Heat-resistant bacteria contamination investigation in Chinese soybean curd industrial processing using high-throughput gene sequencing and MALDI-TOF-MS

Abstract Background The accurate identification of carbapenem resistance mechanisms is decisive for the appropriate selection of antibiotic regimens. Numerous methods can detect carbapenemase-producing carbapenem-resistant bacteria (CPCR). However, non-CPCR (NCPCR) are routinely assumed to display porin loss as a diagnosis of exclusion. No further confirmatory tests are performed since the gold standard (sodium dodecylsulfate polyacrylamide gel electrophoresis, SDS–PAGE) is laborious and time consuming. We propose a test for rapid and easy detection of porin loss by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Methods Clinical meropenem-resistant Enterobacterales strains (10 CPCR, 10 NCPCR) and control strains recommended by EUCAST (5 carbapenemase-producing, one with porin loss, one-negative control) were analyzed. Membrane proteins were extracted by successive centrifugation of bacterial suspensions (McFarland 0.5) and addition of ethanol, formic acid and acetonitrile. MALDI-TOF MS of the protein extracts was performed on a 96-spot target (Bruker Daltonics, Germany). Peaks between 35 and 40 kDa were analyzed for the presence of porins and compared with the bands observed in the SDS–PAGE of the protein extracts. Results Within the molecular weight range of 35–40 kDa, the MALDI-TOF MS-based method revealed peaks in all CPCR isolates corresponding to those observed in the carbapenemase-producing control strains. In contrast, the control strain with porin loss as well as all CNCR isolates showed a lower quantity of peaks in this range. All peaks observed correlated with the bands observed in the SDS–PAGE of the protein extracts at the corresponding molecular weight (Figure 1). Conclusion Yielding results that reliably correspond to the current gold standard, we propose a method for accelerated detection of porin loss as an alternative to the diagnosis of exclusion usually made in routine settings. With a processing time of approximately 20 minutes, the method can be easily implemented in the clinical setting. Applying this MALDI-TOF MS-based approach, valuable information will be provided about a resistance mechanism that otherwise remains unexplained. Disclosures All authors: No reported disclosures.

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16S rDNA high-throughput sequencing and MALDI-TOF MS are complementary when studying psychrotrophic bacterial diversity of raw cows' milk

International Dairy Journal ◽

10.1016/j.idairyj.2019.06.001 ◽

2019 ◽

Vol 97 ◽

pp. 86-91 ◽

Cited By ~ 12

Author(s):

Dong Zhang ◽

Jon Palmer ◽

Koon Hoong Teh ◽

Patrick Biggs ◽

Steve Flint

Keyword(s):

Bacterial Diversity ◽

16S Rdna ◽

High Throughput ◽

High Throughput Sequencing ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Tof Ms

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Determination of Drug Efflux Pump Efficiency in Drug-Resistant Bacteria Using MALDI-TOF MS

Antibiotics ◽

10.3390/antibiotics9100639 ◽

2020 ◽

Vol 9 (10) ◽

pp. 639 ◽

Cited By ~ 1

Author(s):

Wen-Jung Lu ◽

Hsuan-Ju Lin ◽

Pang-Hung Hsu ◽

Hong-Ting Victor Lin

Keyword(s):

Minimum Inhibitory Concentration ◽

Inhibitory Concentration ◽

Resistant Bacteria ◽

Pump Efficiency ◽

Drug Efflux ◽

Maldi Tof Ms ◽

E Coli ◽

Maldi Tof ◽

Conventional Methods ◽

Tof Ms

Multidrug efflux pumps play an essential role in antibiotic resistance. The conventional methods, including minimum inhibitory concentration and fluorescent assays, to monitor transporter efflux activity might have some drawbacks, such as indirect evidence or interference from color molecules. In this study, MALDI-TOF MS use was explored for monitoring drug efflux by a multidrug transporter, and the results were compared for validation with the data from conventional methods. Minimum inhibitory concentration was used first to evaluate the activity of Escherichia coli drug transporter AcrB, and this analysis showed that the E. coli overexpressing AcrB exhibited elevated resistance to various antibiotics and dyes. Fluorescence-based studies indicated that AcrB in E. coli could decrease the accumulation of intracellular dyes and display various efflux rate constants for different dyes, suggesting AcrB’s efflux activity. The MALDI-TOF MS analysis parameters were optimized to maintain a detection accuracy for AcrB’s substrates; furthermore, the MS data showed that E. coli overexpressing AcrB led to increased ions abundancy of various dyes and drugs in the extracellular space at different rates over time, illustrating continuous substrate efflux by AcrB. This study concluded that MALDI-TOF MS is a reliable method that can rapidly determine the drug pump efflux activity for various substrates.

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High-throughput identification of the microbial biodiversity of cocoa bean fermentation by MALDI-TOF MS

Letters in Applied Microbiology ◽

10.1111/lam.12621 ◽

2016 ◽

Vol 63 (5) ◽

pp. 347-355 ◽

Cited By ~ 16

Author(s):

S. Miescher Schwenninger ◽

S. Freimüller Leischtfeld ◽

C. Gantenbein-Demarchi

Keyword(s):

High Throughput ◽

Cocoa Bean ◽

Microbial Biodiversity ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Cocoa Bean Fermentation ◽

Tof Ms

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Development of an Optimized MALDI-TOF-MS Method for High-Throughput Identification of High-Molecular-Weight Glutenin Subunits in Wheat

Molecules ◽

10.3390/molecules25184347 ◽

2020 ◽

Vol 25 (18) ◽

pp. 4347

Author(s):

You-Ran Jang ◽

Kyoungwon Cho ◽

Se Won Kim ◽

Susan B. Altenbach ◽

Sun-Hyung Lim ◽

...

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

High Throughput ◽

Glutenin Subunits ◽

Maldi Tof Ms ◽

Molecular Weights ◽

Maldi Tof ◽

Rp Hplc ◽

Group 2 ◽

Tof Ms

Because high-molecular-weight glutenin subunits (HMW-GS) are important contributors to wheat end-use quality, there is a need for high-throughput identification of HMW-GS in wheat genetic resources and breeding lines. We developed an optimized method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to distinguish individual HMW-GS by considering the effects of the alkylating reagent in protein extraction, solvent components, dissolving volume, and matrix II components. Using the optimized method, 18 of 22 HMW-GS were successfully identified in standard wheat cultivars by differences in molecular weights or by their associations with other tightly linked subunits. Interestingly, 1Bx7 subunits were divided into 1Bx7 group 1 and 1Bx7 group 2 proteins with molecular weights of about 82,400 and 83,000 Da, respectively. Cultivars containing the 1Bx7 group 2 proteins were distinguished from those containing 1Bx7OE using well-known DNA markers. HMW-GS 1Ax2* and 1Bx6 and 1By8 and 1By8*, which are difficult to distinguish due to very similar molecular weights, were easily identified using RP-HPLC. To validate the method, HMW-GS from 38 Korean wheat varieties previously evaluated by SDS-PAGE combined with RP-HPLC were analyzed by MALDI-TOF-MS. The optimized MALDI-TOF-MS method will be a rapid, high-throughput tool for selecting lines containing desirable HMW-GS for breeding efforts.

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