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Multicenter Evaluation Supports Accuracy of the Beckman Coulter Gram-Negative Identification Panel ECCMID 2016 with Improved Database for Clinically Significant Hindler J1, Lewinksi, M, Schreckenberger P2, Beck C3, Nothaft D3, Bobolis J3, Carpenter D3, Mann L, Madriaga OM3, Wong T3, Smoot L3 1UCLA David Geffen School of Medicine, Los Angeles, CA, 2Loyola University Medical Center, Maywood, IL, and 3Beckman Coulter Microbiology, West Sacramento, CA

INTRODUCTION METHODS (Continued) RESULTS (Continued) Incorrect Results (Table 3) A multicenter study was performed to evaluate the performance of the • MicroScan Dried Gram-negative Identification panels were assessed at Fermenter Results (Table 2) There were 8 incorrect results out of a total of 609 clinical and stock MicroScan Gram-negative Identification (NID) panels’ updated organism 16-18 hours. If any of the following criteria were met, reagents were Fermenters were identified correctly 99.2% (417/420) and only 3 isolates. database and revised performance matrix on the WalkAway system and the added to the VP, IND, NIT, and TDA tests and the results reported: isolates generated an incorrect identification. autoSCAN-4 instrument. • GLU, SUC, or SOR were positive Table 2. Fermenter Total Performance Table 3. Total Isolates Incorrectly Identified • ARG and OF/G were positive Reference Identification NID Panel Identification The Gram-negative organism database was revised and includes an • ARG and CET were positive B. cepacia complex 85.92% additional 39 new taxa – 22 fermentative and 17 non-fermentative • LYS or ORN were positive Achromobacter species R. pickettii 10.23% Correct ID (with organisms – along with updated nomenclature. A multicenter study was Correct ID Incorrect ID B. gladioli 3.85% • FD64 and OF/G were positive (oxidase-negative isolates only) additional tests) performed to evaluate the accuracy of the updated NID database and • Panels which could not be read at 16-18 hours were rechecked at 21 Organism Total Cupriavidus spp. 64.36% performance matrix. Achromobacter xylosoxidans A. faecalis 18.48% hours. If the criteria above were met, reagents were added and the P. aeruginosa 17.16% results reported. N % N % N % D. acidovorans 36.44% • Panels for which the criteria did not apply at 21 hours were re- S. putrefaciens 22.83% METHODS incubated an additional 24 hours, after which reagents were added and caviae complex ^ 1 1 100 0 0.0 0 0.0 Achromobacter xylosoxidans B. bronchiseptica 22.83% the results reported. Cupriavidus spp. 13.37% complex 1 1 100 0 0.0 0 0.0 Myroides spp. 4.54% Study Design Data Analysis (Tables 1 and 2) • Data analysis included the following calculations for clinical and frozen A. lwoffii group 93.35% Clinical and Stock Isolate Testing complex ^ 1 1 100 0 0.0 0 0.0 Achromobacter xylosoxidans P. oryzihabitans 5.67% • MicroScan Dried Gram-negative Identification panels were tested at stock isolates included in the study: Citrobacter amalonaticus 5 5 100 0 0.0 0 0.0 A. baumannii/haemolyticus 0.97% two sites. • Number and percent species correct ID Citrobacter braakii ^ 4 4 100 0 0.0 0 0.0 Enterobacter aerogenes Very Rare Biotype • A total of 609 clinical and frozen stock isolates: • Number and percent species correct ID, with additional tests (e.g., E. amnigenus 1 97.71% egg yolk agar, motility, growth at 42°C, production of H2S) ^ 15 14 93.3 1 6.7 0 0.0 C. sedlakii 1.12% • 420 fermentative Gram-negative isolates (Fermenters) complex • 189 non-fermentative Gram-negative isolates (Non-Fermenters) • Number and percent species incorrect ID (or Very Rare Biotype) 14 14 100 0 0.0 0 0.0 E. cloacae 1.03% E. cancerogenous 0.14% • Reference testing was performed following the sites’ current protocols. Cronobacter sakazakii 1 1 100 0 0.0 0 0.0 Enterobacter hormaechei E. cloacae 99.99% Sequencing of 16S rDNA and MALDI-TOF analysis were included for Citrobacter werkmanii ^ 3 3 100 0 0.0 0 0.0 Pseudomonas putida group P. aeruginosa 99.99% discrepant isolates. RESULTS Enterobacter aerogenes 25 23 92.0 1 4.0 1+ 4.0 Panels Quality Control Enterobacter amnigenus 1 1 1 100 0 0.0 0 0.0 • MicroScan Dried Gram-negative Identification panels contained Non-Fermenter Results (Table 1) All Quality Control (QC) strains tested were within specification. modified conventional and chromogenic tests for the identification of Non-Fermenters were identified correctly 97.4% (184/189) and only 5 Enterobacter amnigenus 2 1 1 100 0 0.0 0 0.0 isolates generated an incorrect identification. fermentative and non-fermentative Gram-negative bacilli. Enterobacter cloacae 36 35 97.2 0 0.0 1 2.8 Additional New Taxa (Table 4) Quality Control Table 1. Non-Fermenter Total Performance 78 76 97.4 2 2.6 0 0.0 There were 39 new taxa added to the Gram-negative ID database: • Quality Control (QC) testing was performed prior to the beginning of Correct ID (with Enterobacter gergoviae 1 1 100 0 0.0 0 0.0 Correct ID Incorrect ID Table 4. New Taxa Added to Gram-Negative ID Database the study using all QC strains and specifications listed in MicroScan Organism Total additional tests) Enterobacter hormaechei ^ 1 0 0.0 0 0.0 1 100 Instructions for Use. N % N % N % New Non-Fermenters New Fermenters Edwardsiella tarda 1 1 100 0 0.0 0 0.0 Achromobacter piechaudii Aeromonas caviae complex 2 Enterobacter hormaechei • Weekly QC testing was performed using the primary QC strains. Alcaligenes faecalis 1 0 0.0 1 100 0 0.0 Hafnia alvei 1 1 100 0 0.0 0 0.0 Achromobacter denitrificans Aeromonas veronii complex 2 Escherichia albertii • The QC strains are below: Achromobacter 21 13 61.9 5 23.8 3 14.3 Acinetobacter junii Citrobacter braakii 3 Leminorella grimontia 4 xylosoxidans/denitrificans 27 26 96.3 1 3.7 0 0.0 Primary Strains Acinetobacter ursingii Citrobacter farmer 3 Leminorella richardii 4 Acinetobacter 60 59 98.3 1 1.7 0 0.0 Bordetella trematum Citrobacter freundii 3 Mannheimia haemolytica ATCC 25922 Escherichia coli baumannii/haemolyticus 22 21 95.5 1 4.5 0 0.0 Morganella morganii 15 14 93.3 1 6.7 0 0.0 Brevundimonas diminuta 1 Citrobacter gilleni 3 Serratia ficaria ATCC 49131 Klebsiella oxytoca complex Brevundimonas vesicularis 1 Citrobacter murliniae 3 Vibrio metschnikovii Acinetobacter lwoffi group ^ 6 6 100 0 0.0 0 0.0 Pantoea agglomerans 2 2 100 0 0.0 0 0.0 ATCC 49128 Pseudomonas putida Burkholderia gladioli Citrobacter rodentium Vibrio furnissii Burkholderia cepacia Providencia alcalifaciens 1 1 100 0 0.0 0 0.0 ATCC 27853 1 1 100 0 0.0 0 0.0 Comamonas testosteroni 1 Citrobacter sedlakii 3 5 complex Additional Strains 56 54 96.4 2 3.6 0 0.0 Pseudomonas alcaligenes 1 Citrobacter werkmanii 3 5 Bordetella trematum ^ 1 1 100 0 0.0 0 0.0 ATCC 49132 multocida 3 3 100 0 0.0 0 0.0 Pseudomonas pseudoalcaligenes 1 Citrobacter youngae 3 5 Chryseomonas indologenes 4 2 50.0 2 50.0 0 0.0 Pseudomonas mendocina ATCC 29213 Staphylococcus aureus 3 3 100 0 0.0 0 0.0 1 – Previously included in Pseudomonas species group Cupriavidus species 1 1 100 0 0.0 0 0.0 Paracoccus yeei ATCC 49138 Shewanella putrefaciens 2 – Previously included in Aeromonas hydrophila complex 1 1 100 0 0.0 0 0.0 Ralstonia mannitolilytica ATCC 49127 Stenotrophomonas maltophilia Myroides species 1 1 100 0 0.0 0 0.0 3 – Previously included in Citrobacter freundii complex 16 16 100 0 0.0 0 0.0 Roseomonas species Pseudomonas aeruginosa 89 87 97.8 2 2.2 0 0.0 4 – Previously included as Leminorella species Shewanella algae Proteus vulgaris 4 4 100 0 0.0 0 0.0 5 – Previously included in group Panel Inoculation, Incubation, and Reading Pseudomonas stutzeri 2 2 100 0 0.0 0 0.0 Wautersiella falsenii • All isolates were subcultured to trypticase soy agar (TSA) with 5% Pseudomonas Salmonella Typhi 2 2 100 0 0.0 0 0.0 12 11 91.7 0 0.0 1 8.3 sheep blood or MacConkey agar and incubated for 18-24 hours at 35- fluorescens/putida 6 6 100 0 0.0 0 0.0 Rhizobium radiobacter 1 1 100 0 0.0 0 0.0 37ºC prior to testing. Isolates from frozen stocks were subcultured Serratia fonticola 1 1 100 0 0.0 0 0.0 CONCLUSION Stenotrophomonas twice before testing. 23 23 100 0 0.0 0 0.0 Serratia liquefaciens 1 1 100 0 0.0 0 0.0 • Inoculum suspensions for each strain were prepared with the direct maltophilia standardization (turbidity standard) method for MicroScan Dried Gram- Sphingomonas paucimobilis 1 1 100 0 0.0 0 0.0 30 30 100 0 0.0 0 0.0 The results of this evaluation with fresh clinical isolates show that the negative Identification panels. Acinetobacter species * 1 1 100 0 0.0 0 0.0 Serratia odorifera 1 1 100 0 0.0 0 0.0 MicroScan Dried Gram-negative Identification panel with an updated • Following inoculation, MicroScan Dried Gram-negative Identification Pseudomonas species * 1 1 100 0 0.0 0 0.0 Pantoea species * 1 1 100 0 0.0 0 0.0 database and performance matrix provides accurate identification results for clinically important Gram-negative bacteria. panels were incubated at 35±2ºC in the WalkAway system. All panels Achromobacter species * 1 0 0.0 0 0.0 1 100 TOTAL 420 408 97.1 9 2.1 3 0.7 were read on the WalkAway system and a subset were read manually TOTAL 189 173 91.5 11 5.8 5 2.6 and on the autoSCAN-4 instrument. Beckman Coulter, the stylized logo and the Beckman Coulter product and service names mentioned herein are trademarks or registered trademarks ^ = New taxa added to the Gram-negative ID database. ^ = New taxa added to the Gram-negative ID database. of Beckman Coulter, Inc. in the United States and other countries. * = Isolates had reference IDs to genus-level only. * = Isolates had reference IDs to genus-level only. + = Very Rare Biotype