MALDI-TOF MS Biomarker Detection Models to Distinguish RTX Toxin Phenotypes of Moraxella bovoculi Strains Are Enhanced Using Calcium Chloride Supplemented Agar

Moraxella bovoculi is the bacterium most often cultured from ocular lesions of cattle with infectious bovine keratoconjunctivitis, also known as bovine pinkeye. Some strains of M. bovoculi contain operons encoding for a repeats-in-toxin (RTX) toxin, which is a known virulence factor of multiple veterinary pathogens. We explored the utility of MALDI-TOF MS and biomarker detection models to classify the presence or absence of an RTX phenotype in M. bovoculi. Ninety strains that had undergone whole genome sequencing were classified by the presence or absence of complete RTX operons and confirmed with a visual assessment of hemolysis on blood agar. Strains were grown on Tryptic Soy Agar (TSA) with 5% sheep blood, TSA with 5% bovine blood that was supplemented with 10% fetal bovine serum, 10 mmol/LCaCl2, or both. The formulations were designed to determine the influence of growth media on toxin production or activity, as calcium ions are required for toxin secretion and activity. Mass spectra were obtained for strains grown on each agar formulation and biomarker models were developed using ClinProTools 3.0 software. The most accurate model was developed using spectra from strains grown on TSA with 5% bovine blood and supplemented with CaCl2, which had a sensitivity and specificity of 93.3% and 73.3%, respectively, regarding RTX phenotype classification. The same biomarker model algorithm developed from strains grown on TSA with 5% sheep blood had a substantially lower sensitivity and specificity of 68.0% and 52.0%, respectively. Our results indicate that MALDI-TOF MS biomarker models can accurately classify strains of M. bovoculi regarding the presence or absence of RTX toxin operons and that agar media modifications improve the accuracy of these models.

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Direct detection of intact Klebsiella pneumoniae carbapenemases produced by Enterobacterales using MALDI-TOF MS

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkaa007 ◽

2020 ◽

Vol 75 (5) ◽

pp. 1174-1181 ◽

Cited By ~ 1

Author(s):

Eun-Jeong Yoon ◽

Eun Hee Lee ◽

Dong Hwi Hwang ◽

Hyukmin Lee ◽

Je-Hyun Baek ◽

...

Keyword(s):

Molecular Mass ◽

Sensitivity And Specificity ◽

Direct Detection ◽

External Validation ◽

Agar Plate ◽

Analytical Sensitivity ◽

Exact Mass ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Tof Ms

Abstract Objectives A MALDI-TOF MS-based identification method for KPC-producing Enterobacterales was developed. Methods The molecular mass of the intact KPC-2 polypeptide was estimated for blaKPC-2 transformants using MALDI Microflex and the exact mass was confirmed by LC and a high-resolution MS/MS system. A total of 1181 clinical Enterobacterales strains, including 369 KPC producers and 812 KPC non-producers, were used to set up the methodology and the results were compared with those from PCR analyses. For external validation, a total of 458 Enterobacterales clinical isolates from a general hospital between December 2018 and April 2019 were used. Results The exact molecular mass of the intact KPC-2 protein was 28 718.13 Da and KPC peaks were observed at m/z 28 708.87–28 728.34 using MALDI Microflex. Most of the KPC-2 (99.1%, 335/338) and KPC-3 (100%, 6/6) producers presented a clear peak via this method, while 12.0% (3/25) of the KPC-4 producers had a peak of weak intensity associated with low levels of gene expression. It took less than 20 min for the entire assay to be performed with colonies on an agar plate. External validation showed that the analytical sensitivity and specificity of the method compared with PCR were 100% (59/59) and 99.50% (397/399), respectively. Conclusions The MALDI-TOF MS-based method for directly detecting the intact KPC protein is applicable to routine tests in clinical microbiology laboratories, supported by its speed, low cost and excellent sensitivity and specificity.

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Porous silicon affinity chips for biomarker detection by MALDI-TOF–MS

Journal of Chromatography B ◽

10.1016/j.jchromb.2008.10.001 ◽

2008 ◽

Vol 875 (2) ◽

pp. 502-508 ◽

Cited By ~ 14

Author(s):

Y CHEN ◽

F BI ◽

S WANG ◽

S XIAO ◽

J LIU

Keyword(s):

Porous Silicon ◽

Biomarker Detection ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Tof Ms

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Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification of Moraxella bovoculi and Moraxella bovis isolates from cattle

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638718789725 ◽

2018 ◽

Vol 30 (5) ◽

pp. 739-742 ◽

Cited By ~ 2

Author(s):

Kara Robbins ◽

Aaron M. Dickey ◽

Michael L. Clawson ◽

John D. Loy

Keyword(s):

Laser Desorption ◽

Laser Desorption Ionization ◽

Ionization Time ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Flight Mass Spectrometry ◽

Moraxella Bovis ◽

Pcr Rflp ◽

Moraxella Bovoculi ◽

Tof Ms

Infectious bovine keratoconjunctivitis (IBK) is an economically significant disease caused by Moraxella bovis. Moraxella bovoculi, although not reported to cause IBK, has been isolated from the eyes of cattle diagnosed with IBK. Identification of M. bovis and M. bovoculi can be performed using biochemical or DNA-based approaches, both of which may be time consuming and inconsistent between laboratories. We conducted a comparative evaluation of M. bovoculi and M. bovis identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with a database provided by Bruker Daltonics (termed the BDAL database), the BDAL database supplemented with spectra generated in our study (termed the UNLVDC database), and with PCR–restriction-fragment length polymorphism (PCR-RFLP) typing. M. bovoculi ( n = 250) and M. bovis ( n = 18) isolates from cattle with or without IBK were used. MALDI-TOF MS using the UNLVDC database correctly identified 250 of 250 (100%) of M. bovoculi and 17 of 18 (94%) of M. bovis isolates. With the BDAL database, MALDI-TOF MS correctly identified 249 of 250 (99%) of M. bovoculi and 7 of 18 (39%) of M. bovis isolates. In comparison, the PCR-RFLP test correctly identified 210 of 250 (84%) of M. bovoculi and 12 of 18 (66%) of M. bovis isolates. Thus, MALDI-TOF MS with the UNLVDC database was the most effective identification methodology for M. bovis and M. bovoculi isolates from cattle.

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