The spectacle microbiota and its hygienic relevance

Abstract

The aim of this thesis was to provide a solid basis for a deeper understanding of the hygienic relevance of spectacles and related ophthalmologically important surfaces. To do so, comprehensive cultivation-dependent and -independent studies on the microbial community composition of worn spectacles, microscope oculars and slit lamps were conducted. Using spectacles from university staff (n = 11) and inhabitants of a nursing home (n = 10) for elderly people, it was shown that all spectacles were contaminated with bacteria, with nosepads and earclips showing the highest density. In particular sites undergoing direct skin contact showed high germ counts and were dominated by staphylococci. The microbial load of the university spectacles was similar to the nursing home ones. However, the latter showed a higher diversity (10 genera, compared to 2 genera at the university environment), presumably due to skin factors changing with age. Using a collection of gram-negative and gram-positive test bacteria (including Staphylococcus epidermidis as the dominant isolate of the study) it was shown that wet cleaning wipes reduced the microbial load on spectacle lenses by about 2 log scales, while dry cleaning was less effective. These results were corroborated in a cleaning experiment with naturally contaminated, worn spectacles. Here, the average bacterial load was significantly (94%) lower compared to the uncleaned university spectacles investigated before. To account for the well-known bias of cultivation, a molecular analysis pipeline based on NGS of 16S rRNA gene amplicons was established. Using this protocol on 30 worn spectacles at three different sampling sites, a remarkable bacterial diversity of 665 bacterial genera was unravelled. In addition, significant differences in community composition between the sampling sites were detected, with the highest bacterial diversity on the lenses. On all spectacle sites, only a few taxa dominated the bacteriota: Staphylococcus, Propionibacterium (Cutibacterium), Corynebacterium, Lawsonella and Streptococcus, in decreasing order. The taxa identified as dominant on spectacle surfaces can be used a test organisms with a high relevance for practice in the development of novel cleaning and coating strategies for spectacles. Bacterial transmission between inanimate surfaces and human beings is more likely if surfaces are touched by different persons. Therefore, 10 microscope oculars from a university laboratory and 46 slit lamps from two eye clinics were included in this thesis as reference surfaces to the previously analysed spectacle surfaces. In the case of the microscope oculars, both cultivation-based and molecular analyses of the microscope microbiota were performed. All oculars were found to be contaminated with bacteria, with a maximum load of 1.7 x 103 CFU cm-². Although selective media for fungal detection were also used, no fungi could be isolated. 64 bacterial genera were detected with cultivation, compared to 227 when based on the sequencing results. The dominant bacterial genera were Cutibacterium (C. acnes), Staphylococcus (S. capitis), Brevibacterium, Paracoccus, Pseudomonas and Acinetobacter. Wet cleaning of microscope oculars with isopropyl alcohol reduced the microbial load by up to two log scales. All investigated slit lamp samples also showed contamination with bacteria originating mostly from human skin, mucosa and probably from eyes. Across all samples, 268 genera were identified, predominantly cutibacteria, staphylococci and corynebacteria. Statistical analysis suggested an exchange of bacteria between the patients’ and examiners’ sites, presumably including a potential pathogen transfer. As staphylococci were among the most abundant taxa on all analyzed optical devices including slit lamps, MRSA (Methicillin-resistant Staphylococcus aureus) was searched for by means of qPCR. However, no MRSA signals above the detection limit were detected. As MRSA is highly prevalent in hospital environments, this is a favorable result from a hygienic point of view. In summary, the studies conducted in the course of this thesis clearly showed that spectacles, microscope oculars and slit lamp surfaces are colonized by a diverse bacterial community, mostly originating from human skin, epithelia and the environment. Many of the detected genera are known to comprise potential pathogens. Successful cultivation of bacteria from the investigated surfaces clearly indicated the presence of viable cells, i.e. cells that can potentially cause infections. Transmission of potential pathogens is more likely to occur if spectacles, microscopes and slit lamps are regularly touched or used by different persons (e.g. ophthalmologists, healthcare workers, opticians, etc.). Consequently, these devices must be regarded as fomites. Regular cleaning significantly reduces the bacterial load and is therefore highly recommended to prevent eye and skin infections. Future research will address more deeply whether spectacles might serve as a reservoir for pathogens in recurring eye infections or function as vehicles to spread antibiotic resistance genes in healthcare environments. To do so, stronger function-oriented analysis methods will be established, such as shotgun metagenomic sequencing. This technique provides a broader and more accurate resolution of microbial diversity and can include non-bacterial microorganisms, in particular viruses. Establishing a metagenomic approach will also provide for more comprehensive detection of bacterial resistance genes and virulence factors on ophthalmologically relevant surfaces.