INTRODUCTION

During their Normal Anatomy classes, medical students spend most of their time in dissecting rooms. During these classes, while remaining in closed compartments, students come into contact with multiple microorganisms dwelling on surfaces that may be both man-made (table tops, trays, anatomical models), and biological (anatomical specimens, including plastinated specimens). In every room, particular surfaces provide specific conditions for the growth of specific microorganisms. These conditions depend on two factors, namely: i) the surface coating material; and ii) the source of microfloral growth (Davis et al., 2012). The literature contains numerous reports on the growth of microfloral species on the surfaces of silicone implants widely used in plastic surgery (Eerenstein et al., 1999; Price et al., 2005; Rodger et al., 2010). However, reports of infections on silicone surfaces of S10-plastinated anatomical specimens are very rare (Prinz et al., 1999). Biodur^TM S10 plastination is a common plastination technique making use of polyalkyl siloxane (Biodur^TM S10 MSDS, Nov. 1998) (Holladay et al., 2001; de Jong and Henry, 2007). At numerous centers, the first steps in plastination are usually made using the silicone technique, as it is relatively simple and associated with low risk of procedural errors (Asadi, 1998; Zhong et al., 2000). The technique was first described by Gunther von Hagens in 1985 (von Hagens, 1985). The main concept consists of the specimen being gradually dehydrated, and subsequently saturated with silicone. At our lab, silicone plastination has been carried out since 2014. Despite their excellent appearance and lack of unpleasant odors, plastinated specimens retain the character of biological specimens, which may provide favorable conditions for potential microbial growth. Due to the rare reports on microfloral species being detected on specimens plastinated using the S10 technique, we decided to carry out a series of microbial assays to determine whether these plastinates are colonized by microfloral species and what species of microorganisms, if any, are present on their surface.

MATERIALS AND METHODS

A total of 20 standard swab collection kits was used in the study.

The study team consisted of 4 individuals. Two individuals were responsible for preparation of surfaces for swab collection, the third person was responsible for swab collection while the fourth individual recorded the code and name of the sample.

The study was carried out according to aseptic principles:

- individuals involved in direct contact with the study samples wore sterile, disposable protection garments including caps and surgical masks;

- before study-related tasks, study team members washed their hands according to a surgical hand washing technique and put on sterile gloves.

Microbial culture swabs were collected from different surfaces classified into 2 categories:

1) Surfaces of specimens subjected to cold-temperature S10 silicone plastination technique in 2014;

2) Surfaces of the practice room and anatomical models.

Swab culture samples were transferred to an analytical lab for culture and identification, using standard techniques. The results were analyzed and compared to data available in the literature.

RESULTS

Microbial assays revealed the presence of Micrococcus species on the surfaces of silicone and plastic anatomical models as well as on the surface of the skull used by the students as a study aid, and Staphylococcus epidermidis on the entrance door handle, as well as Staphylococcus epidermidis and Micrococcus species on the tops of the tables used by the students. No growth was detected for swabs collected from one of the plastic anatomical models, a plastic tray used for handling anatomical preparation, and student chairs. No fungal species were cultured from any of the samples. No microorganisms were cultured from swabs collected from the surfaces of S10-plastinated specimens. The study results are illustrated in Table 1.

DISCUSSION

It is well known that the risk of contamination is particularly high at early specimen preparation stages when the tissues are still fresh and the body fluids are liquid. Contamination may be avoided by means of appropriate prevention measures, careful cleansing and disinfection of working surfaces and instruments, as well as appropriate disposal of tissues remaining after the procedure (Smith and Holladay, 2001). Little information is available on the possibility of plastinated specimens being infected by microbial species after all plastination stages are completed and the specimens are released for educational purposes.

S10 plastination silicone: structure vs susceptibility to infection

Studies on the chemical activity used in S10 cold-temperature plastination technique suggest that the polyalkyl siloxane within Biodur S10 is likely to be a hydroxyl-terminated polydimethylsiloxane or very similar silicone monomer (Holladay et al., 2001). Polydimethylsiloxanes (PDMS) are macromolecular organosilicon polymers with -Si-O- bonding pattern repeated in the molecular chains. The polysiloxane structure is summarized using the formula -[R₂Si-O]- where R is a methyl (alkyl) group; thus, the compounds are classified as alkyl siloxanes (Mojsiewicz-Pieńkowska and Łukasiak, 2003). Silicones, particularly methylsilicone rubbers, are used for production of biomedical materials such as breast implants (Dunn et al., 1992), nasal implants (Deva et al., 1998), ophthalmological implants (Teferra, 2017) and vocal cord implants (Echternach et al., 2008).  Polydimethylsiloxanes were also reported as some of the polymer candidates for the low cost, mass production of bio-microelectromechanical system devices (Bio-MEMS) (Mata et al., 2005). Infections of silicone implant materials have been observed and reported in the literature (Wixtrom et al., 2012).

Microbial growth on plastinated specimen surfaces

In our study, no bacterial flora were detected on the surfaces of plastinated specimens. However, other authors have reported cases of fungal microflora growth observed on S10-plastinated specimens. Infections were manifested by the presence of numerous, rapidly growing, white, green, and black spots on the specimen surfaces. Similar features of fungal infections could even be observed on the wooden material of shelves used for specimen storage. Different fungal species were reported for different types of surfaces: Penicillium janthinellum was observed on kidney, cerebellum and brain stem specimens, Penicillium corylophilum was detected on abdominal sagittal section, stomach, and hand specimens; Aspergillus niger was detected on rotator cuff muscle specimens, Aspergillus flavus was detected on heart specimens, and Aspergillus fumigatus was detected on abdominal transverse section specimens. Notably, fungal flora was detected only within the superficial specimen layers. Careful analysis of deeper tissues revealed no presence of microbial cultures (Prinz et al., 1999).

 Microfloral cultures on plastinated specimens: causes and prevention

Rapid increase in humidity is reported as the main cause behind plastinated specimens becoming infected with fungal microflora (Prinz et al., 1999). No precise guidelines regarding the humidity and temperature conditions for the storage of such specimens are available in the literature. At our department, plastinated specimens are stored in facilities at constant temperature of 21° C and humidity of 45%; the conditions are subject to continuous monitoring. Since no microbial growth could be observed after 5 years of storage on any of the plastinated specimens, the storage conditions can be considered optimal. In a similar manner, air-conditioned, low-humidity storage conditions have been recommended by other authors as means to prevent fungal contamination of specimens (Prinz et al., 1999). If, however, such a contamination occurs, detailed guidelines describing the procedure of eradicating the infection from the surfaces of plastinated specimens are available in the literature (Prinz et al., 1999).

In our opinion, the instruction to “store in a cool and dry place” is not enough to effectively prevent contamination of plastinates. Temperature and humidity in the storage rooms should be subject to regular monitoring.

Characteristics of microbial species detected on tested surfaces

Micrococcus

The Micrococcus species detected on the anatomical models comprise the natural microflora of skin and mucosal membranes of humans and other mammals (Carr and Kloos, l977). Together with genera Staphylococcus and Planococcus, the Micrococcus species comprise the family of Micrococcaceae which belongs to a group of 17 Gram-positive cocci (Bergey and Holt, 1994). They are aerobic, non-spore-forming bacteria; some may present with cilia (Herbert et al., 1988). They grow in the temperature range of 25°-37° C. All strains are capable of growing in the presence of 5% NaCl while some are capable of growing even in the presence of 10-15% NaCl (Bergey and Holt, 1994).  Micrococcus species may pose a threat to human health only when one’s immunity is impaired. Cases of bacteremia caused by Micrococcus species have been reported as complications of immunodeficiency in some patients (von Eiff et al., 1996).

Staphylococcus

Staphylococcus epidermidis is one of the leading species found in the microbiota of skin and mucosal membranes in humans (Scharschmidt and Fischbach, 2013). They belong to the group of coagulase-negative staphylococci (Becker et al., 2014). S. epidermidis are Gram-positive (Bojar and Holland, 2002). Commensal S. epidermidis are permanent skin residents throughout  human life (Grice and Segre, 2011). In order to survive on the skin surface, S. epidermidis have developed a number of mechanisms to detect and defend against, or to bypass, the human immune system (Kocianova et al., 2005).

1. epidermidis actively supports the skin in its barrier function and complements the body’s innate immunity mechanisms. They produce a number of antimicrobial agents active against pathogenic bacteria (Janek et al., 2016). S. epidermidis may, however, also be responsible for hospital-acquired infections. The pathogenesis of these infections is related mainly to the formation of biofilms on the surfaces of biomaterials introduced into the patient's body (Hidron et al., 2008). S. epidermidis infections develop mainly in immunodeficient patients, patients undergoing immunosuppressive treatment, prematurely born infants, human immunodeficiency virus (HIV)-infected patients, patients undergoing long hospitalization, and critically ill patients (Ghassemi et al., 2015).

The human body is known to provide habitats for different bacterial strains in different body compartments (Costello et al., 2009). When in the dissecting room, students come in contact with many surfaces, potentially leaving behind bacteria typically dwelling on the skin of their hands. When microorganisms are transferred from a human body onto a man-made surface, their presence on that surface is strongly dependent on the contact with humans (Davis et al., 2012). However, atypical strains may also be transferred from man-made surfaces into human systems (Ferier et al., 2010).

Considering the characteristics of the bacteria of Micrococcus species, one may conclude that their presence on the surfaces of training room equipment, bone specimens, and anatomical models is closely correlated with these surfaces coming into contact with human skin. Since the swab collections were taken immediately after completion of classes, and before the rooms were cleaned, the counts of bacteria present on the surfaces were high enough to be detected by the test method.

In the case of S. epidermidis, the structure of the bacterial cell wall, consisting of multiple layers of peptidoglycan (murein) ensures high cell stability so that the bacteria are resistant against desiccation, osmotic shock, and mechanical factors (Bojar and Holland, 2002). Thus, they are capable of surviving for significant periods after being transferred from hands onto external surfaces, allowing us to detect their presence on the door handle and the table top. Other authors have reported on a similar mechanism responsible for transmission of various microfloral species to and from man-made surfaces in closed facilities. For example, Meadow et al. (2014) reported that numerous Lactobacillus strains typical for intestines and vagina were detected on chairs in the lecture rooms of the University of Oregon, USA. Streptococcus species typical for human skin and oral cavity, as well as some Streptococci observed in humans with certain pathological conditions were detected on lecture room desktops. Bacteria typical for human skin were also detected on the room floor, in addition to other species which are typically present in soil rather than human bodies. Sphingomonas and Alicyclobacillus species were detected on lecture room walls (Meadow et al., 2014).

Other authors who studied the microflora present on the desktops at Connecticut schools, grades 7 to 12, observed an absolute prevalence of bacteria and fungi from the genera Streptococcus (≥37%) and Candida (≥ 38%), respectively (Kwan et al., 2018).

Microbial analysis of air within the Louvre Museum in Paris, revealed the presence of 10³/10⁴ Escherichia coli/Aspergillus fumigatus genome equivalents per m³ (Gaüzere et al., 2014).

CONCLUSION

As shown by the study results, no bacterial or fungal growth was observed on the surfaces of specimens plastinated using the S10 cold-temperature technique. Avoidance of microbial growth on the surfaces of plastinated specimens requires: i) strict compliance with the plastination protocol as described by von Hagens; and ii) continuous monitoring of temperature and humidity of the plastinate storage rooms. The analysis of microflora within the dissecting practice rooms revealed the presence of benign Micrococcus species and Staphylococcus epidermidis strains.