Rapid classification of live bacteria in blood using antimicrobial peptide microarrays

The growing threat of antimicrobial resistance has made rapid and sensitive detection of bacterial pathogens in human samples an urgent need. In the case of blood infection or bacteremia, standard pathogen identification protocols are based on a two-step process. First, bacteria are cultured to increase their concentration in the sample. Then, the pathogen is assessed using cultural, molecular methods or mass spectrometry-based identification methods. To detect a larger panel of pathogens with a single, wide-spectrum microarray, we selected natural antimicrobial peptides (AMPs) and designed de novo peptides as powerful ligands for single-step bacterial classification using surface plasmon resonance imaging (SPRi). Human blood samples were spiked with low levels (less than 30 CFU ml^-1) of clinically relevant pathogens: E. coli O157:H7 and E. coli O1:K1:H7 as Gram-negative species, as well as methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) Staphylococcus aureus as Gram-positive species. One-step analysis of the samples on AMP microarrays using SPRi enabled the detection of 2.6 ± 0.2 CFU ml^-1E. coli O157:H7 in less than 10 hours and 2.7 ± 0.4 CFU ml^-1 MRSA in less than 24 hours. Multidimensional analyses of SPRi data showed that a single AMP microarray can efficiently discriminate between E. coli and S. aureus strains. This process is even easier with the help of the de novo KIWI peptide. Our approach, which facilitates faster classification and identification of bacterial pathogens, paves the way for more powerful diagnostic tools in the future.