Abstract
Antimicrobial resistance poses a critical threat to global public health with particular concern focused on the ESKAPE pathogens - Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species – as well as Escherichia coli (E. coli). These pathogens are increasingly associated with multidrug resistance, including resistance to 3rd and 4th generation cephalosporins and carbapenems. The widespread and inappropriate use of antibiotics is the fundamental driver responsible for the global spread of antimicrobial resistance. Antimicrobial stewardship programs offer effective guidelines to curb antibiotic exposure. An important cornerstone to reduce antibiotic usage is the implementation of rapid diagnostic procedures. Improved diagnostic testing will not only address the root cause of resistance development but will also improve treatment outcome, particularly in critical conditions such as sepsis, where the survival rate declines by nearly 8 % every hour. However, in sepsis the challenge lies in the low number of pathogens in the bloodstream, necessitating pathogen enrichment through blood culturing. Depending on the pathogen, blood culturing lasts between 10 hours up to several days. Following enrichment, species identification and phenotypic antimicrobial susceptibility testing are conducted.
In Chapter 2, we reviewed the current literature for alternative enrichment methods that bypass blood culturing by extracting bacteria from whole blood using diverse capture agents often coupled to magnetic beads. These agents include components of the immune system, phage proteins, aptamers, or antimicrobial peptides. The ability to capture live pathogens allows potential combination with routine downstream analyses and facilitates phenotypic antimicrobial susceptibility testing. Our literature research revealed that some capture agents exhibit broad specificity, making them applicable across a diverse range of pathogens, while others demonstrate high specificity for particular species, enabling targeted detection. Despite promising sensitivity and throughput of several capture molecules, none of them seem to possess the optimal combination of stability, efficiency, sensitivity, and cost-effectiveness. Considering the favorable attributes of nanobodies, including their exceptional stability and small size, they unite all features of an ideal capture molecule.
In Chapter 3, we present the identification of E. coli-specific nanobodies designed for the sensitive capture of concentrations below 50 CFU/mL. Aiming for live capture, our strategy centered around targeting the bacterial cell surface. Given the high diversity of lipopolysaccharides, we chose to target the conserved and abundantly present outer membrane proteins OmpA and OmpF. To achieve this, we purified the two most frequently found sequence variants of OmpA of E. coli (EcOmpA), denoted as OmpA-short and OmpA-long isoforms, as well as OmpF. Purified outer membrane proteins were utilized in alpaca immunizations and subsequent phage display selections. Employing the NestLink technology, we identified nanobodies binding in the native cellular context to the outer membrane proteins. Owing to the higher abundance on the cell surface, our nanobody characterization focused on the two isoforms of OmpA. Structure determination using X-ray crystallography revealed interactions of the nanobodies with all four surface-exposed loops of OmpA-short or OmpA-long, respectively. Flow cytometry confirmed cellular context binding and determined an E. coli species coverage of 91 % within the 661k database containing 85,680 E. coli OmpA sequences. These nanobodies were then applied to establish a magnetic capture assay for the efficient enrichment of pathogenic E. coli from buffer solution. To bridge the O-antigen layer present in clinical strains, we engineered linkers of increasing length and adjusted growth conditions and buffers. Thereby we demonstrated efficient capture of a broad set of clinical strains.
Having identified specific E. coli binders, we aimed to expand our repertoire to generate nanobodies against pathogenic Klebsiella pneumoniae (K. pneumoniae). These nanobodies, directed against OmpA of K. pneumoniae, termed KpOmpA, posed additional challenges due to the thick layer of capsular polysaccharides in hypervirulent K. pneumoniae strains. To overcome potential steric hindrances, we optimized our selection protocol. This involved investigation into the effects of phage display selection on cells, along with the determination of the optimal target-to-nanobody library ratios in our NestLink protocol to identify rare cellular context binders.
Our engineered and characterized nanobodies demonstrated high specificity and sensitivity for the surface staining and capture of live E. coli and K. pneumoniae, offering potential applications in diagnostics. While we have successfully managed to efficiently capture these pathogens from buffer solution, further exploration is required to validate their applicability in blood samples. Utilizing these nanobodies as an alternative pathogen enrichment method in bloodstream infection diagnostics, the time-consuming blood culturing step could be omitted or shortened. This would not only accelerate the diagnostic process but would also minimize the antibiotic exposure to patients, thereby mitigating the emergence and spread of antimicrobial resistance. Beyond diagnostics, the identified nanobodies could serve as carriers of therapeutic warhead structures, find applications in water or food surveillance, or be utilized for the staining of clinical E. coli and K. pneumoniae isolates in basic research.
Sorgenfrei, Michèle