attractive limit of detection of L. pneumophila estimated at 2 × 10 2 CFU/mL. The target bacteria were captured four times more efficiently than P. fluorescens, B. subtilis, and E. coli, which is highly promising for environmental monitoring.

Selective Detection of Legionella pneumophila Serogroup 1 and 5 with a Digital Photocorrosion Biosensor Using Antimicrobial Peptide-Antibody Sandwich Strategy

“…Therefore, the amide-related peaks in the FTIR spectra (1605 cm −1 , 1655 cm −1 , 1734 cm −1 , and 3218 cm −1 ) confirm the AMP immobilization on the GaAs surface. Furthermore, the XPS amide-related peak at 288.08 eV observed for our samples [ 40 ] corroborates the successful immobilization of peptides, in agreement with Corrales-Ureña and colleagues [ 59 ].…”

“…The immobilization of AMPs on GaAs surface was verified using FTIR-absorbance measurements followed by the same procedure as reported by Islam, Hassen, Tayabali, and Dubowski [ 40 ]. The absorbance band at 1605 cm −1 that is well known for C=O stretching could be assigned to the amide II [ 50 , 51 ] (see Table S1 for FTIR absorbance bands reported in literature).…”

“…We have explored warnericin RK AMP for the application in a DIP biosensor and demonstrated rapid detection of L. pneumophila at 10 3 CFU/mL [ 39 ]. In a follow up publication [ 40 ], we reported that a cysteine-modified warnericin RK AMP (Cys RK AMP) biosensing architecture increased sensitivity to 200 CFU/mL.…”

Evaluation of a Most Probable Number Method for Detection and Quantification of Legionella pneumophila

“…In addition, some viable but non-culturable bacteria (VBNC) could not be detected by the plate culture method. This is one disadvantage because these VBNC microorganisms may acquire the ability to grow and proliferate under favorable conditions, which may lead to false negative results [ 19 ]. In general, the plate culture method has some limitations in daily monitoring.…”

Investigation of Conditions for Capture of Live Legionella pneumophila with Polyclonal and Recombinant Antibodies

“…Furthermore, living bacteria could be further characterised to provide a second level of authentication or additional information such as antimicrobial sensitivity. As an alternative to antibodies, we have also employed capture with short-ligand and sandwich antimicrobial peptide architectures that allowed detection of L. pneumophila at 1–2 × 10 2 CFU/mL [ 26 ].…”