Rapid first-line discrimination of methicillin resistant Staphylococcus aureus strains using MALDI-TOF MS
Introduction
Staphylococcus aureus (S. aureus) is a primary cause of nosocomial infection. In many countries methicillin-resistant S. aureus (MRSA) is very common and often accounts for 25–50% of all S. aureus identified in the laboratory. It is well-documented that infections caused by MRSA are associated with increased mortality and morbidity compared to infections caused by methicillin-susceptible S. aureus (MSSA) strains (Cosgrove et al., 2003). Early identification of an outbreak is essential for rapid and successful control.
Implementing control measures in a suspected outbreak involves sampling and culture of swab samples from the nose and other surveillance samples potentially from many patients and staff members, as well as cohort isolation of patients with contact exposure to the index patients and extraordinary decontamination of the outbreak ward(s) (Muto et al., 2003). The cost of prevention and control of nosocomial outbreaks of MRSA is therefore high (Gould et al., 2010).
A reliable discrimination of MRSA isolates is essential to identify or rule out an outbreak. At present, molecular typing methods, such as S. aureus protein A (spa) typing, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) are generally used for this purpose. These methods are relatively time-consuming and rarely performed routinely in smaller laboratories (Koreen et al., 2004, Struelens et al., 2009, Weller, 2000).
Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) has recently become a standard method in many microbiological laboratories for identification of bacterial pathogens cultured in patient samples. Identification of cultured organisms applying MALDI-TOF MS is based on the generation of mass spectra obtained from colony material, which are compared to the spectra of known species in a reference library (Clark et al., 2013, Nomura, 2015). At present, few studies using MALDI-TOF MS as a method of discriminating between different clonal complexes and spa types of MRSA and S. aureus have been published (Böhme et al., 2012, Josten et al., 2013, Lasch et al., 2014, Wolters et al., 2011). All of these studies used methods involving protein extraction which is time-consuming and labour-intensive, making them less useful as part of laboratory routines. None of the previous assays included internal quality control of the spectra obtained.
We present a simple and rapid MALDI-TOF MS-based method that is suitable for use as a daily routine for first-line discrimination of MRSA strains in a routine laboratory.
Section snippets
Study design
Retrospective and prospective study material comprising clinical MRSA isolates recovered from various clinical specimens from in- and out-patients in our uptake area during the period 2009–2015 was included (N = 600).
In the retrospective part of the study, mass spectra were obtained from 426 randomly selected representatives of frozen strains kept in a local retrospective collection representing clinical isolates from January 2009 to December 2013. An observer analysed these spectra in order to
MALDI-TOF peaks and groups
We identified 17 peak clusters, including 17 standard peaks and 26 marker peaks (Table 1, Table 2). MALDI-TOF groups were divided into three main groups (A–C) based on the very prominent peak cluster (5032/5027/5002) and isolates with the s-peak at m/z 5032 were assigned to an A group. Most isolates belonged to the A group, including all CCs except CC22. Subdivision of the A group into 22 subgroups was based on the pattern of s- and m-peaks of 14 other peak clusters.
Spectra obtained from each
Discussion
We present data on mass spectra from 600 clinical MRSA isolates, representing 16 different clonal complexes including CC1, CC5, CC8, CC12, CC22, CC30, CC45, CC59, CC72, CC80, CC88, CC97, CC130, ST152/377, CC398 and CC509, and 89 different spa types.
We identified and included 43 discriminatory peaks in our grouping scheme, forming 26 different MALDI-TOF groups. As far as we know, 27 of 43 peaks have never previously been described as useful for the purposes of discrimination, whereas we
Acknowledgements
We thank Anders Rhod Larsen, The Staphylococcal Laboratory at Statens Serum Institut, Copenhagen, for routine spa typing of all isolates, and laboratory technician Mette Skov Laursen for conducting much of the laboratory work in the prospective part of the study.
No external financial support was received.
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Current address: Department of Clinical Microbiology, Odense University Hospital, Denmark.