Ann Lab Med.  2019 Jan;39(1):43-49. 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

Affiliations
  • 1Department for Infectious Diseases, Microbiology and Hygiene, University Hospital of Heidelberg, Germany. irene.burckhardt@med.uni-heidelberg.de

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.

Keyword

Laboratory automation; Methicillin-resistant Staphylococcus aureus; Multi-drug resistant gram-negative bacteria; Vancomycin-resistant enterococci; Chromogenic media; Workflow

MeSH Terms

Automation
Automation, Laboratory*
Bacteria*
Gram-Negative Bacteria
Humans
Kinetics*
Methicillin-Resistant Staphylococcus aureus
Vancomycin-Resistant Enterococci

Figure

  • Fig. 1 Growth kinetics: time to result (=first growth, single colonies, typical morphology) for clinical strains and ATCC strains (MRSA, MDRGN, VRE, 108/mL). Red lines represent imaging times for patient sample evaluation.Abbreviations: ATCC, American Type Culture Collection; MRSA, methicillin-resistant Staphylococcus aureus; MDRGN, multi-drug resistant gram-negative bacteria; VRE, vancomycin-resistant enterococci.


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