Notes

These early systems included the nine-test Enterotube (Roche, Basel, Switzerland) and two-tube R-B systems for identifying members of Enterobacteriaceae. These systems offered several advantages over conventional testing, namely, all inocula for a set of tests performed sequentially were prepared from a single colony, preparation costs and the amount of storage space required for standard reagents and media were reduced, less incubation space was required, and in some instances identification times were reduced. However, biochemical reactions observed with commercial systems often did not correlate well with conventional test results, and because of the limited number of tests employed in these systems, the percentage of strains correctly identified to the species level was less than satisfactory. These kits were rapidly followed by the landmark API 20E strip test (bioMérieux-Vitek, Hazelwood, Mo.), which was a micromethod employing 20 different biochemical tests. The 20E strip generated a septyl (7-digit) code in 18 to 21 h based upon biochemical reactions plus a screening oxidase test. The numeric code could then be located in a logbook that converted septyl codes into a final bacterial identification. The system identified species in the family Enterobacteriaceae and common nonfermenters, such as Pseudomonas aeruginosa. Although a manual method, the API 20E strip test was so advanced for its time that it is still commonly used in clinical microbiology labs throughout the world and is considered by many to be the “gold standard” commercial system against which all other such systems are measured (16).

In 1978, MicroScan (now Dade Behring MicroScan, West Sacramento, Calif.) developed the first combination panel providing both organism identification and susceptibility profile determination (MIC determination) simultaneously. The 1980s saw continued improvements in diagnostic bacteriology with the introduction of automation into the laboratory (23). In 1983, MicroScan released the AS-3/touchSCAN system, the first widely accepted automated system for microbial identification and drug susceptibility testing in the clinical laboratory. Other systems, such as the Vitek AutoMicrobic system, quickly followed (23). These automated systems transformed processes for bacterial identification by reducing the time required to identify rapidly growing bacteria to as little as 2 to 4 h, in contrast to the 1 or more days that had been required previously for a final identification by conventional test methodologies.

The use of molecular biology and molecular techniques as an aid to bacterial taxonomy and identification was in its infancy in the 1960s. Early studies using DNA base composition could clearly distinguish between genomes that were unrelated based upon differences in G+C content (in moles percent) (21). However, the first major leap in molecular taxonomy and identification applicable to diagnostic microbiology occurred with the introduction of DNA-DNA hybridization studies pioneered by Don Brenner and his colleagues at the Walter Reed Army Institute of Research and later at the Centers for Disease Control and Prevention (3). The value of DNA hybridization was that it provided a quantitative definition of what constituted a species, ∼70% or greater DNA-DNA relatedness with a ▵Tm of 5°C or lower (26, 30). In cases where new species were identified via DNA hybridization, it was also observed that in most instances the results of simple biochemical tests would clearly separate newly recognized or redesignated genera or species from other established groups. This allowed for taxonomic advances made through DNA hybridization studies to be easily adapted to the diagnostic laboratory through the use of new phenotypic identification schemes. However, not all newly recognized taxa that had been identified by molecular techniques could be readily identified by biochemical tests in clinical laboratories.

The downside of DNA hybridization is that it is an expensive, technically complex, and labor-intensive procedure that, at its zenith, was restricted to a small number of research or public health centers around the world. Today, very few laboratories perform DNA hybridization by classic methods. The world of molecular taxonomy was revolutionized, however, in the mid-1980s with the advent of full sequence analysis of molecular chronometers such as rRNA (21). By the mid-1990s, sequencing of the small subunit (16S) rDNA genes had become commonplace, considered a standard tool of microbial taxonomists not only for elucidating phylogenetic relatedness but also as a means of bacterial identification (15, 21). The automation of 16S rDNA gene sequencing with such instruments as the ABI Prism 377 DNA sequencer (Applied Biosystems, Foster City, Calif.) allowed for a quick comparative analysis of published sequences deposited in microbial genome databases (14). Today, bacterial strains that defy identification by conventional commercial methodologies are often subjected to 16S rDNA sequence analysis so that a useful label can be placed on the isolate in question (31)