# Metagenomic Analysis of Bacterial Diversity on Reusable Tourniquets in Hospital Environments
## 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 healthcarecosts. 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]. [PERSON] 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 165 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.