Microbiome Population Adaptation Study

The investigators will accomplish our research aims by collecting cutaneous microbiome samples from 50 persons that have undergone the Osseointegration (OI) surgery at eight timepoints, prospectively. The investigators will also collect control samples to correct for turnovers in species compositions that may naturally occur and to compare the residual limb microbiome to the sound contralateral limb. The investigators will sequence the bacterial community using universal bacterial primers. Using these sequences, The investigators will borrow from ecological theory and calculate the alpha and beta diversity. The alpha diversity will determine the species and abundance of each species that are present, while the beata diversity will allow us to compare how species assemblages and frequencies change between time points. Then, the investigators will take a phylogenetic modeling approach to determine if particular species assemblages correlate with rates of wound healing. The investigators will construct phylogenies from the sequences at the different time points and "paint" the rate of wound healing along the phylogeny (e.g., improved, stagnated, worsened). Using Akaike and Bayesian information criterion, the investigators can determine which phylogenetic model best explains the patterns the investigators see across patients. Lastly, the investigators will quantify soft tissue stability and health and correlate this with the homeostasis of the microbial community. Specifically, the investigators will determine if redundant soft tissue leads to altered microbial communities that can impact the rate of wound healing. Finally, the investigators will further stratify these data to compare microbial communities between the sexes, upper versus lower limbs, and proximal versus distal amputations. This work will allow us to better treat infections after OI surgery and can shed light on wound healing process so that the investigators can better treat limb loss patients and the military community as a whole.

Patients that are undergoing to have OI surgery will be asked if they would like to participate in the research study. All the data collected for the study is research related and will be collected on the case report forms ( CRFs) /source documents and will be entered into the Henry Jackson Foundation (HJF) REDCap.

Below is a breakdown of the research-related study procedures. All of these procedures will be done for research purposes, none of them are for the standard of care. The investigators will coincide study visit with clinical visits/ follow ups. The investigators will collect the following information from the patients/clinician:

Baseline/Pre-operative Surgery Stage I

Demographics (DOB, sex, height, weight, BMI, length of residual limb, amputated side, side indicated for surgery, date of amputation, reason for amputation, details of reason for amputation, issues with prior/current prosthesis) Medication: The investigators will collect detailed information about the medications (names, indication, dose, frequency, route. etc.) that the patients are currently taking (including over-the counter medications, vitamins, or herbal treatment).

Hygiene Questionnaire Biome sample (Osseointegrated and control site) Flora Data Collection Sample Questionnaire Flora Data Collection Sample Questionnaire-Control Holger Score System Questionnaire ( It is a score that the investigators is given after they evaluate the participant's skin condition).

Clinical Assessment

Pre-operative Surgery Stage II, Post-Operative 3 Weeks, 3 Months, 6 Months, and 12 Months Follow-Ups from Surgery Stage II

Medication: The patients will be asked about any changes to the medications that they are taking currently.

Hygiene Questionnaire Biome sample (Osseointegrated and control site) Flora Data Collection Sample Questionnaire Flora Data Collection Sample Questionnaire-Control Holger Score System Questionnaire

Post-Operative Surgery Stage II (in the OR)

Flora Data Collection Sample Questionnaire Flora Data Collection Sample Questionnaire-Control Biome sample (Osseointegrated and control site) Holger Score System Questionnaire

24 Months Post-Operative Surgery Stage II

Medication: The patients will be asked about any changes to the medications that they are taking currently.

Hygiene Questionnaire Biome sample (Osseointegrated and control site) Flora Data Collection Sample Questionnaire Flora Data Collection Sample Questionnaire-Control Holger Score System Questionnaire Thighplasty Questionnaire Clinical Assessment

Data collected will include flora samples collections and soft tissue envelop analysis

Flora samples collections

Biome samples will be collected /swabbed by one of the study team members. The swab area of skin or abutment will be about 2 cm x 2 cm.

Samples collecting swabbing sites For unilateral amputation patients Amputated limb: Implant surface, aperture site, the midpoint between amputated site and groin or axillary crease, and amputation site Contralateral limb as a control: as same level as amputated limb (compatible location of swabbing sites to the imputed limb) For the bilateral amputation patients Both amputated limbs: Implant surface, aperture site, the midpoint between amputated site and groin or axillary crease (as control) Anytime a participant has an infection and comes in outside of research-related timepoints The investigators will collect a Biome sample (Osseointegrated and control site), Flora Data Collection Sample Questionnaire, Flora Data Collection Sample Questionnaire-Control, and clinical assessment.

Sample collection swabbing procedures

Flora is the microorganisms that exist in all living things. The investigators will use the swabbing method to collect the participant's skin biome/flora samples on their Osseointegrated and control site. The fresh swab will be in a moistening buffer such as saline or distilled water. One of the study team members will use one hand to stretch the skin site taut. With the other hand, hold the swab so the shaft is parallel to the skin surface. Apply firm pressure and rub the swab back and forth rigorously 50 times (for 30 seconds). The swab will be put in the tube buffer and will be taken to the freezer as soon as possible (within 30 min of collecting). This is a noninvasive procedure that will cause no pain but may cause discomfortable at the collecting site. If the participants ask us to stop to collection the samples because they experience any pain, The investigators will stop to collection the flora samples.

The participant's skin flora samples will be collected at Baseline/Pre-operative Surgery Stage I, Pre-operative Surgery Stage II, Immediate post-operative Surgery Stage II (in the Operation room), Post-Operative 3 Weeks, 3 Months, 6 Months, 12 Months, and 24 months Follow-Ups from Surgery Stage II.

Swab samples processing, storing, and analysis

The swab needs to go in the tube buffer and be taken to the freezer ASAP (within 30 min of collecting). These biome samples will be stored at the -20 º the Surgical Critical Care Initiative (SC2i) freezer located in building#9. Once the investigators have accumulated enough samples, the investigators will send these samples to Zymo Research Corporation for sequencing on dry ice so the samples remain frozen. After the analysis of these samples by Zymo, the results will be sent back as text and a fasta file. The text files will be the results (alpha and beta diversity, species identified, etc.) and the fasta file will be the actual genetic sequence. Zymo will dispose of all unused samples after three months once the study has been completed

The soft tissue envelope analysis

The soft tissue envelope will be measured for redundancy and scored using the Modified Holger Score System. The investigators will document soft tissue redundancy and hypermobility, drainage and irritability, and active infection. Clinical data being collected are demographics (DOB, sex, height, weight, BMI, length of residual limb, amputated side, side indicated for surgery, date of amputation, reason for amputation, details of reason for amputation, issues with prior/current prosthesis), other relevant past and present medical conditions and medications. Questionnaires that will be collected are hygiene (how many times a day do the participants clean participants' residual limb, what product(s) do the participants use to clean their residual limb, do participant apply any product(s), like lotion, after the participants have cleaned their residual limb, if yes what product(s), flora data collection sample questionnaire (date of assessment, flora sample site, flora sample control site, what site collecting from (implant surface, aperture site, contralateral limb) and person collecting sample), Holger Score System (normal skin, reddish, red and moist, granulation tissues and skin complication leading to removal of the abutment, skin overgrowth), Clinical Assessment (soft tissue-redundancy, hypermobility, drainage irritable), wound grade (improved, stagnated and worsened) and thighplasty questionnaire (did the patient have to have a thighplasty).

Eligibility Criteria:
Inclusion Criteria:

* Any adult male or female that will undergo an OI procedure between the ages of 18-75.
* Defense Enrollment Eligibility Reporting System (DEERs) eligible

Exclusion Criteria:

* Inability to provide self-consent