Metagenomic Analysis of Bacterial Diversity on Reusable Tourniquets in Hospital Environments

Abstract

Background: Reusable tourniquets are commonly used to aid venipuncture and blood collection. However, inadequate disinfection may lead to bacterial contamination and increase the risk of healthcare-associated infections (HAIs). Tourniquets can function as fomites, facilitating the spread of pathogenic bacteria. This study assessed microbial contamination of reusable tourniquets in the emergency department and operating theatre, focusing on clinically relevant HAI-associated pathogens. Methods: Tourniquets from four hospital departments (emergency: adult observation and resuscitation; surgical theatres: pediatric and adult general surgery) were sampled at three time points (n = 12). DNA was extracted and analyzed via 16S rRNA sequencing using NGS technology to identify microbial contamination and taxonomic composition. Results: Sequencing revealed 131 bacterial species across the 12 tourniquets, including clinically important pathogens. Among the top ten HAI-associated groups, Klebsiella spp. were detected in 5/12 samples, Enterococcus spp. in 9/12, Staphylococcus aureus in 1/12, Pseudomonas aeruginosa in 9/12, and Acinetobacter spp. in 10/12. No Escherichia coli, Clostridium difficile, coagulase-negative staphylococci, Proteus spp., or Enterobacter spp. were found. Emergency department tourniquets showed higher bacterial loads; operating theatres had greater species diversity. Conclusions: Reusable tourniquets harbor significant bacterial contamination. Considering disinfection challenges and the lack of guidelines, single-use tourniquets should be considered to reduce HAI risk.

1. Introduction

Numerous reports by the WHO and other organizations have indicated a widening endemic burden of healthcare-associated infections (HAI), including antimicrobial-resistant infections, that harm patients daily in the health care sector in all countries, regardless of their income status. The most significant of these potentially preventable infections are bloodstream infections and other infections related to the use of intravascular catheters [1]. Contaminated medical equipment, including reusable tourniquets, can serve as a significant vector for pathogen transmission. Hospital-acquired infections (HAIs) remain a major challenge in healthcare settings, causing increased morbidity, mortality, and healthcare costs. Previous studies have identified bacterial colonization on frequently used hospital equipment; however, data on tourniquet contamination remain limited [2].

Reusable tourniquets are widely used in health care environments for practices, including intravenous (IV) cannulation, diagnostic blood sample withdrawal, and medication infusion. Specifically, these devices are elastic material placed on a patient’s limb to promote vein distension by locating 10–15 cm proximal to the desired insertion site. Reusable tourniquets, despite routine use and direct contact with the patient’s skin, are frequently neglected in infection control measures. The World Health Organization in “Guidelines for the Prevention of Bloodstream and Other Infections Associated with the Use of Intravascular Catheters” recommends that the tourniquet be disinfected before use, but this may not be practical; alternatively, a disposable tourniquet can be used if available [1]. Parreira et al. reported that the application of disposable tourniquets and bandages with reinforced edges reduced the rate of PIVC infection from 44.1% to 17.9%, with statistical significance (p = 0.014) [3]. It is important to emphasize that the problem of reusable tourniquet contamination is highly relevant in clinical practice. Studies have shown that the contamination rate of reusable tourniquets ranges from 9% to as high as 100%, with many analyses reporting rates exceeding 70% [4]. Such high levels of contamination highlight how widespread and underestimated this issue is in everyday healthcare settings. The most frequently isolated bacteria on tourniquet surfaces are staphylococci, especially coagulase-negative staphylococci and Staphylococcus aureus, but other pathogens, such as Bacillus, members of the Enterobacteriaceae family, Enterococcus, and even antibiotic-resistant strains like MRSA, are also present [2,4]. Reusable tourniquets can act as fomites, serving as vehicles for pathogen transmission between patients, often via the hands of healthcare workers [2,5]. The design of tourniquet elastic materials and surfaces that are difficult to clean thoroughly makes effective disinfection challenging, and routine procedures are often insufficient [2]. For these reasons, an increasing number of international organizations recommend the use of disposable tourniquets or the implementation of rigorous disinfection protocols to reduce the risk of cross-infection. Including these aspects underscores the need for research on microorganisms present on reusable tourniquets and highlights the impact of this issue on patient safety and the quality of healthcare [6,7].

This study expands on our previous work [8] by using 16S rRNA metagenomic sequencing to provide a comprehensive analysis of bacterial diversity on reusable tourniquets, revealing taxa that could not be detected by culture-based methods. The aim of this study was to characterize the bacterial species present on reusable tourniquets used in the emergency department and operating rooms, assessing differences in bacterial load and species diversity by location and time of use using 16S rRNA V3-V4 amplicon sequencing. Overall, this study aims to inform infection control practices by identifying potential microbial transmission risks associated with reusable tourniquets and highlighting the need for optimized sterilization protocols to reduce HAIs.

2. Materials and Methods

2.1. Study Design and Settings

The cross-sectional study was conducted between July 2024 and February 2025 in Gdansk. The collection of material for this study took place in a hospital with the status of a tertiary hospital in northern Poland. The researchers followed the guidelines of Strengthening the reporting of molecular epidemiology for infectious diseases (STROME-ID): an extension of the STROBE statement [9].

Reusable tourniquets were selected from two distinct clinical Units within the hospital: the emergency department—“SR” tourniquets labeling and the operating theater—“SBO” tourniquets labeling (

Table 1. Sampling design and observation timeline in the emergency department and the operating theater.

Sample Name	Observation Time	Area
SR3_1	Indefinite	Adult observation
SR3_2	14 days
SR3_3	28 days
SR6_1	Indefinite	Resuscitation area
SR6_2	14 days
SR6_3	28 days
SBO7_1	Indefinite	Pediatric surgery
SBO7_2	14 days
SBO7_3	28 days
SBO10_1	Indefinite	Adult general surgery
SBO10_2	14 days
SBO10_3	28 days

“SR” labeling applies to emergency department tourniquets, and “SBO” labeling applies to operating theater tourniquets.

). These settings were chosen based on their high patient turnover and frequent use of tourniquets, representing areas with elevated potential for microbial contamination and cross-transmission. The chosen departments differ substantially in their aseptic procedures and disinfection protocols. The operating theater adheres to stricter infection control standards and routine sterilization practices due to the invasive nature of procedures conducted there. In contrast, the emergency department operates under high-pressure, fast-paced conditions where aseptic protocols may be less stringent or inconsistently applied. Inclusion criteria: tourniquets intended for repeated use (reusable), constructed from polyester-elastane fabric, actively used in emergency department settings, and actively used in operating theater settings. Exclusion criteria: single-use (disposable) tourniquets, tourniquets composed of latex, thermoplastic elastomer (TPE), polyurethane (PU), or synthetic rubber, or tourniquets used in hospital units other than the emergency department or operating theater.

2.2. Sample Collection and Processing

A reusable tourniquet material made of porous, flexible fabric was microbiologically analyzed. This study included 12 tourniquets collected from the emergency department and the operating theater. The detailed procedure for sample collection has been described in a previous publication [9]. In short, in the laboratory, plastic elements were cut off from the fabric part of each tourniquet. The fabric part of the tourniquet was placed in a bag with nutrient broth and shaken in a homogenizer. The resulting suspension was inoculated onto microbiological media. After incubation at 37 °C under aerobic conditions, bacterial colonies were counted. Cultures grown in nutrient broth were, after 72 h, transferred to sterile 15 mL tubes (Falcon type, Genoplast, Rokocin, Poland) and centrifuged at 5000 g for 10 min. The resulting pellet was preserved at −80 °C at the Department of Immunobiology and Environmental Microbiology, Medical University of Gdansk, Gdansk, Poland.

2.3. DNA Extraction and Sequencing

ExtractMe total DNA purification columns (Blirt, Gdańsk, Poland) were used to isolate the genetic material, and the manufacturer’s instructions were followed. Purified DNA, suspended in TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA), was assessed for concentration and presence of contaminants using a UV-VIS NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA, USA). The resulting genetic material in the nutrient broth was preserved at −20 °C. Samples were subjected to sequencing of the 16S rRNA amplicon, covering the V3–V4 region. The selected fragment (V3–V4) of the 16S rRNA gene was amplified using specific primers, followed by sequencing using NGS technology (Macrogen Europe BV, Amsterdam, The Netherlands). The resulting sequences were bioinformatically pre-analyzed, where, after purification and filtering, they were assigned to taxa based on comparison with databases (NCBI by BLAST v.4.4.2).

2.4. Data Analysis

Data analysis was performed in the R programming environment using RStudio v.3.6.0. The workflow included data preprocessing, visualization, and clustering. Core libraries included dplyr, ggplot2, pheatmap, and ensemble. Analyses in Figure 1 and Figure 2, concerning microbial species diversity at the family and genus level, were performed using cluster analysis with Ward’s method and squared Euclidean distance. The heatmap blocks were adjusted according to the clustering-based division of the microorganism.

2.5. Ethical Consideration

The study design has been registered at ClinicalTrials.gov (NCT06566495) and has received approval from the Independent Bioethics Committee for Research of the Medical University of Gdansk (approval number: KB/45/2024, 29 February 2024).

3. Results

3.1. Microbial Composition on the Surface of Reusable Tourniquets

The 16S rRNA sequencing revealed a diverse microbial composition on the surface of reusable tourniquets across different hospital departments. The microbial composition of reusable tourniquets was dominated by Proteobacteria, Firmicutes, Actinobacteria, and the Bacteroidetes family, with Staphylococcus, Pseudomonas, Acinetobacter, and Enterococcus being the most frequently detected genera (Figure 1 and Figure 2). Differences in bacterial composition were observed between emergency departments and operating theatres, with higher bacterial diversity in tourniquets from the operating theatre setting.

Figure 2 illustrates the diversity of bacteria in individual tourniquets, based on the number of species occurring within a specific genus. Additionally, data on the colony-forming units per cm² of material part of tourniquets, sourced from an earlier publication [9], have been included.

High diversity was observed in samples with both low and severe bacterial contamination. For example, samples SBO10_1 (53 CFU/cm²) and SBO7_2 (22 CFU/cm²) contained, respectively, 33 and 25 genera. Similarly, high bacterial diversity was recorded in a heavily contaminated tourniquet, SBO7_3 (>677 CFU/cm²), with 23 genera. Conversely, low species diversity was also observed across both lightly and heavily contaminated samples. For instance, SBO10A_2 (4 CFU/cm²) contained only five different genera, and SR6_2 (551 CFU/cm²) had just two (Staphylococcus and Pseudomonas). There was no correlation between microbiological contamination of the sample (CFU) and species diversity. Spearman’s rank correlation analysis showed a value of 0.0897 with p = 0.7815. Therefore, it can be concluded that there is no clear explanation, and both contamination and species diversity appear to be random.

Cluster analysis using the Ward method, based on the number of species occurring within different genera in the samples, allowed for the division of the studied samples into three groups. Group A consists of three samples characterized by the highest diversity of species and genera. These tourniquets originate only from the operating theatre, with an average of 27 different genera per sample. The genera common to all samples in this group include Pseudomonas, Shewanella, Acinetobacter, Aeromonas, Bacillus, Serratia, Staphylococcus, Klebsiella, Stenotrophomonas, Enterobacter, and Enterococcus. Genera found only in group A are Shewanella, Rahnella, Buttiauxella, Citrobacter, Vibrio, Rhizobium, Sphyngobacterium, Timonella, Paracoccus, Paenarthrobacter, Microvirgula, Lysinibacillus, Clostridium, Delftia, Yersinia, Janthinobacterium, Pseudoescherichia, Silvania, Scandinavium, Raoultella, Providencia, Proteus, Phenylobacterium, Pantoea, Obesumbacterium, Leclercia, Hauxiibacter, Aeribacillus, Cutibacterium, and Comamonas. Group B includes four tourniquets, with half originating from the operating theatre and half from the emergency unit. The average number of genera in this group was 6.5, rounded to 6. The genera common in all samples in this group are Pseudomonas, Acinetobacter, and Enterobacter. Group C consists of five tourniquets, four of which were taken from the emergency unit and one from the operating theatre. The average number of different bacterial genera in this group was 7.2, rounded to 7. The only genus present in all tourniquets within this group was Staphylococcus.

The bacterial diversity across the tested hospital units was measured using the Shannon Diversity Index, which shows slight variations; the operating theater had the highest diversity (2.29), suggesting more species are equally distributed, while the emergency department shows slightly lower diversity (2.17), potentially indicating dominance of specific species.

3.2. Identification of Pathogenic Bacteria

3.2.1. Pathogenic Bacteria Cause HAI

Based on data from the ECDC report on healthcare-associated infections (HAIs) in Europe in the years 2022–2023 [10], ten bacterial species and groups most frequently responsible for HAI were selected. The presence of several of these organisms was confirmed in our research, including Klebsiella spp. (Klebsiella pneumoniae 5/12 and Klebsiella aerogenes 1/12), Enterococcus spp. (Enterococcus faecalis), Acinetobacter spp. (Acinetobacter baumannii 9/12 and Acinetobacter lwoffii 3/12), as well as Staphylococcus aureus and Pseudomonas aeruginosa. Detailed data are presented in

Table 2. Occurrence of pathogens responsible for Healthcare-Associated Infections (HAIs) on tourniquets.

Microorganism	Percent of HAI Cases in Europe 2022–2023 [cyt]	Number of Tourniquets All	Number of Tourniquets Emergency Department	Number of Tourniquets Operating Theater
E. coli	12.7%	0/12	0/6	0/6
Klebsiella spp.	11.7%	5/12	2/6	3/6
Enterococcus spp.	10%	9/12	4/6	5/6
Staphylococcus aureus	9%	1/12	0/6	1/6
Clostridium difficile	8%	0/12	0/6	0/6
Pseudomonas aeruginosa	7.9%	9/12	4/6	5/6
Coagulase-negative staphylococci	5.8%	11/12	6/6	5/6
Proteus spp.	3.2%	0/12	0/6	0/6
Acinetobacter spp.	3.2%	10/12	4/6	6/6
Enterobacter spp.	3%	0/12	0/6	0/6

.

3.2.2. Bacteria Pathogenic to Humans Found on Reusable Tourniquets

On the surface of reusable tourniquets, our study found bacteria that are pathogenic to humans. On the surface of reusable tourniquets from the emergency department the following pathogenic bacteria were found: Staphylococcus aureus, Staphylococcus hominis, Enterococcus faecalis, Enterococcus hirae, Enterobacter quasihormaechei, Klebsiella pneumoniae, Klebsiella variicola, Pseudomonas aeruginosa, Pseudomonas fulva, Acinetobacter baumannii, Brevibacterium casei, Corynebacterium fournieri, Staphylococcus caprae, Acinetobacter lwoffii, Serratia liquefaciens, Pseudomonas luteola, Acinetobacter pittii, Acinetobacter radioresistens, Pseudomonas juntendi, Staphylococcus warneri, Staphylococcus saprophyticus, Roseomonas gilardii, Massilia timonae, and Moraxella osloensis. Reusable tourniquets from the operating theatre showed Enterobacter quasihormaechei, Acinetobacter baumannii, Acinetobacter pittii, Acinetobacter radioresistens, Pseudomonas aeruginosa, Pseudomonas juntendi, Staphylococcus hominis, Staphylococcus taiwanensis, Enterococcus faecalis, Enterobacter wuhouensis, Klebsiella pneumoniae, Klebsiella variicola, Serratia fonticola, Vibrio cholerae, Vibrio metschnikovii, Brevibacterium casei, and Enterococcus hirae (Figure 3).

Samples from the emergency department (SR series) generally showed high bacterial loads of individual species, which could be linked to the rapid, frequent handling of tourniquets in a high-turnover environment. Conversely, samples from the operating theater (SBO series) displayed a broader range of bacteria species, likely due to different handling and environmental exposure patterns, suggesting distinct cleaning or contamination processes in these departments (Figure 3).

4. Discussion

Key Findings and Departmental Variability

Studies have demonstrated the presence of microorganisms on hospital tourniquets that are among the most common causative agents of healthcare-associated infections (HAIs), including K. pneumoniae, K. aerogenes, E. faecalis, A. baumannii, A. lwoffii, S. aureus, and P. aeruginosa. According to the European Centre for Disease Prevention and Control (ECDC) reports from 2022 and 2023, in Poland, Klebsiella species are responsible for approximately 16% of all reported HAIs, making them the leading cause of hospital-acquired infections nationwide. Enterococcus spp. accounts for 11.6% of cases, S. aureus for 8.8%, P. aeruginosa for 7%, and Acinetobacter spp. for 5.7%. Klebsiella spp. are most associated with urinary tract infections. Enterococcus spp. is frequently linked to surgical site infections, while S. aureus is predominantly involved in bloodstream infections and surgical site infections. P. aeruginosa and Acinetobacter spp. are primarily associated with pneumonia and lower respiratory tract infections [10]. The clinical significance of Klebsiella pneumoniae is further underscored by the increasing prevalence of carbapenem-resistant strains (CRKP), which are associated with severe outcomes, limited treatment options, and high mortality rates, particularly among ICU patients [11].

In the presented study, most tourniquets were contaminated by pathogens of the genus Acinetobacter (including Acinetobacter baumannii and Acinetobacter lwoffii), which were detected in 10 out of 12 samples. Although Acinetobacter is not the leading cause of hospital-acquired infections, data from the ECDC report raise concern, indicating that 82.9% of Acinetobacter strains isolated from healthcare-associated infections exhibit resistance to carbapenems (CAR-R) [10]. A study made by Duszynska and Litwin in the intensive care unit (Wrocław, Poland) revealed that 32% of healthcare-associated infections were caused by A. baumannii. During the study period, a statistically significant increase in infections caused by this bacterium was observed, from 16.5% of HAIs in 2011 to 41% in 2016. Notably, 73.8% of these cases were associated with ventilator-associated pneumonia. The study also found that 98.36% of the isolates were multidrug-resistant (MDR), highlighting the serious clinical challenge posed by this species, particularly in units treating immunocompromised patients [12]. A study conducted in southern Poland between 2016 and 2019 in an intensive care unit involving 3028 patients revealed that the two main causative agents of infections were K. pneumoniae (16.3%) and A. baumannii (14.4%). A. baumannii was primarily responsible for pneumonia and lower respiratory tract infections (34.9%), while K. pneumoniae was most commonly associated with urinary tract infections (20.2%—the second most frequent cause after Escherichia coli) as well as bloodstream infections (16.7%) [13]. In a study conducted in Poland between 2014 and 2019 by Rafa and Kołpa in neurosurgery departments of two hospitals, A. baumannii was responsible for 8.8% of all healthcare-associated infections, with pneumonia being the predominant type, accounting for 21.7% of cases in Hospital A [14]. In a study by Wałaszek and Słowik, conducted between 2014 and 2018 on orthopedic patients (6261 patients, 111 cases of healthcare-associated infections), Acinetobacter baumannii was identified as the causative agent in 4.5% of the infections [15].

The next most frequently identified pathogens on the examined tourniquets were E. faecalis and P. aeruginosa, both detected on 9 out of 12 samples. In studies conducted by Rafa and Wałaszek (2021) [13], E. faecalis was responsible for 7.1% of HAIs, mostly urinary tract infections (14.5%), and bloodstream infections (8.3%). P. aeruginosa accounted for 6.9% of infections in the intensive care unit, including 11.5% of urinary tract infections [13]. Enterococcus spp. was also identified as a causative agent of surgical site infections in the study by Wałaszek and Słowik, accounting for 5.4% of cases, primarily following hip prosthesis implantation procedures [15]. P. aeruginosa was also identified in neurosurgical patients by Rafa and Kołpa in 2022 [14]. It accounted for 4% of all cases in that study and was primarily associated with surgical site infections [14].

In the presented study, Klebsiella was detected in 5 out of 12 tourniquets. In the research conducted by Rafa and Wałaszek (2021), Klebsiella pneumoniae was responsible for 16.3% of reported healthcare-associated infections (HAIs), primarily associated with urinary tract infections (UTIs) [13]. In the study by Wałaszek and Słowik, Klebsiella pneumoniae caused 10.8% of infections [15]. Additionally, in the 2022 study by Rafa and Kołpa, Klebsiella accounted for 12% of infections, mainly postoperative wound infections and UTIs [14].

Staphylococcus aureus was detected on only one tourniquet, and it was associated with skin and soft tissue infections in 42.8% of cases in studies conducted in the Polish ICU [12]. Among neurosurgical patients in a study conducted by Rafa and Kołpa (2022), Staphylococcus aureus was responsible for 11.5% of infections, primarily bloodstream infections and surgical site infections [14].

The presence of pathogens associated with healthcare-associated infections on tourniquets represents a significant challenge in the treatment of patients in Polish hospitals. Infections caused by these bacteria have been documented across various hospital departments and patient populations.

A total of 12 bacterial species potentially capable of antibiotic resistance were identified on the surface of reusable tourniquets. These pathogens pose a significant risk, as they are resistant to multiple classes of antibiotics, complicating the treatment of healthcare-associated infections. The identified potentially antibiotic-resistant species include Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterococcus faecalis, Stenotrophomonas maltophilia, Enterobacter quasihormaechei, Acinetobacter lwoffii, Klebsiella variicola, Shewanella algae, Serratia liquefaciens, Serratia fonticola, and Staphylococcus aureus. Additionally, 16 bacterial species capable of forming biofilms were identified on tourniquet surfaces. Biofilm formation is a significant survival strategy that enhances bacterial persistence and resistance to antimicrobial treatments, including disinfectants. Among them, Acinetobacter baumannii can exhibit resistance to carbapenems (e.g., imipenem, meropenem), cephalosporins (e.g., ceftriaxone), aminoglycosides (e.g., gentamicin), fluoroquinolones (e.g., ciprofloxacin), tetracyclines, and sometimes polymyxins, making it challenging to treat with conventional antibiotics. Additionally, A. baumani forms robust biofilms on surfaces, which increase its resistance to disinfectants and its ability to survive on medical devices. It is also known to survive on dry surfaces, such as medical tourniquets, for extended periods [16]. Pseudomonas aeruginosa is known for resistance to carbapenems (e.g., meropenem), aminoglycosides, cephalosporins, fluoroquinolones, and occasionally polymyxins, due to its intrinsic and acquired resistance mechanisms. It forms biofilms, complicating removal through standard cleaning protocols, and can survive in both moist and dry environments, making it a persistent contaminant on medical equipment [17]. Klebsiella pneumoniae produces extended-spectrum beta-lactamases (ESBLs) and carbapenemases, resulting in resistance to beta-lactam antibiotics, including cephalosporins and carbapenems. Additionally, resistance to aminoglycosides may occur via other mechanisms, such as the presence of the armA gene [18]. It also forms biofilms that enhance its survival on surfaces, adding to the difficulty of complete disinfection. While both Enterococcus faecalis and Enterococcus faecium can exhibit resistance to vancomycin (VRE), aminoglycosides, and certain beta-lactams, the vast majority of VRE isolates are E. faecium, which tends to be more antibiotic-resistant than E. faecalis [19]. It was also proven that it can withstand a range of pH and temperature conditions, increasing its resilience in healthcare settings [19]. Stenotrophomonas maltophilia displays intrinsic resistance to carbapenems, aminoglycosides, beta-lactams, and fluoroquinolones, which limits available treatment options [20]. It is known to form biofilms on various surfaces, complicating elimination. Environmental Adaptability: It is capable of surviving in diverse conditions, including low-oxygen environments, which aids in its persistence on reusable medical equipment. Staphylococcus aureus, especially methicillin-resistant strains (MRSA), exhibits resistance to nearly all beta-lactam antibiotics, including penicillins and cephalosporins, and frequently shows resistance to macrolides, lincosamides, aminoglycosides, fluoroquinolones, and tetracyclines (Tong et al., 2015) [21]. The presence of S. aureus, A. baumannii, P. aeruginosa, and E. faecalis on reusable tourniquets aligns with previous studies, which have also reported contamination of these devices with potential healthcare-associated pathogens [22,23]. Such bacteria are known to cause catheter-related bloodstream infections (CRBSI) and peripheral venous catheter-related bloodstream infections (PVCR-BSI), both of which are significant causes of morbidity in hospitalized patients. Lastly, CRBSI and PVCR-BSI are most associated with S. aureus (including MRSA), coagulase-negative Staphylococci, Enterococcus spp., Klebsiella spp., and P. aeruginosa. The contamination of reusable tourniquets with these microorganisms poses a potential risk for cross-contamination during vascular access procedures. Improper handling of tourniquets may transfer bacteria to the patient’s skin or catheter insertion site, increasing the likelihood of infection [24,25,26].

These findings underscore the challenges in maintaining sterile conditions with reusable medical devices such as tourniquets, especially given the presence of multidrug-resistant and biofilm-forming pathogens that are difficult to eliminate. These results highlight the necessity for rigorous disinfection protocols or consideration of single-use alternatives to minimize healthcare-associated infection risks.

Given these findings, our study reinforces the urgent need for single-use tourniquets or the development of validated disinfection protocols for reusable devices. Current manufacturer guidelines provide inconsistent recommendations regarding tourniquet decontamination, and, in some cases, fail to mention disinfection procedures at all. To reduce the risk of healthcare-associated infections (HAIs), it is critical to establish evidence-based infection control measures that include tourniquets as potential vectors of pathogen transmission.

Furthermore, the detection of multidrug-resistant species, including Stenotrophomonas and Acinetobacter, highlights the risk posed by persistent contamination of reusable medical equipment. Future research should evaluate the efficacy of various disinfection methods and assess the long-term microbial burden on reusable tourniquets in different clinical settings. While this study focused on reusable tourniquets as potential reservoirs for pathogens, it is important to acknowledge that other frequently handled medical devices similarly contribute to nosocomial transmission. Medication administration tools—including pillboxes, medication carts, and dispensing systems—have been documented as significant fomites carrying multidrug-resistant organisms. For instance, a study of medication preparation areas found that 33% of surfaces harbored pathogenic bacteria, with contamination patterns directly correlating with handling frequency. Like tourniquets, these devices experience high-touch contact without consistent disinfection protocols between uses. This parallel underscores a systemic challenge in healthcare settings: porous materials and complex surfaces on commonly reused equipment create persistent reservoirs for pathogens. Our findings on tourniquet contamination should, therefore, inform broader infection control strategies targeting all high-risk fomites, particularly those with textured surfaces that impede effective cleaning [27].

5. Limitations

The use of 16S rRNA sequencing provides taxonomic identification but does not differentiate between live and dead bacteria or determine antibiotic resistance profiles. The absence of negative controls (e.g., unused tourniquets) limits direct comparisons of disinfection efficacy. However, this aligns with this study’s focus on contamination dynamics in actively used clinical devices. Finally, variations in environmental conditions and handling practices across hospital departments may have influenced the level of microbial contamination. In this study, we did not monitor the way staff handled the tourniquets, which also included the possible use of disinfectants. Additionally, the relatively small sample size (n = 12) may limit the generalizability of our findings to other clinical settings. Future studies should include larger sample sizes and systematically document handling and disinfection protocols to better assess the factors influencing tourniquet contamination. Moreover, while switching to single-use tourniquets can reduce the risk of cross-contamination, this approach presents additional challenges. Disposable tourniquets are typically made from non-biodegradable plastics, contributing to environmental pollution and increased carbon footprint. On the other hand, insufficiently disinfected reusable tourniquets may facilitate the transmission of healthcare-associated infections (HAIs), which are associated with substantial treatment costs. The economic burden of managing a single HAI episode may far exceed the cost of implementing disposable alternatives. Therefore, future research should also consider the environmental impact of disposable tourniquets and perform comprehensive cost–benefit analyses to guide infection control strategies in different healthcare settings.

6. Conclusions

Metagenomic analysis of reusable tourniquets revealed a consistent presence of clinically relevant pathogens (HAI), including Klebsiella spp., Enterococcus spp., Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter spp.

Tourniquets sampled from operating theatres showed greater taxonomic diversity, suggesting variability in contamination dynamics driven by departmental usage patterns and disinfection practices. Cluster and diversity analyses further indicated that the highest genera diversity was exclusive to operating theatre tourniquets, although high contamination was not always associated with increased diversity.

The frequent detection of potentially biofilm-forming and antibiotic-resistant bacteria underscores the limitations of current decontamination protocols. The findings strongly support the implementation of standardized sterilization procedures or the adoption of single-use tourniquets to mitigate the risk of cross-contamination and reduce the transmission of multidrug-resistant pathogens in clinical settings. However, the feasibility of widespread single-use tourniquet adoption requires careful consideration of resource constraints, particularly in low-income settings where cost and supply chain limitations may pose significant barriers. Healthcare systems should balance infection control priorities with local economic realities when implementing these recommendations.

7. Implications for Practice

Reusable tourniquets represent an underestimated vector for the transmission of healthcare-associated pathogens, including multidrug-resistant and biofilm-forming bacteria. The findings underscore the necessity of integrating tourniquet management into infection prevention strategies. Transitioning to single-use devices or establishing standardized, evidence-based disinfection protocols is essential to limit microbial persistence and cross-contamination in high-risk clinical settings. Incorporating these practices may contribute to a measurable reduction in the incidence of hospital-acquired infections, particularly in departments with high patient turnover and invasive procedures.