Use of the MicroSeq 500 16S rRNA Gene-Based Sequencing for Identification of Bacterial Isolates That Commercial Automated Systems Failed To Identify Correctly

Abstract

Reliable automated identification and susceptibility testing of clinically relevant bacteria is an essential routine for microbiology laboratories, thus improving patient care. Examples of automated identification systems include the Phoenix (Becton Dickinson) and the VITEK 2 (bioMérieux). However, more and more frequently, microbiologists must isolate "difficult" strains that automated systems often fail to identify. An alternative approach could be the genetic identification of isolates; this is based on 16S rRNA gene sequencing and analysis. The aim of the present study was to evaluate the possible use of MicroSeq 500 (Applera) for sequencing the 16S rRNA gene to identify isolates whose identification is unobtainable by conventional systems. We analyzed 83 "difficult" clinical isolates: 25 gram-positive and 58 gram-negative strains that were contemporaneously identified by both systems-VITEK 2 and Phoenix-while genetic identification was performed by using the MicroSeq 500 system. The results showed that phenotypic identifications by VITEK 2 and Phoenix were remarkably similar: 74% for gram-negative strains (43 of 58) and 80% for gram-positive strains were concordant by both systems and also concordant with genetic characterization. The exceptions were the 15 gram-negative and 9 gram-positive isolates whose phenotypic identifications were contrasting or inconclusive. For these, the use of MicroSeq 500 was fundamental to achieving species identification. In clinical microbiology the use of MicroSeq 500, particularly for strains with ambiguous biochemical profiles (including slow-growing strains), identifies strains more easily than do conventional systems. Moreover, MicroSeq 500 is easy to use and cost-effective, making it applicable also in the clinical laboratory.Since the time when microbial identification (ID) was performed by using tube tests, much progress has been made. Initially, to assist microbiologists, miniaturized ID systems appeared, followed by innovative automatic ID systems such as VITEK 2 (bioMérieux, Marcy l'Étoile, France) and Phoenix (Becton Dickinson Microbiology Systems, Cockeysville, Md.) (1, 9). These are new, fully automated systems that provide accurate and reproducible IDs, as well as antimicrobial susceptibility tests. The systems possess either sophisticated software to identify microorganism phenotypes or "advanced expert systems" able to elaborate and validate the antimicrobial susceptibilities of the isolates (1, 2, 6, 7, 9, 10). In spite of the undoubtedly innovative results obtained with the widespread use of these automated systems, they do have some drawbacks, particularly when microbiologists need to identify microorganisms exhibiting biochemical features that do not fit into any known patterns of genus and species. These unusual isolates are quite common, especially when we consider that more and more strains isolated from patients that have undergone longterm antimicrobial therapy (such as hematological patients and those in intensive care units) can lose their typical b...

Comparative analyses of phenotypic methods and 16S rRNA, khe, rpoB genes sequencing for identification of clinical isolates of Klebsiella pneumoniae

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Escherichia coli Variants in Periprosthetic Joint Infection: Diagnostic Challenges with Sessile Bacteria and Sonication

“…In addition to the diagnostic systems API20E and Vitek2, 16S rRNA (500-bp) partial gene sequencing (ABI Prism 3130; Applied Biosystems Inc., Foster City, CA) was applied (7).…”

16S rRNA Gene Sequencing in Routine Identification of Anaerobic Bacteria Isolated from Blood Cultures

“…Conventional phenotypic identification of anaerobic bacteria to the species level can be difficult and laborious in the clinical microbiology laboratory. Commercial phenotypic systems like the Rapid ID 32A or Vitek 2 ANC card (bioMérieux, Marcy-l'Etoile, France) are faster but limited by the taxa included in the system database and, in most cases, only reliable to the genus level (6,8,11,12,14). In the last few years, the use of 16S rRNA gene sequencing as an alternative method for species identification has increased, while the time and costs (Ϸ$35 to $40) have been reduced (7,8,14,15).…”

“…Commercial phenotypic systems like the Rapid ID 32A or Vitek 2 ANC card (bioMérieux, Marcy-l'Etoile, France) are faster but limited by the taxa included in the system database and, in most cases, only reliable to the genus level (6,8,11,12,14). In the last few years, the use of 16S rRNA gene sequencing as an alternative method for species identification has increased, while the time and costs (Ϸ$35 to $40) have been reduced (7,8,14,15). The method has been applied for anaerobic bacteria, including isolates from blood cultures, in some studies but not in a routine prospective design (8,10,15,16).…”