ReviewBioassays: The best alternative for conventional methods in detection of Legionella pneumophila
Introduction
Given the fact that microbial diseases are one of the main causes of death in developing countries and the world, their precise identification can be very important. From a very long time ago, various methods have been developed in relation to the method of identifying bacterial pathogens has been used. Most of the methods used to identify bacteria to date are microscopic methods, culture methods and serology tests [1]. Given that the aforementioned methods had many problems and limitations, with the advancement of molecular techniques, the field was expanded rapidly, following the invention of the Polymerase Chain Reaction by Kary Mullis [2]. Various forms of PCR were invented that could largely eliminate the limits of routine tests, such as Rea-Time PCR, but these tests also had limitations, including the high cost and complexity of doing it. So that these tests were needed for experienced and professional people [3]. Over time, scientists were looking for more modern and better techniques to replace routine methods until biosensors were invented. Biosensors are analytical tools that can detect biological compounds such as proteins, enzymes, cells, and DNA. Biosensors have many advantages, among which the most important ones are the low cost, and high sensitivity and the specificity of using them [4]. With reference to the above, the use of biosensors, especially in relation to the identification of fastidious bacteria, is increasing rapidly. Because hard-growing bacteria grow slowly and have complex nutritional needs, the use of biosensors can be the best alternative to old methods [5].
Section snippets
Fastidious bacteria
A fastidious organism is any organism that has a complex nutritional requirement. In other words, a fastidious organism will only grow when specific nutrients are included in its diet, the more restrictive term fastidious microorganism is often used in the field of microbiology to describe microorganisms that will grow only if special nutrients are present in their culture medium [6]. In this overview, we try to summarize all the conventional methods used to detect Legionella pneumophila and
Clinical manifestations
Legionnaires' disease is a typical pneumonia that might clinically resemble pneumococcal or other bacterial pneumonias [14]. Initial findings seemed to indicate a distinct clinical syndrome, [19] yet some prospective studies have shown that Legionnaires' disease and pneumococcal pneumonia might have some similar clinical and radiographic findings. [15,[20], [21], [22]] Symptoms range from mild disease to severe pneumonia requiring hospital admission [15]. The identification of Legionella at the
Culture
Legionella spp are Gram-negative bacteria with strict growth requirements. They grow on various solid-selective and nonselective media. Legionella colonies are usually detectable after days 3–5 of incubation. Young colonies are 0.5–1 mm in diameter, self-contained, flat, and smooth, with a typical ground-glass appearance and an iridescent hue. When a colony is suspected to be Legionella, it should be Gram stained to check for small to filamentous Gram-negative rods and plated onto two different
Trend in conventional techniques
The traditional methods for pathogen detection include microscopy, culture and serology. Microscopy is simple, easy to use and a versatile technique. Culture is the gold standard for diagnosis of many microorganisms, e.g., Mycobacterium tuberculosis and serology forms the strength of diagnosis in some of the diseases, e.g., syphilis. The traditional methods of detection are inexpensive but prolonged methods. Microscopy has limited sensitivity in many settings and the interpretation may be
Why biosensors?
Biosensors are analytical devices that convert a biological response into an electrical signal. Typically biosensors must be highly specific, independent of physical parameters such as pH and temperature and should be reusable. The term “biosensor” was coined by Cammann, 1 and its definition was introduced by IUPAC [47]. Biosensors consist of two main components: a bioreceptor or biorecognition element, which recognizes the target analyte and a transducer, for converting the recognition event
Immobilization
Immobilization of enzymes is an important feature in designing the biorecognition part of enzyme based biosensors. Enzyme immobilization appears as a key factor to develop efficient biosensors with appropriate performances such as good operational and storage stability, high sensitivity, high selectivity, short response time and high reproducibility [60].
Entrapment enzymes
Entrapment, which consists of the physical retention of the enzyme in the inner cavities of a porous matrix deposited onto the electrode surface, has been also used as immobilization technique in microbial biosensors [61]. Immobilized in three-dimensional matrices such as an electropolymerized film, an amphiphilic network composed of polydimethylsiloxane (PDMS), a photopolymer, a silica gel, a polysaccharide or a carbon paste can be performed. This immobilization is easy to perform. Enzyme,
Electrochemical biosensors
In electrochemical biosensors, bioreceptor can be a nucleic acid (DNA, PNA, RNA and aptamer etc.), enzyme, antibody, cell and microorganism [62,63]. The immobilization of a nucleic acid molecule onto a matrix requires complete retention of their biological recognition properties in the active state. The desired probes are attached at one terminus onto the surface [45].This allows for the highest degree of thermodynamic freedom that significantly speeds up the hybridization rate. Binding should
Optical biosensors
Among various sensor systems, optical biosensors allow easy-to-use, rapid, portable, multiplexed, and cost-effective diagnosis. Here, we review current trends and advances in pathogen-diagnostic optical biosensors. The technological and methodological approaches underlying diverse optical-sensing platforms and methods for detecting pathogenic microorganisms are reviewed, together with the strengths and drawbacks of each technique. Finally, challenges in developing efficient optical biosensor
Conclusion
Undoubtedly, the rapid, specific and precise detection of legionella pneumophila is one of the most important challenges in medical bacteriology. To date, various tests have been developed to identify Legionella pneumophila, but as mentioned in the text, these tests have many limitations. The most difficult problem is the incubation time of more than a few days. A very interesting point is that, despite the length of a long cultivation, cultivation is still considered as golden standard till
Acknowledgements
We gratefully acknowledge the partial financial support by Tabriz University of Medical Sciences, Tabriz, Iran. This study was supported by Tabriz University of Medical Science as PhD thesis of A. Mobed.
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