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Quantitative matrix-assisted laser desorption ionization-time of flight mass spectrometry for rapid resistance detection.

ABSTRACT: Antibiotic resistance in Gram-negative microorganisms is an increasing health care problem. The rapid detection of such resistance is crucial for starting an early specific therapy and to enable initiation of the required hygiene measures. With continued emphasis on reducing the cost of laboratory testing, only economical/low-cost approaches have a chance of being implemented. During recent years, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been developed to be a standard method in microbiology laboratories for the rapid and cost-efficient identification of microorganisms. Extending the usage of MALDI-TOF MS in the clinical microbiology laboratory to the area of resistance testing is an attractive option. Quantitative MALDI-TOF MS using an internal standard facilitates the measurement of the quantity of peptides and small proteins within a spectrum. These quantities correlate to the number of microorganisms and therefore to the growth of a microorganism. The comparison of growth in the presence or absence of an antibiotic allows for analysis of the susceptibility behavior of a strain. Here, we describe a novel method and its application in the analysis of 108 Klebsiella sp. isolates. After 1 h of incubation at a meropenem concentration of 8 ?g/ml, a sensitivity of 97.3% and a specificity of 93.5% were achieved (compared to Etest results).

SUBMITTER: Lange C

PROVIDER: S-EPMC4313279 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Quantitative matrix-assisted laser desorption ionization-time of flight mass spectrometry for rapid resistance detection.

Lange Christoph C Schubert Sören S Jung Jette J Kostrzewa Markus M Sparbier Katrin K

Journal of clinical microbiology 20140917 12

Antibiotic resistance in Gram-negative microorganisms is an increasing health care problem. The rapid detection of such resistance is crucial for starting an early specific therapy and to enable initiation of the required hygiene measures. With continued emphasis on reducing the cost of laboratory testing, only economical/low-cost approaches have a chance of being implemented. During recent years, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has bee ...[more]

PMID: 25232164

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Project description:BACKGROUND:Accurate and rapid identification of dipteran vectors is integral for entomological surveys and is a vital component of control programs for mosquito-borne diseases. Conventionally, morphological features are used for mosquito identification, which suffer from biological and geographical variations and lack of standardization. We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for protein profiling of mosquito species from North India with the aim of creating a MALDI-TOF MS database and evaluating it. METHODS:Mosquito larvae were collected from different rural and urban areas and reared to adult stages. The adult mosquitoes of four medically important genera, Anopheles, Aedes, Culex and Armigerus, were morphologically identified to the species level and confirmed by ITS2-specific PCR sequencing. The cephalothoraces of the adult specimens were subjected to MALDI-TOF analysis and the signature peak spectra were selected for creation of database, which was then evaluated to identify 60 blinded mosquito specimens. RESULTS:Reproducible MALDI-TOF MS spectra spanning over 2-14 kDa m/z range were produced for nine mosquito species: Anopheles (An. stephensi, An. culicifacies and An. annularis); Aedes (Ae. aegypti and Ae. albopictus); Culex (Cx. quinquefasciatus, Cx. vishnui and Cx. tritaenorhynchus); and Armigerus (Ar. subalbatus). Genus- and species-specific peaks were identified to create the database and a score of > 1.8 was used to denote reliable identification. The average numbers of peaks obtained were 55-60 for Anopheles, 80-100 for Aedes, 30-60 for Culex and 45-50 peaks for Armigeres species. Of the 60 coded samples, 58 (96.67%) were correctly identified by MALDI-TOF MS with a score > 1.8, while there were two unreliable identifications (both Cx. quinquefasciatus with scores < 1.8). CONCLUSIONS:MALDI-TOF MS appears to be a pragmatic technique for accurate and rapid identification of mosquito species. The database needs to be expanded to include species from different geographical regions and also different life-cycle stages to fully harness the technique for entomological surveillance programs.

Project description:Streptococcus suis, particularly S. suis serotype 2 (SS2), is an important zoonotic pathogen causing meningitis in humans worldwide. Although the proper classification of the causative and pathogenic serotype is salutary for the clinical diagnosis, cross-reactions leading to the indistinguishability of serotypes by the current serotyping methods are significant limitations. In the present study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of extracted peptides was developed to improve the classification of serotype of S. suis. The peptide mass fingerprint (PMFs) database of S. suis was generated from the whole-cell peptides of 32 reference strains of S. suis isolates obtained from pigs. Thirty-two human S. suis isolates from clinical cases in Thailand were used to validate this alternative serotyping method in direct comparison to the multiplex (m)PCR approach. All reference strains, representing 32 serotypes of S. suis, exhibited their individual PMFs patterns, thus allowing differentiation from one another. Highly pathogenic SS2 and SS14 were clearly differentiated from the otherwise serologically closely related SS1/2 and SS1, respectively. The developed MALDI-TOF-MS serotyping method correctly classified the serotype in 68.8% (22/32) of the same serotype isolates generated from the PMFs database; while the validity for the clinical human isolates was 62.5% (20/32). The agreement between the MALDI-TOF-MS and mPCR serotyping was moderate with a Kappa score of 0.522, considering that mPCR could correctly serotype up to 75%. The present study demonstrated that PMFs from the developed MALDI-TOF-MS-based method could successfully discriminate the previously indistinguishable highly pathogenic SS2 and SS14 from SS1/2 and SS1, respectively. Moreover, this serotyping method distinguished pathogenic SS6, and so is an alternative approach of choice to rapidly and reliably serotype clinically pathogenic S. suis isolates.

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Quantitative matrix-assisted laser desorption ionization-time of flight mass spectrometry for rapid resistance detection.

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Quantitative matrix-assisted laser desorption ionization-time of flight mass spectrometry for rapid resistance detection.

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