Search of Streptomycin-Resistant Bacteria in Creek Water and Application of MALDI-TOF MS to Grouping of the Isolated Bacteria

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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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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An improved MALDI-TOF MS data analysis pipeline for the identification of carbapenemase-producing Klebsiella pneumoniae

Journal of Clinical Microbiology ◽

10.1128/jcm.00800-21 ◽

2021 ◽

Author(s):

Eva Gato ◽

Ignacio Pedro Constanso ◽

Ana Candela ◽

Fátima Galán ◽

Bruno Kotska Rodiño-Janeiro ◽

...

Keyword(s):

Data Analysis ◽

Klebsiella Pneumoniae ◽

Operating Procedure ◽

Standard Operating Procedure ◽

Resistant Bacteria ◽

Analysis Pipeline ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Data Analysis Pipeline ◽

Tof Ms

The increasing emergence of carbapenemase-producing Klebsiella pneumoniae (CPK) is a global health alarm. Rapid methods that require minimum sample preparation and rapid data analysis are urgently required. MALDI-TOF MS has recently been used by clinical laboratories for identification of antibiotic resistant bacteria; however, discrepancies have arisen regarding biological and technical issues. The aim of this study was to standardize an operating procedure and data analysis for identification of CPK by MALDI-TOF MS. To evaluate this approach, a series of 162 K. pneumoniae (112 CPK and 50 non CPK), were processed in the MALDI BioTyper system (Bruker Daltonik, Germany) following a standard operating procedure. The study was conducted in two stages, the first denominated the “Reproducibility stage” and the second, “CPK identification”. The first stage was designed to evaluate the biological and technical variation associated with the entire analysis of CPK and the second stage, to assess the final accuracy of MALDI-TOF for the identification of CPK. Therefore, we present an improved MALDI-TOF MS data analysis pipeline using neural network analysis implemented in Clover MS data analysis software (Clover Biosoft, Spain), that is designed to reduce variability, guarantee inter-laboratory reproducibility and maximize the information selected from the bacterial proteome. Using the Random Forest (RF) algorithm, 100% of CPK producing isolates were correctly identified when all the peaks in the spectra were selected as input features and TIC normalization was applied. Thus, we have demonstrated that real-time direct tracking of CPK is possible using MALDI-TOF MS.

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

LWT ◽

10.1016/j.lwt.2021.111618 ◽

2021 ◽

pp. 111618

Author(s):

Lijun Zhao ◽

Lirong Jia ◽

Bingcun Ma ◽

Wei Zhong ◽

Ying Huang ◽

...

Keyword(s):

High Throughput ◽

Gene Sequencing ◽

Resistant Bacteria ◽

Maldi Tof Ms ◽

Industrial Processing ◽

Heat Resistant ◽

Maldi Tof ◽

Soybean Curd ◽

Tof Ms

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MALDI-TOF MS Based Bacterial Antibiotics Resistance Finger Print for Diabetic Pedopathy

Frontiers in Chemistry ◽

10.3389/fchem.2021.785848 ◽

2022 ◽

Vol 9 ◽

Author(s):

Haojie Sun ◽

Peng Lai ◽

Wei Wu ◽

Hao Heng ◽

Shanwen Si ◽

...

Keyword(s):

Drug Resistance ◽

Diabetic Foot ◽

Maldi Ms ◽

Resistant Bacteria ◽

Maldi Tof Ms ◽

Finger Print ◽

Maldi Tof ◽

Diabetic Foot Infections ◽

Ms Analysis ◽

Tof Ms

Diabetes mellitus has become a major global health issue. Currently, the use of antibiotics remains the best foundational strategy in the control of diabetic foot infections. However, the lack of accurate identification of pathogens and the empirical use of antibiotics at early stages of infection represents a non-targeted treatment approach with a poor curative effect that may increase the of bacterial drug resistance. Therefore, the timely identification of drug resistant bacteria is the key to increasing the efficacy of treatments for diabetic foot infections. The traditional identification method is based on bacterial morphology, cell physiology, and biochemistry. Despite the simplicity and low costs associated with this method, it is time-consuming and has limited clinical value, which delays early diagnosis and treatment. In the recent years, MALDI-TOF MS has emerged as a promising new technology in the field of clinical microbial identification. In this study, we developed a strategy for the identification of drug resistance in the diagnosis of diabetic foot infections using a combination of macro-proteomics and MALDI MS analysis. The macro-proteomics result was utilized to determine the differential proteins in the resistance group and the corresponding peptide fragments were used as the finger print in a MALDI MS analysis. This strategy was successfully used in the research of drug resistance in patients with diabetic foot infections and achieved several biomarkers that could be used as a finger print for 4 different drugs, including ceftazidime, piperacillin, levofloxacin, and tetracycline. This method can quickly confirm the drug resistance of clinical diabetic foot infections, which can help aid in the early treatment of patients.

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