Comparison of Phenotypic and Genotypic Techniques for Identification of Unusual Aerobic Pathogenic Gram-Negative Bacilli

Abstract

Rapid and accurate identification of bacterial pathogens is a fundamental goal of clinical microbiology, but one that is difficult or impossible for many slow-growing and fastidious organisms. We used identification systems based on cellular fatty acid profiles (Sherlock; MIDI, Inc., Newark, Del.), carbon source utilization (Microlog; Biolog, Inc., Hayward, Calif.), and 16S rRNA gene sequence (MicroSeq; Perkin-Elmer Applied Biosystems Division, Foster City, Calif.) to evaluate 72 unusual aerobic gram-negative bacilli isolated from clinical specimens at the Mayo Clinic. Compared to lengthy conventional methods, Sherlock, Microlog, and MicroSeq were able to identify 56 of 72 (77.8%), 63 of 72 (87.5%), and 70 of 72 (97.2%) isolates to the genus level (P = 0.002) and 44 to 65 (67.7%), 55 of 65 (84.6%), and 58 of 65 (89.2%) isolates to the species level (P = 0.005), respectively. FourAcinetobacter and three Bordetella isolates which could not be identified to the species level by conventional methods were identified by MicroSeq. In comparison to the full 16S rDNA sequences, the first 527 bp provided identical genus information for all 72 isolates and identical species information for 67 (93.1%) isolates. These data show that MicroSeq provides rapid, unambiguous identification of clinical bacterial isolates. The improved turnaround time provided by genotypic identification systems may translate into improved clinical outcomes.

Basic Concepts of Microarrays and Potential Applications in Clinical Microbiology

“…Specific microbial gene amplification by either a broad-range or a multiplex PCR prior to microarray analysis enhances test sensitivity. The amplification of universal microorganism targets by broadrange PCR followed by sequencing analysis has been considered a standard procedure (190); however, microarrays have amplified using a biotinylated primer and then denatured and hybridized to microspheres tagged with target-specific sequence probes. Probe-target hybridization is measured using a streptavidin-bound green fluorophore.…”

“…on April 30, 2020 by guest http://cmr.asm.org/ emerged as potential tools for bacterial detection and identification given their high parallelism in screening for the presence of a wide diversity of genes. The most commonly used gene targets have been the 16S bacterial and 28S fungal and intergenic transcribed spacers (ITSs) in rRNA genes, and microarray technology has been incorporated to compensate for the time-consuming sequencing identification procedure (190). An oligonucleotide microarray targeting the 16S rRNA gene was developed for the detection of a panel of 40 predominant human intestinal bacterial pathogens in human fecal samples (208).…”

Updated perspectives on emerging vibrios associated with human infections

“…Molecular targets for routine identification of pathogens in clinical microbiology and rapid characterization of bacteria with particular growth requirements or unusual biochemical patterns is a valid alternative approach to culture-based methods (Tang et al 1998).…”

Molecular and phenotypic characterization of nonmotile Gram-negative bacteria associated with spoilage of freshwater fish

“…fragi IFO 3458 was much lower, although this and an environmental strain were the closest organisms. 16S rRNA data are widely used not only to define taxa but also to identify organisms present in clinical, veterinary and environmental samples (Boivin-Jahns et al 1995;Tang et al 1998;Drancourt et al 2000;Maruyama et al 2000).…”