The Role of New Technologies in Medical Microbiological Research and Diagnosis

Microbial culture is exemplified by the Petri dish, a tool that (one century after its invention) still remains one of the “gold standards” for microbiological analysis. However, current trends towards automation, massively paralleled assays, and miniaturization (as well as the observation that we still cannot culture most microorganisms), suggest that new ideas in microbial culture are required. In the Petri dish, nutrient containing agar is typically used as the matrix on which microorganisms are cultured. However, new materials such as nanofibres and nanoporous materials may be better choices as supporting matrixes. Further, emerging techniques in microengineering and the fabrication of low cost materials are helping to create new porous disposables that are of sufficiently low cost that they may be used in the routine microbiology laboratory. These disposables are in turn allowing the development of novel miniature culture methods to take place, methods such as microchemostats, cages for growing microorganisms, and “habitats on a chip”. One particularly useful porous ceramic is Porous Aluminium Oxide (PAO), which can be utilized to generate highly subdivided culture chips that possess up to one million separate, miniaturized, growth areas. Indeed, this material has applications in microbiological diagnostics, microbiological research and industrial microbiology. In this chapter, the applications, advantages, and limitations of porous matrixes and accompanying culture chips will be examined. It is expected that these advances will yield significant improvements in microbial culture when compared to the classical Petri dish.

Biosensors utilizing peptide and protein probes offer the potential to provide rapid, highly sensitive, specific, and economical infectious disease diagnostics. In this respect, the following chapter describes recent advances in peptide-based biosensor technologies, including mass perturbance, electrical perturbance, and optical methods. Further, the applicability of these biosensors in the diagnosis of microbial infections in both laboratory and field settings is described. The chapter also discusses current, competing technologies to peptide-based biosensors, as well as point-of-care testing (with an emphasis on the comparison of influenza diagnostics). In addition, the chapter illustrates; 1) the positive impact that rapid diagnosis from biosensors could have on pandemic disease surveillance, 2) describes the different types of peptide-based probes (including antibodies and oligopeptides), and 3) presents an internationally approved clinical method for determining limits of detection for biosensors. The chapter concludes with predictions on the limitations of peptide-based biosensors, and the promising technological advances that will allow full potential of biosensor applications to be achieved within the field of medical microbiological diagnosis.

In medical microbiology, the identification of microorganisms in clinical specimens is a key step for successful therapy. In the last few years, new technologies have emerged for routine identification, among which matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), a technology that appears very promising as it is currently becoming established in microbiological laboratories worldwide. MALDI-TOF MS allows the identification of microorganisms - bacteria as well as fungi - by so called intact-cell mass spectrometry, and the comparison of a sample’s mass spectrum to reference mass spectra in a database. The key factors to the success of this technology are: i) the fact that a uniform sample preparation procedure is utilized for many different types of microorganisms, ii) the short time to a result, and 3) the comparatively low cost per analysis. Additionally, mass spectrometry based identification can be readily expanded to different microbiological fields, including food, industrial and veterinary microbiology.

In this chapter, the basic principles of MALDI-TOF MS are briefly described, followed by an introduction to intact-cell mass spectrometry of microorganisms and mass based identification. Further, limits of the technology are reviewed in the light of expected future developments. Finally, possible consequences of the broad introduction of MALDI-TOF MS based on microbial identification systems for practical and theoretical issues of medical microbiology are discussed.

During the last few years, there have been enormous strides in the ability of microbiologists to analyse complete microbial genomes, the amount of information obtained from these sequences being quite astonishing, not least with respect to deciphering the role of microbial interactions within an environmental context. However, the measurement of metabolic changes could offer even deeper insights into biological mechanisms (as compared to simple DNA sequencing alone), by actually defining and interpreting the responses of microbial systems to environmental and/or genetic modifications. In this respect, (meta-) metabolomics is a recent discipline that attempts to study metabolites and their concentrations, interactions and dynamics at a global level within complex samples. It constitutes one of the tools of the post-genomic era, all of which are concerned with the study of the different functional levels of biological systems, i.e. the (meta-) transcriptome, the (meta-) proteome and the (meta-) metabolome.

The analysis of small metabolites is important because these molecules participate in the metabolic reactions necessary for the normal functioning, maintenance and growth of a cell. In this context, the primary goal of this chapter is to provide a general overview of the techniques, problems and prospects of microbial (meta-) metabolomics with respect to medical microbiological research and diagnosis. A key objective is to show how the fingerprinting analysis of intra- and extracellular metabolites can be used as a reflection of metabolic microbial activities that impact on microbial cell physiology, microbemicrobe interactions, microbe-host interactions, and on the analysis of whole microbial communities.

This chapter describes the use of electronic noses in the field of clinical microbiology. These devices can be used to detect volatile organic compounds directly from clinical materials and can also be applied to monitor the production of volatiles during the process of microbiological culture. Various electronic nose appliances have been developed, but most need rigorous normalization and standardization each and every time that the sensors are renewed. In this chapter, the authors focus on a recently developed, patented micro-technology that does not require regular normalization. Using metal oxide sensors and electronic nose technology allows the dynamic analysis of volatile molecule production during bacterial fermentation to be performed, which will facilitate the real-time detection and identification of live bacteria present within clinical specimens.

The social and financial impact of infectious diseases are unfortunately still very relevant today, and the development of new diagnostic tests with improved detection characteristics continues to be an important research priority. Currently, there is a demand for accurate and specific diagnostic tests that can be performed at the point-of-care without the need for dedicated equipment or highly trained personnel. In this respect, advances in the field of nanotechnology are already being utilized by several research groups around the world, and with very encouraging results. In fact, nanoparticles can now be conjugated to oligonucleotides, antibodies, and peptides, facilitating multi-labeling and hence multi-target detection, which (in the context of diagnostic applications) allows the genetic or immunogenic “footprint” of a microbial pathogen to be determined. Further, there exists a range of metal or polymer nanoparticle materials to choose from, and their choice is dependant on the properties of the material required. Here, we provide a concise description of the applications of 2 such materials, colloidal gold and quantum dots, which have already been utilized in several different applications that target pathogen detection. Emphasis is placed on the principles behind these novel applications, and the way in which the properties of nanoparticles are being used in the development of future microbiological applications.

"LAMP" or Loop-mediated Isothermal Amplification is a simple, rapid, specific and costeffective nucleic acid amplification method that is characterized by the use of 6 different primers (3 different primer pairs) that are specifically designed to recognize 8 distinct regions on the target gene. LAMP amplification takes place at a constant temperature via a strand displacement reaction. Amplification and detection of genes can be completed in a single step, by incubating a mixture of samples, primers, a DNA polymerase (possessing DNA strand displacement activity) and accompanying substrates, at a constant temperature of 60 - 65°C. The high amplification efficiency of the LAMP process means that the presence of amplified products can be monitored by the naked eye either through visual turbidity or visual fluorescence. LAMP technology is emerging as a promising nucleic acid amplification tool that offers rapid, accurate, and cost-effective diagnosis of infectious diseases. The technology has already been developed into commercially available detection kits for a variety of pathogens including bacteria and viruses. Further, the combination of LAMP and novel microfluidic technologies may facilitate the realization of gene-based microbiological point-of-care testing systems, which may be available in both developed and developing countries in the near future.

The rapid and reliable diagnosis of bacterial infection is crucial for three reasons: 1) delays in the identification of bacteraemia during the first 6 hours after hospital admission are associated with higher mortality rates; 2) delays in pathogen identification are associated with an increased utilization of hospital resources (e.g. for ICU treatment); and 3) the treatment of viral illnesses and non-infective causes of inflammation with antibiotics (because of inaccurate diagnoses) contributes to the development of antibiotic resistance, toxicity, and allergic reactions, all leading to increased medical costs. Therefore, the development of rapid and reliable diagnostic tests is an essential prerequisite for more accurate diagnosis and the effective use of antibiotics within hospitals and general practitioners’ surgeries. One of the most promising technologies likely to impact on rapid and reliable diagnostic testing involves the flow cytometric quantitative analysis of new specific and sensitive cell surface markers (receptors) of bacterial infection on phagocytes. As an example of the usefulness of this method, this chapter presents an outline of how flow cytometric quantitative receptor analysis can be utilised for the clinical differential diagnosis of hospitalized febrile patients suspected of having an illness of microbiological origin.

Bead-based flow cytometry (xMAP® and xTAG® Technology, Luminex Corporation) is a recently developed technology that allows for simultaneous quantification of multiple antibodies, antigens or oligonucleotides in a single sample. The newest generation of analyzers allow multiplexing of up to 500 unique tests within a single sample, which renders this technique much less timeconsuming than conventional testing methodsologies. Numerous applications have already been developed for this technology in both medical microbiological research and diagnosis, with the number of reported applications still growing rapidly. Bead-based flow-cytometry has already firmly established itself as a supplemental and/or alternative technology to the more traditional routine diagnostic and research microbiology test platforms.