Microbiological Diagnostics of Bacterial Pathogens Aims Tasks and Current Limitations

The microbiological diagnostics of bacteria has historically focused on the detection and identification of bacteria by providing a differentiation at the species level. In the case of medical microbiology a further emphasis is on determination of the antibiotic resistance pattern revealed by the isolate under investigation.

Three important fields of microbiological bacterial diagnostics are: (1) medical and food microbiology, (2) environmental microbiology, and (3) the detection of biological warfare agents. These fields differ considerably with regard to the diversity of bacteria to be detected and the time scale for diagnosis [9]. Methods for the detection and identification of microorganisms applied in medical microbiology and food technology are directed towards the reliable detection and/or identification at the species/subspecies/strain level of one or a few microbes out of many that may be present in the diagnostic sample [10, 11]. The further major aspect of clinical microbiology is characterization of the antibiotic resistance pattern revealed by an isolated bacterium. Microbial diagnostic methods in environmental and industrial microbiology, on the other hand, are applied to obtain a picture of the structure ofthe entire microbial community under analysis [12, 13]. Requirements for this class of diagnostic methods are the parallel detection of many microbes at the level of the species, genus, or even higher taxon, and the potential for some level of quantification. The detection of bioweapons is characterized by the need for fast and unambiguous detection of a circumscriptive panel of agents and rapid genetic correlation of their virulence traits [14, 15]. A detailed analysis of the molecular fine structure of the agent in question may give further clues to its source and origin.

In medical microbiology the workflow and interpretation of diagnostic results differ depending on the source of the sample. In samples drawn from primarily sterile body sites, e.g., blood or cerebrospinal fluid, the presence of any bacteria is meaningful so long as contamination during harvesting of the sample has been excluded. Fast and reliable differentiation of the bacteria together with a rapid diagnostic process is crucial in these often life-threatening infections. A different diagnostic scenario is faced when the samples, e.g., sputum or stool samples, are obtained from primarily nonsterile body sites of the skin or mucosa. Here, the diagnostic procedure is directed towards identification of a bacterial pathogen among the varied multitude of nonpathogenic bacteria comprising the normal flora. This task is hampered by the facts that (a) "nonpathogenic" bacteria of the normal flora may cause disease in certain circumstances, e.g., in immunocompro-mised patients, (b) bacterial pathogens in diagnostic samples are often a minor subset compared to the bacteria of the concomitant normal flora, and (c) facultative pathogenic bacteria, e.g., E. coli, may cause disease depending on their geno-mic virulence gene repertoire. These virulence traits, however, often remain undetected in a laboratory routine that uses primarily biochemical and serological tests for the identification and characterization of the cultured bacteria. A further drawback of the traditional microbiological diagnostic procedure relates to the detection of fastidious bacteria and of those microorganisms which despite all efforts cannot be cultured. The isolation of others requires laborious procedures that cannot be routinely performed in small laboratories, and, more importantly, the recovery of a large number of organisms commonly involved in human and veterinary infections cannot be achieved in a short time. Consequently, information is not provided rapidly enough to affect initial treatment decisions. In some instances, culture and subsequent identification methods are limited in sensitivity, specificity, or both. During the past 10 years many efforts have focused on the development of new microbiological methods to improve the sensitivity of conventional culture, shorten detection times, and detect nonculturable, fastidious or slow-growing microorganisms.

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