Microbial Contamination in Deceased Musculoskeletal Tissue Donors: A Large‐Scale US Analysis of Infection Risk

ABSTRACT

Background

Microbial contamination of musculoskeletal tissues recovered from deceased donors poses a risk for donor‐derived infections and may lead to tissue exclusion under regulatory standards. Despite stringent aseptic protocols, recovery and processing contamination remains a critical challenge. This study presents a large‐scale analysis of microbial prevalence and distribution in deceased tissue donors in the United States.

Methods

We conducted a retrospective analysis of 1 186 392 musculoskeletal tissue recovery swabs collected from 69 920 deceased donors between 2019 and 2024. Growth‐positive cultures underwent organism identification using MALDI‐TOF mass spectrometry and biochemical methods. Microbial findings were stratified by anatomical recovery zone, and organisms considered potentially disqualifying for transplantation were assessed according to American Association of Tissue Banks (AATB) standards.

Results

Microbial growth was observed in 12.4% of swabs, with 64.8% of donors having at least one growth‐positive sample. Among positive swabs, 92.4% yielded bacteria, 5.0% fungi, and 2.6% yielded both. Organisms considered potentially disqualifying were identified in 5.0% of donors, most commonly Clostridium species and Streptococcus pyogenes. The prevalence of virulent organisms varied by anatomical recovery zone, with the highest rates observed in lower extremity and pelvic sites and the lowest in thoracic and vertebral tissues.

Conclusion

In this large national cohort, potentially virulent microorganisms were identified in a clinically meaningful proportion of deceased tissue donors, with substantial anatomical variability in contamination risk. These findings reinforce the importance of stringent microbiological screening to reduce the risk of recipient infection while supporting safe and effective tissue transplantation.

This large‐scale study of over one‐million musculoskeletal swabs highlights anatomical variability in microbial contamination of musculoskeletal tissues and identifies high‐risk recovery zones for virulent organisms.

Abbreviations

AATB

Association of Advancing Tissue and Biologics

CGTP

Current Good Tissue Practice

TSB

Tryptic Soy Broth

FTM

Fluid Thioglycollate Medium

1. Introduction

The Association of Advancing Tissue and Biologics (AATB) estimates that 3.3 million allografts are distributed by US tissue banks annually [1]. Microbiological contamination remains one of the most critical risks to be managed in tissue banking practices, particularly during the recovery and processing of donor tissue [2]. Bacterial contamination of tissue allografts has been documented to cause fatal and nonfatal infections in recipients of contaminated cartilage, tendon, and other tissues [3, 4, 5]. FDA surveillance data from the MedWatch adverse event reporting system indicate that serious infections following human tissue transplantation have continued to be reported both before and after implementation of Current Good Tissue Practice (CGTP) regulations. Between January 2001 and June 2010, reports of posttransplant infections increased following CGTP implementation, with bacterial pathogens remaining the most frequently reported organisms [6]. Among post‐CGTP reports, approximately one‐third of cases resulted in hospitalization, and a smaller proportion resulted in death [6]. Although MedWatch reports do not establish causality and may include routine postoperative infections, these data underscore the ongoing need for robust donor screening, processing controls, and post‐market surveillance to mitigate infectious risks associated with human tissue products.

A recent report highlighted the ongoing risk of donor‐derived infections in tissue transplantation, including low‐frequency but high‐consequence pathogens such as Mycobacterium tuberculosis, underscoring the importance of surveillance‐based approaches to identifying infectious risks that may not be fully mitigated by donor screening alone [7]. WHO guidelines emphasize that aseptic techniques are critical to prevent microbial contamination during tissue handling and recovery [8]. Efforts to control microbial contamination include preparation of the skin immediately prior to recovery of tissue and astute technical recovery procedures using aseptic techniques to minimize risk of contamination [9]. US tissue banks use Zone Recovery as a technique to reduce the potential spread of microorganisms from one region of the body to another.

Microbiological monitoring is critical during the recovery and processing of tissue‐based products, as the presence of microorganisms can compromise product integrity and increase the risk of posttransplant complications [3]. Swab samples of allograft musculoskeletal tissue are commonly collected for bacterial and fungal bioburden testing to assess the microbial burden, identify potentially pathogenic organisms, and evaluate the effectiveness of aseptic recovery practices [10]. The presence of certain microorganisms may lead to the exclusion of tissues from transplantation or necessitate additional processing steps, including validated sterilization methods.

Microbial testing for musculoskeletal tissue donation in the United States is widely described in the AATB standards. Common practices include 7‐day and 14‐day recovery swab cultures to detect the presence of microorganisms within tissues. These standards (E2.800) identify Clostridium species and Streptococcus pyogenes as organisms of particular concern due to their virulence and potential to cause serious infections in recipients [8]. In addition, tissue processors may designate other bacterial species and fungal organisms as clinically significant based on their specific processing methods, sterilization protocols, and risk mitigation strategies. In this study, we performed a large retrospective analysis encompassing over one million musculoskeletal tissue pre‐processing swab results to evaluate the prevalence and distribution of microbial species, including those classified as potentially pathogenic, to gain a better understanding of contamination trends and risks.

2. Methods

Swabs collected from musculoskeletal tissue recovery procedures by tissue processors were submitted for bioburden assessment. Upon receipt, swabs were inoculated into Tryptic Soy Broth (TSB) and Fluid Thioglycollate Medium (FTM) to promote microbial growth. TSB cultures were incubated at 20°C–25°C, and FTM cultures at 30°C–35°C, for either 7 or 14 days, in accordance with standard microbiological testing protocols.

Cultures exhibiting turbidity (growth) were subjected to Gram staining and subsequently plated onto a variety of solid media to facilitate organism isolation and identification. Microorganism identification was performed using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS, Bruker; 40 Manning Road, Billerica, MA 01821, USA) and the API biochemical identification system (bioMérieux; 376 Chemin de l'Orme, 69280 Marcyl’Étoile, France). The schema of data analysis is depicted in Figure 1.

FIGURE 1.

Data analysis process.

All data used in this analysis were obtained from our Laboratory Information Management System and analyzed using Python 3.10.0 and Microsoft Excel. A total of 1 186 392 swabs from various recovery zones were processed between 2019 and 2024. Of these, 147 522 swabs demonstrated microbial growth and were included in the detailed analysis. Stratification of the data was performed based on tissue recovery zones as defined in Table 1. Statistics and analysis of the data were performed using a chi‐square goodness‐of‐fit test.

TABLE 1.

Tissue donor swab sampling and microorganism growth overview.

Swab collection		# of swabs evaluated	% Growth a	% Virulent organisms
Zone	Sites
Hemi‐pelvis/ilium, left	Hemi‐pelvis, iliac crest, and ilium	58 727	57.16	4.1
Hemi‐pelvis/ilium, right	Hemi‐pelvis, iliac crest, and ilium	58 425	55.03	4.5
Lower extremity, left	Achilles tendon w/ calc, anterior tibialis, anterior/posterior tibialis, fibula, peroneus longus/brevis, tibia, talus, patellar tendon, fascia, and femur	420 032	17.18	1.5
Lower extremity, right	Achilles tendon w/ calc, anterior tibialis, anterior/posterior tibalis, fibula, peroneus longus/brevis, tibia, talus, Patellar tendon, fascia, femur	420 867	23.04	1.8
Upper extremity, left	Humerus, radius, and ulna	103 017	23.23	1.8
Upper extremity, right	Humerus, radius, and ulna	102 210	17.18	1.8
Vertebrae	Spine, vertebral bodies	7300	69.95	5.8
Thoracic	Scapula, costal cartilage, rib, and sternum	15 814	57.27	3.8

Note: Percentages displayed are per recovery zone from growth‐positive swabs.

^a 12.43% of all swabs evaluated had microorganism growth.

3. Results

3.1. Study Cohort and Overall Microbial Growth

A total of 1 186 392 musculoskeletal tissue recovery swabs obtained from 69 920 deceased tissue donors between 2019 and 2024 were included in the analysis. At least one growth‐positive swab was identified in 64.8% of donors (Figure 1). Overall, 12.4% of all swabs demonstrated microbial growth during incubation (n = 147 522).

Among growth‐positive swabs, 92.4% yielded bacterial isolates, 5.0% yielded fungal isolates, and 2.6% demonstrated mixed bacterial and fungal growth (Figure 1). These findings reflect the heterogeneous microbial burden encountered during musculoskeletal tissue recovery.

3.2. Microbial Growth by Anatomical Recovery Zone

Microbial growth rates varied substantially by anatomical recovery zone (Table 1). The vertebral/spinal recovery zone demonstrated the highest percentage of growth‐positive swabs tested (69.9%), followed by thoracic tissues (57.3%) and hemi‐pelvic/ilium sites (55.0%–57.2%). In contrast, upper and lower extremity recovery zones demonstrated lower rates of growth, ranging from 17.2% to 23.2%.

Stratification by recovery zone revealed distinct microbial distribution patterns (Figure 2). The organisms most frequently identified across all zones included Escherichia coli, Staphylococcus epidermidis, Enterococcus faecalis, Staphylococcus aureus, Cutibacterium acnes, Streptococcus agalactiae, coagulase‐negative Staphylococcus species, Enterococcus faecium, and Clostridium species. Many of these organisms are consistent with endogenous gastrointestinal flora or skin commensals.

FIGURE 2.

The heatmap visualization of growth‐positive swabs displays prevalent organisms isolated. The top twenty organisms identified from each zone were included, in addition to the virulent organisms, such as Clostridium species and S. pyogenes. Other organisms identified at lower percentages were excluded in this visual. Heatmap was generated using non‐generative AI (GitHub Copilot‐assisted) to automate and streamline the creation of this figure.

C. acnes (14.7%) and S. epidermidis (18.4%) were particularly prevalent in thoracic and upper extremity recovery zones, whereas E. coli predominated in all zones and especially in hemi‐pelvic and ilium zones. Enterococcus species were consistently detected across multiple anatomical zones.

Polymicrobial growth was more common in certain recovery sites. Swabs collected from vertebral, hemi‐pelvic, and iliac crest zones were significantly more likely to demonstrate polymicrobial growth compared with upper or lower extremity sites (p < 0.0001). Nearly 50% of vertebral swabs exhibited polymicrobial growth, compared with approximately 20% of swabs from upper extremity sites.

3.3. Detection of Potentially Virulent Organisms

Organisms considered potentially disqualifying for transplantation under current AATB standards were identified in 3612 donors, representing 5.0% of all donors evaluated. The distribution of these organisms by anatomical recovery zone is summarized in Table 1.

For donors in which virulent organisms were detected, 61% only had this growth in a single recovery zone (Figure 3a). Detection in multiple zones occurred less frequently; 22% of donors had virulent organisms identified in two recovery zones, and widespread detection across multiple sites was uncommon. On average, 27% of virulent organism detections occurred in lower extremity recovery zones, followed by hemi‐pelvic and ilium sites.

FIGURE 3.

(a) Number of donors that had virulent organisms identified across multiple recovery zones. (b) The prevalence of virulent organisms by recovery zone location.

Among virulent organisms, Clostridium species were the most frequently identified across all recovery zones (Figure 3b). The total number of swabs in which a virulent organism was recovered is displayed in the bar graph by recovery zone location. However, the absolute percentage of virulence is lower for some zones, as there was a higher total number of swabs tested for these sites. Lower extremity recovery zones demonstrated the highest overall prevalence of virulent organisms, including fungal isolates. In contrast, virulent fungi were rarely identified in vertebral, hemi‐pelvic, or thoracic recovery zones.

4. Discussion

Infection acquired through bacterial contamination of donor allografts may result in postoperative complications or death. In this large retrospective analysis of over 69 000 deceased tissue donors, we demonstrate that while microbial contamination of musculoskeletal recovery swabs is common, organisms considered potentially disqualifying under current AATB standards were identified in approximately 5% of donors. Importantly, the prevalence of these organisms varied by anatomical recovery zone, with the highest prevalence observed in the lower extremity and pelvic sites. Although aseptic processing of tissue minimizes bacterial contamination, it cannot eradicate contamination with organisms or disinfectant‐resistant spores, particularly in tissue that is heavily contaminated at the time of recovery [11].

Although most microorganisms identified were consistent with skin commensals or endogenous flora, a subset of organisms, including Clostridium species and S. pyogenes, are associated with severe recipient infections and have been implicated in fatal transplant‐transmitted events. Identification of these organisms in recovery cultures underscores the continued importance of microbiological screening as a critical safeguard in tissue transplantation. While aseptic recovery techniques and validated decontamination processes reduce microbial burden, they cannot fully eliminate the risk posed by heavily contaminated tissues or spore‐forming organisms, particularly when contamination is present at the time of recovery.

The anatomical variability observed in this study has direct implications for recovery practices and infectious risk assessment. Lower extremity and hemi‐pelvic recovery zones demonstrated the highest prevalence of potentially virulent organisms, whereas thoracic and vertebral tissues exhibited lower rates. These patterns reflect differences in skin flora density, proximity to the gastrointestinal tract, environmental exposure, and the technical complexity of recovery procedures. Recognition of high‐risk anatomical zones may support refinement of recovery protocols, including enhanced site preparation or targeted microbiological surveillance, to further reduce contamination risk.

A systematic review of 68 studies demonstrated that variability in donor preparation, recovery environment, and tissue handling affects contamination rates, underscoring the need for standardized recovery protocols [12]. Similarly, an investigation of postmortem bone donors reported microbial growth in 45% of allografts, with contamination risk significantly associated with prolonged postmortem interval and donor trauma [13]. Together, these findings reinforce that microbial contamination is influenced by modifiable procedural factors and highlight the importance of ongoing laboratory surveillance and trend analysis to inform targeted improvements in recovery practices.

From a transplant infectious disease perspective, our findings reinforce the importance of distinguishing low‐pathogenicity organisms from those with clinically meaningful risk to recipients. Detection of potentially virulent organisms was typically confined to one or two recovery zones, and widespread contamination across all sampled sites was uncommon. This observation suggests that contamination is often localized rather than systemic, with implications for tissue selection, processing decisions, and risk stratification. Continued efforts to contextualize microbiological findings may help balance recipient safety with allograft availability.

Our findings are consistent with prior reports examining microbial contamination in musculoskeletal tissue donors. A 10‐year retrospective study from Australia reported that 2.3% of donors (n = 5726) were excluded due to positive microbiological testing, and a subset of recipients who developed postoperative infections had received grafts with positive recovery or processing cultures [14]. In addition, a landmark New England Journal of Medicine report following a fatal case of Clostridium sordellii infection emphasized that preprocessing cultures could reduce but not eliminate the risk of allograft‐derived infection, particularly for tissues not amenable to sterilization [15]. Together, these studies underscore that even low‐frequency contamination events may carry significant clinical consequences, reinforcing the importance of ongoing surveillance and risk mitigation.

The present study also highlights the value of large‐scale surveillance data in informing tissue safety practices. Prior investigations of musculoskeletal allografts have been limited by smaller sample sizes. By leveraging a national dataset spanning multiple tissue processors and recovery settings, our analysis provides a comprehensive assessment of microbial contamination prevalence and organism distribution in US practice. These data complement prior reports linking donor‐derived infections to contaminated allografts and reinforce longstanding recommendations for rigorous recovery‐site asepsis and microbiological monitoring to reduce transplant risk.

Several limitations should be acknowledged. This was a retrospective analysis relying on laboratory culture data without linkage to recipient outcomes; therefore, the presence of microorganisms does not equate to confirmed donor‐derived infection. Detailed donor‐level variables such as postmortem interval, recovery environment (e.g., morgue vs. shared recovery cases with Organ Procurement Organizations), and specific recovery techniques were not available and may influence contamination rates. Variations in disinfection protocols, gowning practices, and aseptic techniques may exist amongst tissue processing entities. In addition, differences in personnel training and technical proficiency could affect recovery outcomes. Future studies incorporating these factors are essential to define sources of bioburden and guide targeted improvements. Despite these limitations, the scale and consistency of the data set strengthen the generalizability of the findings and the importance of understanding the microbial burden of anatomical recovery zones as a critical part of transplant disease prevention.

In conclusion, this large‐scale analysis demonstrates that potentially disqualifying microorganisms were detected in a meaningful minority of deceased tissue donors, with substantial anatomical variability in contamination risk. Per AATB standards for microbial testing, these findings underscore the continued importance of targeted recovery practices and ongoing surveillance to mitigate donor‐derived infection risk in tissue transplantation. As transplantation practices evolve, data‐driven approaches to infection prevention will remain essential to ensuring recipient safety while maximizing the availability of life‐enhancing allografts.

Author Contributions

S.D. and A.M. were responsible for the study conception, design, and wrote the original draft. L.B. and A.M. performed the material preparation, data collection, and analysis. K.E. performed the critical review of intellectual content. All authors have read and agreed to the final version of the manuscript.

Funding

The authors have nothing to report.

Ethics Statement

Ethical approval was not sought for the present study because data presented here were collected retrospectively; deceased donors consented at the time of donation to be screened for infectious diseases and the presence of microorganisms as part of the routine donation process. This data is not sensitive and has no risk for compromising the identity/safety of patients.

Conflicts of Interest

The authors declare no conflicts of interest.