Since most d-amino acids (DAAs) are utilized by bacterial cells but not by mammalian eukaryotic hosts, recently DAA-based molecular imaging strategies have been extensively explored for noninvasively differentiating bacterial infections from the host’s inflammatory responses. Given glutamine’s pivotal role in bacterial survival, cell growth, biofilm formation, and even virulence, here we report a new positron emission tomography (PET) imaging approach using d-5-[11C]­glutamine (d-[5-11C]-Gln) for potential clinical assessment of bacterial infection through a comparative study with its l-isomer counterpart, l-[5-11C]-Gln. In both control and infected mice, l-[5-11C]-Gln had substantially higher uptake levels than d-[5-11C]-Gln in most organs except the kidneys, showing the expected higher use of l-[5-11C]-Gln by mammalian tissues and more efficient renal excretion of d-[5-11C]-Gln. Importantly, our work demonstrates that PET imaging with d-[5-11C]-Gln is capable of detecting infections induced by both Escherichia coli (E. coli) and methicillin-resistant Staphylococcus aureus (MRSA) in a dual-infection murine myositis model with significantly higher infection-to-background contrast than with l-[5-11C]-Gln (in E. coli, 1.64; in MRSA, 2.62, p = 0.0004). This can be attributed to the fact that d-[5-11C]-Gln is utilized by bacteria while being more efficiently cleared from the host tissues. We confirmed the bacterial infection imaging specificity of d-[5-11C]-Gln by comparing its uptake in active bacterial infections versus sterile inflammation and with 2-deoxy-2-[18F]­fluoroglucose ([18F]­FDG). These results together demonstrate the translational potential of PET imaging with d-[5-11C]-Gln for the noninvasive detection of bacterial infectious diseases in humans.

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Data supporting the concept that microbial biofilms are a major cause of non-healing ulcers remain limited. A porcine model was established where delayed healing resulted from methicillin-resistant Staphylococcus aureus (MRSA) infection in full-thickness wounds. At the end of one study a wound remaining open was sampled and a MRSA strain was isolated. This pig-passaged strain was used as the inoculating strain in several subsequent studies. The resulting MRSA wound infections exhibited a greater, more stable tissue bioburden than seen in studies using the parent strain. Furthermore, wounds infected with the passaged strain experienced a greater delay in healing. To understand whether these changes corresponded to an increased biofilm character of the wound infection, wound biopsy samples from studies using either the parent or passaged MRSA strains were examined microscopically. Evidence of biofilm was observed for both strains, as most samples at a minimum had multiple isolated, dense microcolonies of bacteria. However, the passaged MRSA resulted in bacterial colonies of greater frequency and size that occurred more often in concatenated fashion to generate extended sections of biofilm. These results provide a model case in which increasing biofilm character of a wound infection corresponded with a greater delay in wound healing.

Examination of clinical samples indicates bacterial biofilms are present in the majority of chronic wounds, and substantial evidence suggests biofilms contribute significantly to delayed healing. Bacteria in biofilms are highly tolerant of antimicrobials, and little data exist to guide the choice of anti-biofilm wound therapy. Cadexomer iodine (CI) was recently reported to have superior efficacy compared to diverse wound dressings against Pseudomonas aeruginosa biofilms in an ex vivo model. In the current study, the strong performance of CI vs. P. aeruginosa biofilm was confirmed using colony and colony drip-flow in vitro wound biofilm models. Similar in vitro efficacy of CI was also demonstrated against mature Staphylococcus aureus biofilms using the same models. Additionally, the rapid kill of mature S. aureus and P. aeruginosa colony biofilms was visualized by confocal microscopy using Live/Dead fluorescent stains. Superior in vitro efficacy of CI vs. staphylococcal biofilms was further demonstrated against methicillin-resistant S. aureus (MRSA) using multiple biofilm models with log reduction, Live/Dead, and metabolic endpoints. Comparator antimicrobial dressings, including silver-based dressings used throughout and other active agents used in individual models, elucidated only limited effects against the mature biofilms. Given the promising in vitro activity, CI was tested in an established mouse model of MRSA wound biofilm. CI had significantly greater impact on MRSA biofilm in mouse wounds than silver dressings or mupirocin based on Gram-stained histology sections and quantitative microbiology from biopsy samples (>4 log reduction in CFU/g vs. 0.7-1.6, p < 0.0001). The superior efficacy for CI in these in vitro and in vivo models suggests CI topical products may represent a better choice to address established bacterial biofilm in chronic wounds.

A wound biofilm model was created by adapting a superficial infection model. Partial-thickness murine wounds were inoculated with methicillin-resistant Staphylococcus aureus (MRSA). Dense biofilm communities developed at the wound surface after 24 h as demonstrated by microscopy and quantitative microbiology. Common topical antimicrobial agents had reduced efficacy when treatment was initiated 24 h after inoculation compared to 4 h after inoculation. This model provides a rapid in vivo test for new agents to treat wound biofilm infections.