Device-independent, real-time identification of bacterial pathogens with a metal oxide-based olfactory sensor

M. Bruins 0 A. Bos 0 P. L. C. Petit 0 K. Eadie 0 A. Rog 0 R. Bos 0 G. H. van Ramshorst 0 A. van Belkum 0 0 G. H. van Ramshorst Erasmus MC, Department of Surgery, University Medical Centre , 's Gravendijkwal 230, 3015 CE Rotterdam, The Netherlands A novel olfactory method for bacterial species identification using an electronic nose device called the MonoNose was developed. Differential speciation of microorganisms present in primary cultures of clinical samples could be performed by real-time identification of volatile organic compounds (VOCs) produced during microbial replication. Kinetic measurements show that the dynamic changes in headspace gas composition are orders of magnitude larger than the static differences at the end of fermentation. Eleven different, clinically relevant bacterial species were included in this study. For each of the species, two to eight different strains were used to take intra-species biodiversity into account. A total of 52 different strains were measured in an incubator at 37C. The results show that the diagnostic specificities varied from 100% for Clostridium difficile to 67% for Enterobacter cloacae with an overall average of 87%. Pathogen identification with a MonoNose can be achieved within 6-8 h of inoculation of the culture broths. The diagnostic specificity can be improved by broth modification to improve the VOC production of the pathogens involved. - Bacterial identification in the medical microbiology laboratory is still firmly based on old-fashioned biochemical reactions. From the ages of Pasteur and Koch onwards, medical microbiologists have relied on classical culture-based methods in order to confirm bacterial infections and identify the pathogens involved. Most infectious agents are still primarily detected using classical methods that involve liquid or solid semisynthetic growth media [1]. Serological methods for the detection of such pathogens are often lacking in sensitivity and specificity and data generated by the novel generation of expensive molecular tests need to be interpreted with equal caution [2]. Even so, given the clinical impact of bacterial infections in general, methods that reliably speed up the diagnostic process and limit costs are still eagerly awaited. Culture has the major advantage that living organisms are obtained for downstream characterisation, including antimicrobial susceptibility testing and epidemiological typing of the organism. This is an argument in favour of new methods that combine classical culture with procedures that enhance the speed of bacterial species identification. Currently, microbiology laboratories employ classical, growthbased, fermentative species-identification schemes that can be performed either manually or in an automated fashion using instruments such as the bioMrieux VITEK or the Beckon-Dickinson Phoenix. Molecular nucleic acid amplification tests and biophysical procedures, including mass spectrometry, could encompass novel, more real-time methods of bacterial species identification, although this would require additional handling of positive cultures [3]. In conclusion, real-time species identification during primary cultivation of clinical samples would be of added value for controlling costs and optimising patient care in clinical institutions. Odour-based assays could potentially fill this diagnostic niche. General interest in the classification of microorganisms on the basis of odour production has recently increased because of the introduction of so-called electronic nose devices. Differential speciation of micro-organisms present in primary cultures of clinical samples could be performed by real-time identification of volatile organic compounds (VOCs) produced during microbial replication. Classical culture has been combined with such olfactory measurements in the past [4]. It needs to be emphasised that these experiments always involved cultivation endpoint measurements. However, by continuous sampling of a cultures headspace, the kinetics of the synthesis of VOCs can be monitored in real time. The complex bacteriological VOC signals can be defined by a gas measurement technique involving metal-oxide (MO) sensors as used in the present study. We have developed a broadly applicable, inexpensive and highly responsive sensor system, called the MonoNose, which uses real-time VOC pattern recognition and the matching of the measured dynamic olfactory pattern with previously identified reference patterns. This system is analogous to the kinetics of human odour recognition [5]. The MonoNose technology facilitates the timely identification of bacterial species and, thereby, the clinical differentiation of medically relevant pathogens. In this article, we show that several bacterial species can be distinguished on the basis of MonoNose-mediated identification of growth stage-specific VOC production. Materials and methods Electronic nose (MonoNose) A set of 30 custom-designed electronic nose devices were manufactured by C-it (Zutphen, The Netherlands). These MonoNoses employ a single metal oxide-type semiconductor gas sensor. A scheme of a MonoNose and the 30 hand-manufactured devices in operation in an incubator are depicted in Fig. 1. The sensor system itself and the accompanying real-time pattern recognition algorithms are described in more detail elsewhere (manuscript submitted). For each experiment a MonoNose device was fitted with a disposable sterile syringe needle and a sterile HEPA filter with 45-m pore size to prevent crosscontamination. Humidity influences the semiconductor response values [6]. All devices were operated in an incubator at a constant ambient temperature of 37C in order to keep the relative humidity in the headspace constant during all experiments. Eleven different, clinically relevant bacterial species were included in this study. For each of the species two to eight individual, genotypically distinct strains were used to take intra-species biodiversity into account. The 52 strains used are listed in Table 1. The strains were obtained from both commercial sources (American Type Culture Collection; ATCC) and Erasmus MC reference collections. For all strains, the nature of the species was reconfirmed by Fig. 1 a Schematic representation of a MonoNose device for measuring bacterial volatile organic compound (VOC) production in the broth headspace over prolonged periods of time. The sensor is a commercially available metal oxide-based micro-device. b Experimental set-up with 30 MonoNose devices in operation in an incubator. The sensors are serially connected and all data are assembled on a simple portable computer. The vials in the photo are standard BDBACTECPlus-Anaerobic/F disposable bottles Table 1 Overview of the bacterial species and strains used ATCC13047, B33386, B33449 ATCC13311, ATCC14028, ATCC49416 857S, 863S, 865S, 920S, ATCC25923, ATCC29213, ATCC29737, ATCC33862 VITEK analysis before inclusion in the present study. Culture b (...truncated)