
==== Front
J Antimicrob Chemother
J Antimicrob Chemother
jac
Journal of Antimicrobial Chemotherapy
0305-7453
1460-2091
Oxford University Press UK

10.1093/jac/dkae278
dkae278
Supplement Paper
AcademicSubjects/MED00740
AcademicSubjects/MED00290
AcademicSubjects/MED00230
Validation of short culture method for rapid bacterial identification of blood cultures via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
Fitts Eric C Department of Pathology & Laboratory Medicine, School of Medicine, Emory University, Atlanta, Georgia, USA

Dent E Alexander Department of Pathology & Laboratory Medicine, School of Medicine, Emory University, Atlanta, Georgia, USA

Burd Eileen M Department of Pathology & Laboratory Medicine, School of Medicine, Emory University, Atlanta, Georgia, USA

Corresponding author. E-mail: EBURD@emory.edu
Eric C. Fitts and E. Alexander Dent Equal contribution.

9 2024
19 9 2024
19 9 2024
79 Suppl 1 Faster ID and AST revolution: How to Improve Antibiotic Use in the Critically Ill i9i12
© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.

Abstract

Background

Development of rapid bacterial identification from blood cultures has been an area of intense study in diagnostic microbiology. Shortened turnaround time coupled with antimicrobial stewardship interventions have been shown to improve patient outcomes and decrease healthcare-associated costs.

Objectives

We report the validation of a short incubation method for Gram-positive and Gram-negative bacterial identification utilizing MALDI-TOF MS without additional instrumentation, processing or cost compared with current practice.

Methods

Prospective, observational, single-centre study in a quaternary care academic hospital encompassing 376 blood cultures subjected to bacterial identification after short incubation periods of 3–4 and 6–8 h.

Results

There was 97.5% species-level identification agreement with tests undertaken after 3–4 h incubation with 83.6% isolates identified, and 99.7% species-level identification agreement after 6–8 h incubation with 96.7% isolates identified.

Conclusions

The short incubation method provides a rapid MALDI-TOF MS bacterial identification method, reducing turnaround time by 10–18 h compared with standard practice without additional cost, processing or instrumentation.

bioMérieux 10.13039/501100022110
==== Body
pmcIntroduction

Bacterial identification within clinical infection impacts treatment decisions and has been the focus of test development for decades, with methods both faster and more accurate than previous iterations becoming available commercially every year.1–3 The initial widespread use of analytical profile index (API) strips was largely supplanted by biochemical reaction methods that provided automated bacterial identification within clinical microbiology laboratories.4 The adoption of MALDI-TOF MS for bacterial and yeast identification further improved the time to identification, specificity and accuracy of isolate identification.5 What was once a process requiring 48 to 72 h before bacterial identification confirmation is now routinely performed in 18–24 h following a positive blood culture signal.6

What comes next in this dynamic field of rapid identification is hotly contested. Many novel methods of identification have been proposed, with several now commercially available or in development, including nucleic acid hybridization/amplification-based testing, targeted or metagenomic sequencing, and identification via metabolic products or unique antigens.7–9 Many of these methods require additional instrumentation, increased cost, or both, above the current standard of MALDI-TOF MS on subcultures of positive blood culture. Many of these methods are limited in the number of distinct bacterial genera or species each can identify. Due to the volume of positive blood cultures handled in many clinical laboratories, the cost to implement these novel methods may be exorbitant or unfeasible unless these methods are either targeted via stewardship or restricted to particular patient populations.10,11

To overcome resource limitations, several studies have focused on improving workflows utilizing only technologies that are currently in routine, widespread practice, developing techniques in which either aliquots of positive blood culture bottles or early growth from subcultures are manipulated and identified via MALDI-TOF MS.12,13 These methods report significant reduction in time to identification, anywhere from 12–24 h quicker than routine methods; however, all require additional processing steps and often require additional instrumentation.14,15 The validation of a short culture method of MALDI-TOF MS microbial identification performed without additional processing or cost is reported herein. We compared shortened incubation subcultures for bacterial identification of organisms from positive blood culture bottles within a quaternary care academic medical centre between 3–4 or 6–8 h after inoculation using the FDA-approved workflow of 18–24 h post-inoculation to characterize agreement and validate this method for routine use. The objective of this study was to provide a method validation and illustrate the test characteristics of a modified blood culture bacterial identification method.

Methods

The study was designed as a prospective, observational, single-centre study in a quaternary care academic hospital. Inoculated blood culture bottles were loaded on the BACT/ALERT system (bioMérieux) immediately upon receipt from affiliated hospitals. Positive aerobic and anaerobic blood cultures, as determined by the BACT/ALERT system, were included in the study if Gram staining indicated the cultures were monomicrobial, comprising either Gram-negative rods (GNR) or Gram-positive cocci (GPC) and by the timing of the instrument flagging positive. Cultures were subcultured on 5% sheep blood agar and chocolate agar, with the addition of MacConkey agar for GNR, and incubated aerobically at 37°C in 5% CO2. Subcultured isolates were sampled once at 3–4 h, again at 6–8 h and finally at standard practice between 18–24 h after inoculation. Cultures were excluded from study if they were from a patient with an isolate previously enrolled, if the Gram stain from blood culture showed organisms other than GNR or GPC, if standard practice identified a GPC isolate as an organism other than Staphylococcus sp. or Enterococcus sp., or if the culture was considered a contaminant. At each timepoint, MALDI-TOF MS was performed to identify cultured bacteria per standard practice. In short, bacterial colonies were transferred from media to a MALDI-TOF MS target plate in duplicate. Bacteria were overlayed with 1 µL of α-cyano-4-hydroxycinnamic acid matrix and allowed to dry prior to loading the slide into VITEK MS (bioMérieux) for identification. Results were determined to be valid if the confidence score was >99.9% for at least one position and if both positions provided concordant identifications. For samples with different identifications between duplicates, a second firing of the same slide and additional sampling was performed to resolve identification. Samples with low confidence or multiple identifications (not within the same genus) were considered invalid and the result was recorded as no identification. When standard practice identification was a single species, correct identification by short incubation (SI) required that the same species with no other species be identified. For example, SI identification of Klebsiella variicola/Klebsiella pneumoniae and standard practice identification of K. pneumoniae was considered incorrect for species and correct for genus. However, when standard practice identified multiple species, SI identification was considered correct if it included one or more of the species identified by standard practice (and no species outside the standard practice genus). For example, SI identification of Enterobacter hormaechei and standard practice identification of Enterobacter cloacae/asburiae/hormaechei was considered correct for species and genus.

Statistical analysis was performed using McNemar’s test to determine significance due to difference in two independent proportions. All analyses were performed using R Statistical Software.

Results

A total of 376 specimens were followed for enrolment in the study. There were 33 cultures that had mixed Gram stains or were considered to be contaminated and were excluded from analysis. A further 13 (3.8%) specimens had no identification following standard practice using MALDI-TOF MS alone and were excluded. A total of 330 cultures had appropriate identification by standard practice for comparison; see Table 1 (available as Supplementary data at JAC Online) for a list of organisms identified by short culture incubation). At 3–4 h incubation, 54/330 (16.4%) had no identification, while of those with identifications, 269/276 (97.5%) were concordant with standard practice at species level and 276/276 (100%) were concordant at the genus level (see Table 2 for a list of bacteria identified by standard practice with no identification of at least one isolate at 3–4 h). At 6–8 h incubation, 11/330 (3.3%) had no identification, while of those with identification, 318/319 (99.7%) were concordant with standard practice at the species level and 319/319 (100%) were concordant at the genus level (Figure 1) (see Table 3 for a list of bacteria identified by standard practice with no identification of at least one isolate at 3–4 h). Gram-negative identification demonstrated concordance of 140/142 (98.6%) and 142/142 (100%) for species and genus level, respectively, at 3–4 h incubation, as well as 160/161 (99.4%) and 161/161 (100%) for species and genus level concordance, respectively, at 6–8 h incubation compared with standard practice. Gram-positive identification demonstrated concordance of 130/134 (97.0%) and 134/134 (100%) for species and genus level concordance, respectively, at 3–4 h incubation, as well as 157/158 (99.4%) and 158/158 (100%) for species and genus level concordance, respectively, at 6–8 h incubation compared with standard practice.

Figure 1. Percent of isolates identified by SI at the genus level (a, b) and species level (c, d) for Gram-positive isolates (a, c) and Gram-negative isolates (b, d). This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Short incubation at 3–4 h had significantly greater percent non-identifications versus standard practice compared with the percent of non-identifications at 6–8 h versus standard practice (P < 0.001). Similarly, the percent identification accuracy at species level compared with standard practice was significantly lower in the 3–4 h group compared with the 6–8 h group (P = 0.047). See Table 4 for a list of incorrect species-level identifications compared with standard practice.

Discussion

Our results demonstrate that there is a negligible difference in bacterial identifications performed after SI of 6–8 h compared with current standard practice. Despite a small percent of specimens (3.3%) having no identification compared with standard practice, the specimens that were identified could be considered identified with a high degree of confidence. Integrating this into clinical laboratory workflows would be easily accomplished with the caveat of repeating non-identified specimens after reaching a subculture time of 18–24 h. This follows similar workflows to standard practice, in which a secondary method of identification is performed for cultures that receive low confidence or no identification by MALDI-TOF MS and would thus be equivalent or more efficient than standard practice.

While the 3–4 h SI method provided significantly fewer bacterial identifications, the accuracy of the method at genus level was equivalent to standard practice. Species-level discordance was primarily a consequence of closely related enteric bacteria or species complexes, none of which would impact the antibiotic resistance thresholds currently used to determine susceptibility. However, discordant bacterial identifications between cultures in the same patient, even with same genus, may raise concerns for superimposed or polymicrobial infections, impacting patient care and therapeutic decision-making. While decreased time to identification is enticing, the necessity of repeating identifications as would be required with the 3–4 h SI method is antithetical to the idea of increasing efficiency in the laboratory and streamlined workflows. Laboratories frequently batch plate-reading to ensure that disruptions are minimized and that technologist time is utilized appropriately. The Great Resignation, the phenomenon coined in 2021 to describe the mass resignations experienced in many industries related temporally to the SARS-CoV-2 pandemic and shutdowns, greatly impacted clinical laboratories and as such experienced technologists with plate-reading knowledge are precious, and making efficient use of this resource crucial. The option of utilizing the 6–8 h SI is particularly well suited in hospitals utilizing the three-shift labour model that separates days into 8 h shifts: day, evening and night. This would distribute effort across shifts and reduce the number of cultures needed to be read during any one shift, while maintaining continuity and integrating into existing workflows.

In conclusion, the SI 6–8 h method provided reliable, clinically actionable identification 10–18 h sooner than standard practice. While previous studies that evaluated the utility of molecular diagnostics in identification of organisms from positive blood cultures have shown mixed results for patient outcomes, the studies that illustrated benefits of decreased time to culture identification included: decreased length of stay; earlier initiation of targeted antimicrobial therapy; decreased mortality; and reduced healthcare costs.16 A key variable that leads to these benefits has been identified as partnership with antimicrobial stewardship programmes. Direct interaction between the treating physician and infectious disease specialists or clinical pharmacists to target antimicrobial therapies has been recognized as essential to successfully coupling decreased turnaround time and patient benefit (‘Prospective audit with intervention and feedback’ by IDSA).17–19 A second link in this chain is tying early bacterial identification into that intervention by extending the interaction of the treating physician, ID specialist and clinical pharmacist to include the microbiology laboratory.20–22

A key point in the development of this study was the impact of this method and the maintenance of currently used workflows. While there are several commercial products that can facilitate producing either a liquid colony of bacteria from a positive blood culture or a pellet of bacteria using a combination of centrifugation and wash steps, this approach of SI requires no changes in procedure, additional reagents, or instrumentation, which would allow any laboratory currently performing routine MALDI-TOF identification on subcultured positive blood cultures to implement this method.

Supplementary Material

dkae278_Supplementary_Data

Acknowledgements

We sincerely thank the technologists and staff at the Clinical Microbiology Laboratory at Emory University Hospital for their hard work and dedication, without whom this study would not have been possible.

Funding

This paper was published as part of a supplement financially supported by bioMérieux.

Transparency declarations

E.C.F. and E.M.B. received partial salary support from the sponsor, bioMérieux. Reagents used for the study were provided in part or wholly by the sponsor. Data were provided to and analysed by the sponsor with independent review by authors. The manuscript was prepared and provided to the sponsor for review prior to submission. All other authors have no conflicts to declare.

Supplementary data

Tables S1 to S4 are available as Supplementary data at JAC Online.
==== Refs
References

1 Gaieski  DF, Mikkelsen  ME, Band  RA  et al  Impact of time to antibiotics on survival in patients with severe sepsis or septic shock in whom early goal-directed therapy was initiated in the emergency department. Crit Care Med  2010; 38 : 1045–53. 10.1097/CCM.0b013e3181cc4824 20048677
2 Kumar  A . Antimicrobial delay and outcome in severe sepsis. Crit Care Med  2014; 42 : e802. 10.1097/CCM.0000000000000620
3 Buehler  SS, Madison  B, Snyder  SR  et al  Effectiveness of practices to increase timeliness of providing targeted therapy for inpatients with bloodstream infections: a laboratory medicine best practices systematic review and meta-analysis. Clin Microbiol Rev  2016; 29 : 59–103. 10.1128/CMR.00053-14 26598385
4 D’Amato  RF, Holmes  B, Bottone  EJ. The systems approach to diagnostic microbiology. Crit Rev Microbiol  1981; 9 : 1–44. 10.3109/10408418109104485 7028398
5 Welker  M . Proteomics for routine identification of microorganisms. Proteomics  2011; 11 : 3143–53. 10.1002/pmic.201100049 21726051
6 Clerc  O, Prod'hom  G, Vogne  C  et al  Impact of matrix-assisted laser desorption ionization time-of-flight mass spectrometry on the clinical management of patients with Gram-negative bacteremia: a prospective observational study. Clin Infect Dis  2013; 56 : 1101–7. 10.1093/cid/cis1204 23264363
7 Sullivan  KV . Advances in diagnostic testing that impact infection prevention and antimicrobial stewardship programs. Curr Infect Dis Rep  2019; 21 : 20. 10.1007/s11908-019-0676-7 31044313
8 Bouzid  D, Zanella  MC, Kerneis  S  et al  Rapid diagnostic tests for infectious diseases in the emergency department. Clin Microbiol Infect  2021; 27 : 182–91. 10.1016/j.cmi.2020.02.024 32120036
9 Trotter  AJ, Aydin  A, Strinden  MJ  et al  Recent and emerging technologies for the rapid diagnosis of infection and antimicrobial resistance. Curr Opin Microbiol  2019; 51 : 39–45. 10.1016/j.mib.2019.03.001 31077935
10 Pliakos  EE, Andreatos  N, Shehadeh  F  et al  The cost-effectiveness of rapid diagnostic testing for the diagnosis of bloodstream infections with or without antimicrobial stewardship. Clin Microbiol Rev  2018; 31 : e00095-17. 10.1128/CMR.00095-17.29848775
11 Salvador  BC, Lucchetta  RC, Sarti  FM  et al  Cost-effectiveness of molecular method diagnostic for rapid detection of antibiotic-resistant bacteria. Value Health Reg Issues  2022; 27 : 12–20. 10.1016/j.vhri.2021.07.009 34784543
12 Wimmer  JL, Long  SW, Cernoch  P  et al  Strategy for rapid identification and antibiotic susceptibility testing of Gram-negative bacteria directly recovered from positive blood cultures using the Bruker MALDI Biotyper and the BD Phoenix system. J Clin Microbiol  2012; 50 : 2452–4. 10.1128/JCM.00409-12 22518850
13 Charretier  Y, Dauwalder  O, Franceschi  C  et al  Rapid bacterial identification, resistance, virulence and type profiling using selected reaction monitoring mass spectrometry. Sci Rep  2015; 5 : 13944. 10.1038/srep13944 26350205
14 Lotte  R, Courdurié  A, Gaudart  A  et al  Spontaneous bacterial peritonitis: the incremental value of a fast and direct bacterial identification from ascitic fluids inoculated in blood culture bottles by MALDI-TOF MS for a better management of patients. Microorganisms  2022; 10 : 1188. 10.3390/microorganisms10061188 35744706
15 Zengin Canalp  H, Bayraktar  B. Direct rapid identification from positive blood cultures by MALDI-TOF MS: specific focus on turnaround times. Microbiol Spectr  2021; 9 : e0110321. 10.1128/spectrum.01103-21 34908465
16 Perez  KK, Olsen  RJ, Musick  WL  et al  Integrating rapid pathogen identification and antimicrobial stewardship significantly decreases hospital costs. Arch Pathol Lab Med  2013; 137 : 1247–54. 10.5858/arpa.2012-0651-OA 23216247
17 Bauer  KA, West  JE, Balada-Llasat  JM  et al  An antimicrobial stewardship program’s impact. Clin Infect Dis  2010; 51 : 1074–80. 10.1086/656623 20879856
18 Camins  BC, King  MD, Wells  JB  et al  Impact of an antimicrobial utilization program on antimicrobial use at a large teaching hospital: a randomized controlled trial. Infect Control Hosp Epidemiol  2009; 30 : 931–8. 10.1086/605924 19712032
19 Timbrook  TT, Hurst  JM, Bosso  JA. Impact of an antimicrobial stewardship program on antimicrobial utilization, bacterial susceptibilities, and financial expenditures at an academic medical center. Hosp Pharm  2016; 51 : 703–11. 10.1310/hpj5109-703 27803499
20 Morency-Potvin  P, Schwartz  DN, Weinstein  RA. Antimicrobial stewardship: how the microbiology laboratory can right the ship. Clin Microbiol Rev  2017; 30 : 381–407. 10.1128/CMR.00066-16 27974411
21 Bouza  E, Munoz  P, Burillo  A. Role of the clinical microbiology laboratory in antimicrobial stewardship. Med Clin North Am  2018; 102 : 883–98. 10.1016/j.mcna.2018.05.003 30126578
22 MacVane  SH, Hurst  JM, Steed  LL. The role of antimicrobial stewardship in the clinical microbiology laboratory: stepping up to the plate. Open Forum Infect Dis  2016; 3 : ofw201. 10.1093/ofid/ofw201 27975076
