Bryan Lanning

The peptidoglycan cell wall is a common target for antibiotic therapy, but its structure and assembly are only partially understood. Peptidoglycan synthesis requires a suite of penicillin-binding proteins (PBPs), the individual roles of which are difficult to determine because each enzyme is often dispensable for growth perhaps due to functional redundancy. To address this challenge, we sought to generate tools that would enable selective examination of a subset of PBPs. We designed and synthesized fluorescent and biotin derivatives of the β-lactam-containing antibiotic cephalosporin C. These probes facilitated specific in vivo labeling of active PBPs in both Bacillus subtilis PY79 and an unencapsulated derivative of D39 Streptococcus pneumoniae. Microscopy and gel-based analysis indicated that the cephalosporin C-based probes are more selective than BOCILLIN-FL, a commercially available penicillin V analogue, which labels all PBPs. Dual labeling of live cells performed by saturation of cephalosporin C-susceptible PBPs followed by tagging of the remaining PBP population with BOCILLIN-FL demonstrated that the two sets of PBPs are not co-localized. This suggests that even PBPs that are located at a particular site (e.g., septum) are not all intermixed, but rather that PBP subpopulations are discretely localized. Accordingly, the Ceph C probes represent new tools to explore a subset of PBPs and have the potential to facilitate a deeper understand of the roles of this critical class of proteins.

Introduction: Multiplex nucleic acid diagnostics for blood-borne pathogens have moved closer to clinical application in the two years since we first reviewed this topic.Areas covered: A new emphasis on detecting pathogens directly in a blood sample without culture, coupling PCR amplification to microfluidic devices and higher multiplexing in isothermal amplification are some of the advances. A wholly new approach of correlating host gene expression response with specific infectious agents opens another opportunity for multiplex detection. Established microarrays, which had been the highest multiplicity platform, are being displaced by Next Generation Sequencing (NGS) having potentially no limit to the number of pathogens that it can identify. Greater accessibility of sequencing devices, standardization of bioinformatic analysis pathways and increased acceptance from regulatory authorities are driving this technology.Expert commentary: The landscape of traditional diagnostics for detection of blood-borne pathogens has changed in the last 5 years. There is no doubt that NSG is recognized as a disruptive technology with a growing repertoire of tools, such as subtyping, resistome analysis, etc., available for clinical microbiology. Increasing acceptance indicates the dominating position of NGS as the future of multiplex molecular diagnostics for blood-borne pathogens.