Ann Lab Med 2019; 39(1): 43-49  https://doi.org/10.3343/alm.2019.39.1.43
Shorter Incubation Times for Detecting Multi-drug Resistant Bacteria in Patient Samples: Defining Early Imaging Time Points Using Growth Kinetics and Total Laboratory Automation
Irene Burckhardt, M.D., Katharina Last, M.D., and Stefan Zimmermann, M.D.
Department for Infectious Diseases, Microbiology and Hygiene, University Hospital of Heidelberg, Germany
Corresponding author: Irene Burckhardt, M.D.
https://orcid.org/0000-0002-7610-4530
Department for Infectious Diseases, Microbiology and Hygiene, University Hospital of Heidelberg, Im Neuenheimer Feld 324, Heidelberg 69115, Germany
Tel: +49-6221-5637795
Fax: +49-6221-564343
E-mail: irene.burckhardt@med.uni-heidelberg.de
Received: September 26, 2017; Revised: April 14, 2018; Accepted: August 29, 2018; Published online: January 1, 2019.
© Korean Society for Laboratory Medicine. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: The transition from manual processing of patient samples to automated workflows in medical microbiology is challenging. Although automation enables microbiologists to evaluate all samples following the same incubation period, the essential incubation times have yet to be determined. We defined essential incubation times for detecting methicillin-resistant Staphylococcus aureus (MRSA), multi-drug resistant gram-negative bacteria (MDRGN), and vancomycin-resistant enterococci (VRE).
Methods: We monitored the growth kinetics of MRSA, MDRGN, and VRE between two and 48 hours on chromogenic media to establish the time points of first growth, single colony appearance, and typical morphology for 102, 104, 106, and 108 colony forming units/mL. Subsequently, we imaged plates inoculated with 778 patient samples after 20, 24, and 36 hours.
Results: The first growth, single colony appearance, and typical morphology time points were inoculum-dependent. First growth appeared after 6-18 hours, 4-18 hours, and 8-48 hours for MRSA, MDRGN, and VRE, respectively, and single colonies appeared at 12-18 hours, 6-20 hours, and 12-48 hours, respectively. Typical morphology was visible at 14-22 hours and 12-48 hours for MRSA and VRE, but was not determined for MDRGN. By examining patient samples, ≥98% of MRSA and MDRGN were visible 20 hours after the start of incubation. Following 24 hours of incubation, only 79.5% of VRE were clearly visible on the respective plates.
Conclusions: An incubation time of 20 hours is sufficient for detecting MRSA and MDRGN. VRE growth is much slower and requires additional imaging after 36 hours.
Keywords: Laboratory automation, Methicillin-resistant Staphylococcus aureus, Multi-drug resistant gram-negative bacteria, Vancomycin-resistant enterococci, Chromogenic media, Workflow



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