Direct identification of bacterial and human proteins from infected wounds in living 3D skin models

Abstract

Trauma is one of the leading causes of death in people under the age of 49 and complications due to wound infection are the primary cause of death in the first few days after injury. The ESKAPE pathogens are a group of bacteria that are a leading cause of hospital-acquired infections and a major concern in terms of antibiotic resistance. Here, we demonstrate a novel and highly accurate approach for the rapid identification of ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) directly from infected wounds in 3D in vitro skin models. Wounded skin models were inoculated with bacteria and left to incubate. Bacterial proteins were identified within minutes, directly from the wound, by liquid extraction surface analysis mass spectrometry. This approach was able to distinguish closely related strains and, unlike genomic approaches, can be modified to provide dynamic information about pathogen behaviour at the wound site. In addition, since human skin proteins were also identified, this method offers the opportunity to analyse both host and pathogen biomarkers during wound infection in near real-time. Trauma is one of the leading causes of death in people under the age of 49 1. Complications due to infection are the primary cause of death in patients who survive the first few days after a traumatic injury. In recent military conflicts, over half of the total injuries sustained were the result of trauma to the extremities 2. Over 25% of those patients suffered complications due to infection (rising to 50% for those requiring intensive care), either in the bones (osteomyelitis) or deep-wound infection 2,3. Similar rates of infection are associated with civilian trauma 2. Infectious complications result in significantly higher amputation rates, which are also of greater severity. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) are a group of opportunistic pathogens which account for most hospital-acquired infections, and their antibiotic resistance is rising 4,5. Rapid diagnosis and treatment of wound infection is therefore of utmost importance. Current diagnosis involves visual inspection of the wound for signs of infection (inflammation), followed by collection of swabs or tissues, microbial culture and identification in a clinical laboratory, which can take hours or even days. Mass spectrometry (MS) has emerged as a powerful technique for identification and classification of microorganisms. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS has been in development for that purpose since the late 1990s 6,7 and is now well-established in the clinic 8. In the analysis, patient-derived samples are cultured and individual colonies are smeared onto a MALDI target plate. A matrix is applied and the sample is subjected to MALDI-TOF MS. Identification is achieved by spectral fingerprinting...

Liquid Extraction Surface Analysis Mass Spectrometry of ESKAPE Pathogens

“…LESA MS/MS of nine precursor proteins from K. pneumoniae KP257 resulted in identification of six proteins, two of which (DNA-binding protein HU-α and KPN_00497) were identified in the previous analysis of the in vitro 3D skin models. 21 All six proteins were observed in both biological replicates. The same protein mutation R → K at the position 49 and a signal peptide cleavage (1–19) was observed for the KPN_00497 protein.…”

“…( 20 ) More recent work focused on the analysis of bacteria growing on three-dimensional (3D) living skin equivalents, with the aim of simultaneous identification of bacterial and human skin proteins. 21 In vitro skin models were inoculated with three ESKAPE pathogens, Klebsiella pneumoniae , Pseudomonas aeruginosa , and two different strains of Staphylococcus aureus . In each case, LESA MS analysis showed that one to two bacterial proteins could be detected after 48 h and that both bacterial and human skin proteins were observed in the same mass spectra.…”

“…Our pilot LESA MS study focused on Escherichia coli K12, while later work identified 39 proteins from multiple species including 2 of the ESKAPE pathogens Staphylococcus aureus and Pseudomonas aeruginosa. More recent work focused on the analysis of bacteria growing on three-dimensional (3D) living skin equivalents, with the aim of simultaneous identification of bacterial and human skin proteins …”

An Insight into Biomolecules for the Treatment of Skin Infectious Diseases

“…Here, S. aureus and Pseudomonas aeruginosa bacteria have been identified as the most recurrent pathogens. S. aureus is typically located in the skin superficial layers, while P. aeruginosa is spotted in the innermost regions of the wound bed [95,96]. By controlling infection, antimicrobial agents reduce the tendency for conversion of partial thickness wounds into full thickness wounds.…”

A comparison of the in vitro permeation of 3‐O‐ethyl‐l‐ascorbic acid in human skin and in a living skin equivalent (LabSkin™)

“…The dermal layer comprises viable fibroblasts embedded in a fibrin matrix and an epidermis that is developed by seeding keratinocytes on the dermis (26). LabSkin™ has been used for investigating the wound healing process and also for analysing the skin microbiome (26‐28). In the current work, we aimed to determine the permeation of a model compound, 3‐O‐ethyl‐l‐ascorbic acid (EA), in human epidermis and in LabSkin™.…”