Identification of Gram negative non-fermentative bacteria: How hard can it be?

Author summary Infections caused by Gram negative non fermentative bacteria (GNNF), other than the more commonly described Pseudomonas aeruginosa and Acinetobacter baumannii, constitute an emerging problem. They are not only isolated in nosocomial settings, mainly affecting immunocomprised hosts, but also are opportunistic infections causing invasive disease in rural communities. GNNF bacteria are ubiquitously distributed in the environment and have a high propensity for antimicrobial resistance. Thus, their accurate identification to the species level is important for appropriate patient management. These bacteria are taxonomically heterogeneous, and many isolates are not satisfactorily identified by the standard biochemical assays because of overlapping phenotypic characteristics. In this study we explored using the BD Phoenix automated identification system to improve the identification of GNNF isolates and recognized 92% (187/204) of the previously unidentified isolates; 117 of which were to the species level. Antimicrobial resistance profiles were identified for 50% (94/187) of the isolates identified by the BD Phoenix system. For the remaining 12 unidentified organisms whole genome sequencing was performed. Three web-based programs were used for organism identification and results varied because of differences in the underlying databases used. Introduction The prevalence of bacteremia caused by Gram negative non-fermentative (GNNF) bacteria has been increasing globally over the past decade. Many studies have investigated their epidemiology but focus on the common GNNF including Pseudomonas aeruginosa and Acinetobacter baumannii. Knowledge of the uncommon GNNF bacteremias is very limited. This study explores invasive bloodstream infection GNNF isolates that were initially unidentified after testing with standard microbiological techniques. All isolations were made during laboratory-based surveillance activities in two rural provinces of Thailand between 2006 and 2014. Methods A subset of GNNF clinical isolates (204/947), not identified by standard manual biochemical methodologies were run on the BD Phoenix automated identification and susceptibility testing system. If an organism was not identified (12/204) DNA was extracted for whole genome sequencing (WGS) on a MiSeq platform and data analysis performed using 3 web-based platforms: Taxonomer, CGE KmerFinder and One Codex. Results The BD Phoenix automated identification system recognized 92% (187/204) of the GNNF isolates, and because of their taxonomic complexity and high phenotypic similarity 37% (69/187) were only identified to the genus level. Five isolates grew too slowly for identification. Antimicrobial sensitivity (AST) data was not obtained for 93/187 (50%) identified isolates either because of their slow growth or their taxa were not in the AST database associated with the instrument. WGS identified the 12 remaining unknowns, four to genus level only. Conclusion The GNNF bacteria are of increasing concern in the clinical setting, and our inability to identify these organisms and determine their AST profiles will impede treatment. Databases for automated identification systems and sequencing annotation need to be improved so that opportunistic organisms are better covered.