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Md A Nahid,1 Jaclyn M Griffin,2 Michael B Lustik,3 Jordan J Hayes,3 Keith SK Fong,3 Timothy S Horseman,3 Massimo Menguito,4 Erik C Snesrud5 ,† Jason C Barnhill,4,6 Michael A Washington4 1Department of Dental and Craniomaxillofacial Trauma Research, United States Army Institute of Surgical Research, JBSA Fort Sam, Houston, TX 78234, USA; 2Department of Vascular Surgery, Tripler Army Medical Center, Honolulu, HI, 96859, USA; 3Department of Clinical Investigation, Tripler Army Medical Center, Honolulu, HI 96859, USA; 4Department of Chemistry and Life Science, United States Military Academy, West Point, NY 10996, USA; 5Multidrug-Resistant Organism Repository and Surveillance Network (MRSN), Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA; 6Department of Radiology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA†Dr Erik C Snesrud passed away on February 10, 2020Correspondence: Md A NahidUnited States Army Institute of Surgical Research, JBSA Fort Sam, Houston, TX 78234, USATel +1 210-539-6419Fax +1 210-539-3877Email md.a.nahid.mil@mail.milPurpose: The biology of chronic wounds is complex and many factors act concurrently to impede healing progress. In this study, the dynamics of microflora changes and their antibiotic susceptibility patterns were evaluated longitudinally over 30 days using data from 28 patients with a total of 47 chronic lower extremity wounds.Materials and Methods: In this study, colonized wound isolates were characterized using cultural, biochemical, and VITEK 2 methods. Antibiotic susceptibility patterns of the wound isolates were analyzed using various phenotypic assays. Furthermore, antimicrobial resistance patterns and the presence of mutations were evaluated by a genotypic assay, whole-genome sequencing (WGS).Results: Staphylococcus aureus and Pseudomonas aeruginosa were found to be the most common strains at early time points, while members of Enterobacteriaceae were prevalent at later stages of infection. Antimicrobial resistance testing and whole-genome sequencing revealed that the molecular and phenotypic characteristics of the identified wound pathogens remained relatively stable throughout the study period. It was also noted that Enterobacter and Klebsiella species may serve as reservoirs for quinolone resistance in the Pacific region.Conclusion: Our observations showed that wounds were colonized with diverse bacteria and interestingly their numbers and/or types were changed over the course of infection. The rapid genetic changes that accompanied the first 4 weeks after presentation did not directly contribute to the development of antibiotic resistance. In addition, standard wound care procedures did not appear to select for resistant bacterial strains. Future efforts should focus on defining those genetic changes associated with the wound colonizing microorganisms that occur beyond 4 weeks.Keywords: anti-microbial resistance, antimicrobial susceptibility profiling, non-healing wound, wound microflora, wound healing