Differenzierung von Methicillin-sensiblem und Methicillin-resistentem Staphylococcus aureus anhand flüchtiger organischer Verbindungen mittels Multikapillarsäulen-Ionenmobilitätsspektrometrie und elektronischer Nase „Cyranose 320“

Background: Staphylococcus aureus can be found as a commensal in 20 – 40 % of the population and is one of the most frequent causes of nosocomial infections. Multi- resistant strains have emerged early and are classified into methicillin-sensitive strains (MSSA) and methicillin-resistant strains (MRSA). Resistance to reserve antibiotics is also increasingly developing. S. aureus is responsible for increased morbidity and mortality as well as prolonged hospitalisation periods including isolation measures and associated costs. Microbiological diagnosis using bacterial culture remains the gold standard for germ detection and susceptibility testing albeit being time-consuming and expensive. PCR-based diagnostics ensure quicker results but are uneconomical. Early bacterial detection ensured by screening tests with subsequent isolation or decolonisation measures can lead to a reduction in transmission rates and consequently in morbidity, mortality, and associated costs. Thus, there is a need for cost- and time-effective methods for the detection of MSSA and MRSA with high diagnostic value. Aim: The aim of this study was to determine whether MRSA and MSSA can be differentiated from the culture medium Brain Heart Infusion Broth (BHI) and from each other based on the analysis of volatile organic compounds (VOCs) – which are expressed by organisms as a result of metabolic processes – by using multicapillary column ion mobility spectrometry (MCC-IMS) as well as the electronic nose Cyranose 320. Methods: 20 samples each with MRSA and MSSA obtained from routinely collected screening samples were transferred to the liquid culture medium BHI and diluted to a concentration of 108 CFU/ml. From each sample, 500 μl were collected for measurements. Using the MCC-IMS, 20 MRSA and MSSA samples each and analogously 27 samples of the non-incubated liquid culture medium BHI were analysed by headspace measurements. Each sample measurement was preceded by a blank measurement of the laboratory bottle for reference and to eliminate interfering factors. The peaks resulting from the measurements were visualised and statistically analysed and subsequently allowed differentiation between the groups by assigning specific peaks to the respective samples. By comparison with an existing database, the peaks could be assigned to corresponding organic substances. The Cyranose 320 was used to analyse the headspace of 20 MRSA and MSSA samples each and 20 samples of non-incubated BHI. Each sample was measured 5 times in succession to ensure machine learning. This was followed by linear discriminant analysis and calculation of Mahalanobis distance to differentiate between groups. Leave-one-out cross-validation was performed to determine the cross-validation value. The groups could be distinguished from each other by pattern recognition. Results: Using MCC-IMS, 19 highly significant peaks (p < 0.001) were detected in comparison between the groups MRSA and BHI, showing discrimination with sensitivity and specificity of > 90 % up to 99.9 % each. MSSA can be differentiated from culture medium BHI using 20 highly significant peaks with sensitivity of 92.6 % up to 96.3 % and specificity of 90 % up to 99.9 %. MRSA and MSSA can be differentiated from each other based on 11 highly significant peaks with sensitivity and specificity of 90 % up to 99.9 % each. Two peaks are sufficient to ensure separation of all groups using a two-step decision tree. Cyranose 320 was able to differentiate MRSA from BHI with sensitivity of 96 % and specificity of 94 %. Differentiation of MSSA and BHI was achieved with sensitivity of 81 % and specificity of 75 %. MRSA and MSSA could be differentiated from each other with sensitivity of 100 % and specificity of 91 %. Conclusion: Differentiation between MRSA, MSSA and the liquid culture medium BHI is possible with high sensitivity and specificity by using MCC-IMS as well as Cyranose 320. Further clinical prospective studies will be needed to verify the results and thus find a possible, time- and cost-saving alternative to conventional diagnostics.