High throughput identification of clinical isolates of Staphylococcus aureus using MALDI-TOF-MS of intact cells

Rajakaruna, Lakshani; Hallas, Gillian; Molenaar, Linda; Dare, Diane; Sutton, Helen; Encheva, Vesela; Culak, Renata; Innes, Ingrid; Ball, Graham; Sefton, Armine; Eydmann, Melvin; Kearns, Angela; Shah, Haroun N.

doi:10.1016/j.meegid.2009.01.012

Cited by 60 publications

(46 citation statements)

References 21 publications

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“…In cell extracts, PSM␣2 (m/z 2,307) and PSM␣3 (m/z 2,636) were predominant. The delta-toxin at m/z 3,007 (formylated version) is one of the peaks that was frequently discovered in the early attempts at mass spectrometry of intact S. aureus cells (7,9,10,21). Most useful for typing was the fourth group of signals, which comprised the stress or hypothetical proteins that are present in multiple isoforms.…”

Section: Resultsmentioning

confidence: 99%

Analysis of the Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrum of Staphylococcus aureus Identifies Mutations That Allow Differentiation of the Main Clonal Lineages

et al. 2013

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Staphylococcus aureus causes a wide range of hospital infections. Often, these infections involve epidemic methicillin-resistant S. aureus (MRSA) strains that are transferred by health care workers to patients. In order to detect outbreaks that are caused by epidemic strains, the clinical isolates have to be typed. Multilocus sequence typing (MLST) relies on the sequence analysis of housekeeping genes and is used to allocate the strains to sequence types (ST), which can be grouped into clonal complexes (CC). This method has provided a detailed insight into the population structure of MRSA and methicillin-susceptible S. aureus (MSSA) strains (1). Finer discrimination is achieved by pulsed-field gel electrophoresis (PFGE) and spa typing (2). All of these methods need additional experimentation.In matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), different spectra or signatures of cell extracts (3, 4, 5, 6) or whole cells (7,8,9,10,11,12) could be identified for different strains or groups of strains. An increasing number of laboratories use MALDI-TOF MS for the identification of S. aureus. However, the differences in the signatures of the strains have not been evaluated so far, because the discriminatory threshold of the software used in clinical settings is set up to assign the isolate to a species. To this end, more subtle differences are ignored. Another reason is that, so far, MALDI-TOF MS of whole bacterial cells has been employed in a heuristic manner, and for most species, the identities of the compounds that are detected in the measurements are unknown. Thus, the spectra are not well understood, and the variations in the signatures cannot be interpreted.In principle, two spectra might differ by signal intensity, loss of a signal, or by the shift of a signal. Variations in signal intensity are probably caused by expression differences, which might be directly correlated with culture conditions and, therefore, do not give unambiguous information about a genotype of the strain. The loss of a signal is caused by the total failure to express a protein or peptide, which in turn might indicate a mutation causing a frameshift or stop codon, but might also depend on culture conditions, mutations of regulatory factors, or sample preparation. Thus, there is no clear-cut correlation between the loss of a signal and a genotype. In contrast, peak shifts (i.e., loss of a signal coupled to the appearance of a new signal, both of which are correlated to the same peptide) correspond to point mutations in the genes of the peptides detected in the analysis; the mutation leads to an amino acid exchange that alters the molecular weight of the corresponding gene product.In order to characterize the clonal lineages of S. aureus in the MALDI-TOF MS, this work was aimed at the identification of the peptides that are detected in the spectra and that show mass variations between the clonal complexes of S. aureus. To this end, we analyzed the spectra of 401 S. aureus strains, concentrating on...

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Section: Resultsmentioning

confidence: 99%

Analysis of the Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrum of Staphylococcus aureus Identifies Mutations That Allow Differentiation of the Main Clonal Lineages

et al. 2013

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“…This previous lack of identification was compounded by the high cost and time required for phenotypic tests, as well as the usual poor clinical significance of a majority of CoNS isolated from clinical specimens. Conversely, mass spectrometry is notably able to more accurately carry out S. aureus identification than when using slide or tube coagulase tests, and it is faster and cheaper than any current automated system using phenotypic tests (20,21).…”

Section: Discussionmentioning

confidence: 99%

Implementation of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry in Routine Clinical Laboratories Improves Identification of Coagulase-Negative Staphylococci and Reveals the Pathogenic Role of Staphylococcus lugdunensis

et al. 2015

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The use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for staphylococcal identification is now considered routine in laboratories compared with the conventional phenotypical methods previously used. We verified its microbiological relevance for identifying the main species of coagulase-negative staphylococci (CoNS) by randomly selecting 50 isolates. From 1 January 2007 to 31 August 2008, 12,479 staphylococci were isolated with phenotypic methods, of which 4,594 were identified as Staphylococcus aureus and 7,885 were coagulase negative staphylococci. Using MALDI-TOF MS from 1 January 2011 to 31 August 2012, 14,913 staphylococci were identified, with 5,066 as S. aureus and 9,847 as CoNS. MALDI-TOF MS allowed the identification of approximately 85% of the CoNS strains, whereas only 14% of the CoNS strains were identified to the species level with phenotypic methods because they were often considered contaminants. Furthermore, the use of MALDI-TOF MS revealed the occurrence of recently characterized Staphylococcus species, such as S. pettenkoferi, S. condimenti, and S. piscifermentans. Microbiological relevance analysis further revealed that some species displayed a high rate of microbiological significance, i.e., 40% of the S. lugdunensis strains included in the analysis were associated with infection risk. This retrospective microbiological study confirms the role of MALDI-TOF MS in clinical settings for the identification of staphylococci with clinical consequences. The species distribution reveals the occurrence of the recently identified species S. pettenkoferi and putative virulent species, including S. lugdunensis. C oagulase-negative staphylococci (CoNS) are normal inhabitants of the human skin that lately have revealed themselves as significant etiological agents causing nosocomial infections, particularly in medical devices (1-4). Until recently, the phenotypic identification of Ͼ40 currently recognized CoNS species remained a challenge, because the technique was labor-intensive and uncommon CoNS were usually not identified (5, 6). Until recently, CoNS identification at the species level was performed only for clinical isolates that were considered clinically significant according to clinical or microbiological criteria. Meanwhile, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a reliable, fast, and cost-effective alternative for bacterial species identification and is now widely used in microbiology laboratories (7,8). This method allows the identification of all staphylococci isolated in the clinical setting, thus rendering the use of well-known tests, such as free or bound coagulase tests, unnecessary. Previous reports have shown that MALDI-TOF MS is a reliable method to identify Staphylococcus at the species level; however, questions regarding the impact of such methods in clinical settings remain uncertain (9). Staphylococcus epidermidis is probably the major CoNS species causing nosocomial inf...

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“…Quality-controlled databases of reference spectra including many relevant micro-organisms were established from the first 'proof of principle' experiments to integrate this method into clinical laboratories. To date, several commercial databases and non-commercial approaches for bacterial identification have been evaluated (Carbonnelle et al, 2007;Friedrichs et al, 2007;Rajakaruna et al, 2009;Seng et al, 2009). Nevertheless, these experiments have varied, particularly with regard to the sample preparation protocols: either formic acid extraction or direct smear preparation.…”

Section: Introductionmentioning

confidence: 99%

“…Nevertheless, these experiments have varied, particularly with regard to the sample preparation protocols: either formic acid extraction or direct smear preparation. Moreover, the samples were measured with different mass spectrometer instruments and the spectra generated were compared with varying ranges of proteomic fingerprints (Friedrichs et al, 2007;Rajakaruna et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

Evaluation of species-specific score cut-off values for various Staphylococcus species using a MALDI Biotyper-based identification

Richter

Hollstein

Woloszyn

et al. 2012

Journal of Medical Microbiology

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Although previous studies investigating the MALDI Biotyper database (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based identification) have proven its high accuracy for bacterial identification, the studies differed in sample preparation, number of replicates, quantity of shots and target types used. In particular, the score cut-off values of special importance for reliable species identification varied. The aim of the present study was to identify species-specific differences in the mean score values for staphylococci. Cut-off values recommended by the manufacturer were adapted using the 20th percentile to rule out unknown score-modifying factors, even though the specificity was high and the lowest cut-off values would also yield an accurate result. Whilst correct species diagnosis was obtained in 97.32 % of samples (1382/1420), only 220 of all duplicates (15.49 %) revealed a score of ¢2.3, whilst 968 (68.17 %) had a score between 2.0 and 2.299, and 194 (13.66 %) had a score of ,2.0. Ten of 21 species had a calculated 20th percentile of ,2.0 and one species of ,1.7. In conclusion, the use of species-specific cut-off values improves the relative sensitivity of species identification in staphylococci.

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High throughput identification of clinical isolates of Staphylococcus aureus using MALDI-TOF-MS of intact cells

Cited by 60 publications

References 21 publications

Analysis of the Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrum of Staphylococcus aureus Identifies Mutations That Allow Differentiation of the Main Clonal Lineages

Analysis of the Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrum of Staphylococcus aureus Identifies Mutations That Allow Differentiation of the Main Clonal Lineages

Implementation of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry in Routine Clinical Laboratories Improves Identification of Coagulase-Negative Staphylococci and Reveals the Pathogenic Role of Staphylococcus lugdunensis

Evaluation of species-specific score cut-off values for various Staphylococcus species using a MALDI Biotyper-based identification

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