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

Antibiotics ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 639 ◽  
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.

Investigation of High-Risk ST131 Clone in Extended Spectrum β-Lactamase–Producing Escherichia coli Isolates in Children

2021 ◽  
Author(s):  
Mehmet E. Bulut ◽  
Gülen Hürkal ◽  
Nazan Dalgıç
Keyword(s):  
Escherichia Coli ◽  
High Risk ◽  
Pediatric Patients ◽  
Pediatric Population ◽  
Maldi Tof Ms ◽  
E Coli ◽  
Maldi Tof ◽  
Extended Spectrum ◽  
St131 Clone ◽  
Tof Ms

Abstract Objective Antimicrobial resistance poses a serious threat to children's health. In recent years, high-risk Escherichia coli ST131 has become an important target for global surveillance studies. The E.coli ST131 clone is associated with extended spectrum β-lactamase (ESBL) production, as well as multidrug resistance and treatment failure. Studies on this clone in the pediatric age group are limited. We aim to investigate the rate of high-risk E. coli ST131 clone in ESBL-positive E. coli isolates obtained from pediatric patients. Methods A total of 292 ESBL-positive E. coli isolates from clinical samples of pediatric patients was included in the study. MALDI-TOF MS system was used for bacterial identification. Susceptibility tests were performed using BD Phoenix automated system. ST131 detection was done by MALDI-TOF-MS. Fisher's exact test was used to compare the groups (significance <0.05). Results A total of 292 isolates was analyzed. The high-risk ST131 clone was detected in 117 (40%) of the 292 ESBL-positive isolates. ST131 rates were found to be significantly higher in children under the age of 5 years compared with children over the age of 5 years (49.3 vs. 31.1%, p = 0.0019). Ciprofloxacin resistance was higher in ST131 isolates (45.6 vs. 31.7%; p < 0.05). Conclusion The rate of the ST131 clone was found to be high in the pediatric population. The significantly high rate of resistance to ciprofloxacin, which is not commonly used in the pediatric population, in ST131 isolates reveals the importance of the spread of high-risk clones for the development of resistance.

scholarly journals 2155. Accelerated Confirmation of Porin Loss in Carbapenem-Resistant Enterobacterales: A MALDI-TOF Mass Spectrometry-Based Approach

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S731-S731
Author(s):  
Carlos Correa-Martinez ◽  
Evgeny A Idelevich ◽  
Karsten Becker
Keyword(s):  
Mass Spectrometry ◽  
Molecular Weight ◽  
Gold Standard ◽  
Sodium Dodecylsulfate ◽  
Resistant Bacteria ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Carbapenem Resistant ◽  
Sds Page ◽  
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.

scholarly journals Detection of Carbapenemase-Producing Enterobacteriaceae in the Baltic Countries and St. Petersburg Area

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Anastasia Pavelkovich ◽  
Arta Balode ◽  
Petra Edquist ◽  
Svetlana Egorova ◽  
Marina Ivanova ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cost Effective ◽  
Maldi Tof Ms ◽  
E Coli ◽  
Maldi Tof ◽  
Baltic Countries ◽  
Exact Data ◽  
Cost Effective Method ◽  
The Baltic ◽  
Tof Ms

The spread of carbapenemase-producing Enterobacteriaceae is a global problem; however, no exact data on the epidemiology of carbapenemase in the Baltic countries and St. Petersburg area is available. We aimed to evaluate the epidemiology of carbapenemase-producingEscherichia coliandKlebsiella pneumoniaein the Baltic States and St. Petersburg, Russia, and to compare the different methods for carbapenemase detection. From January to May 2012, allK. pneumoniae  n=1983andE. coli  n=7774clinical isolates from 20 institutions in Estonia, Latvia, Lithuania, and St. Petersburg, Russia were screened for carbapenem susceptibility. The IMP, VIM, GIM, NDM, KPC, and OXA-48 genes were detected using real-time PCR and the ability to hydrolyze ertapenem was determined using MALDI-TOF MS. Seventy-seven strains were found to be carbapenem nonsusceptible. From these, 15K. pneumoniaestrains hydrolyzed ertapenem and carried theblaNDMgene. All of these strains carried integron 1 and most carried integron 3 as well as genes of the CTX-M-1 group. No carbapenemase-producingE. coliorK. pneumoniaestrains were found in Estonia, Latvia, or Lithuania; however, NDM-positiveK. pneumoniaewas present in the hospital in St. Petersburg, Russia. A MALDI-TOF MS-based assay is a suitable and cost-effective method for the initial confirmation of carbapenemase production.

scholarly journals Mass Spectrometry Proteotyping-Based Detection and Identification of Staphylococcus aureus, Escherichia coli, and Candida albicans in Blood

2021 ◽  
Vol 11 ◽  
Author(s):  
Nahid Kondori ◽  
Amra Kurtovic ◽  
Beatriz Piñeiro-Iglesias ◽  
Francisco Salvà-Serra ◽  
Daniel Jaén-Luchoro ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Candida Albicans ◽  
Blood Cultures ◽  
Blood Samples ◽  
Maldi Tof Ms ◽  
E Coli ◽  
Maldi Tof ◽  
Detection And Identification ◽  
Tof Ms

Bloodstream infections (BSIs), the presence of microorganisms in blood, are potentially serious conditions that can quickly develop into sepsis and life-threatening situations. When assessing proper treatment, rapid diagnosis is the key; besides clinical judgement performed by attending physicians, supporting microbiological tests typically are performed, often requiring microbial isolation and culturing steps, which increases the time required for confirming positive cases of BSI. The additional waiting time forces physicians to prescribe broad-spectrum antibiotics and empirically based treatments, before determining the precise cause of the disease. Thus, alternative and more rapid cultivation-independent methods are needed to improve clinical diagnostics, supporting prompt and accurate treatment and reducing the development of antibiotic resistance. In this study, a culture-independent workflow for pathogen detection and identification in blood samples was developed, using peptide biomarkers and applying bottom-up proteomics analyses, i.e., so-called “proteotyping”. To demonstrate the feasibility of detection of blood infectious pathogens, using proteotyping, Escherichia coli and Staphylococcus aureus were included in the study, as the most prominent bacterial causes of bacteremia and sepsis, as well as Candida albicans, one of the most prominent causes of fungemia. Model systems including spiked negative blood samples, as well as positive blood cultures, without further culturing steps, were investigated. Furthermore, an experiment designed to determine the incubation time needed for correct identification of the infectious pathogens in blood cultures was performed. The results for the spiked negative blood samples showed that proteotyping was 100- to 1,000-fold more sensitive, in comparison with the MALDI-TOF MS-based approach. Furthermore, in the analyses of ten positive blood cultures each of E. coli and S. aureus, both the MALDI-TOF MS-based and proteotyping approaches were successful in the identification of E. coli, although only proteotyping could identify S. aureus correctly in all samples. Compared with the MALDI-TOF MS-based approaches, shotgun proteotyping demonstrated higher sensitivity and accuracy, and required significantly shorter incubation time before detection and identification of the correct pathogen could be accomplished.

scholarly journals Analysis of the Degradation of Broad-Spectrum Cephalosporins by OXA-48-Producing Enterobacteriaceae Using MALDI-TOF MS

2019 ◽  
Vol 7 (12) ◽  
pp. 614
Author(s):  
Marina Oviaño ◽  
María Rosario Rodicio ◽  
Jürgen J. Heinisch ◽  
Rosaura Rodicio ◽  
Germán Bou ◽  
...  
Keyword(s):  
Broad Spectrum ◽  
Negative Control ◽  
Point Of View ◽  
Reaction Products ◽  
Beta Lactam ◽  
Maldi Tof Ms ◽  
E Coli ◽  
Maldi Tof ◽  
Lactam Ring ◽  
Tof Ms

The objective of the study was to evaluate the activity of OXA-48 against different broad-spectrum cephalosporins and to identify the reaction products by MALDI-TOF MS. The action of OXA-48 on cefotaxime, ceftazidime, and ceftriaxone was assessed by this method, using an Escherichia coli J53 transconjugant carrying only the ~62 Kb IncL plasmid containing the blaOXA-48 gene, and the same strain without any plasmid was included as a negative control. In addition, a collection of 17 clinical OXA-48-producing Enterobacteriaceae, which were susceptible to broad-spectrum cephalosporins, was evaluated. MALDI-TOF MS-based analysis of the E. coli transconjugant carrying the blaOXA-48-harboring plasmid, and also the clinical isolates, showed degradation of cefotaxime into two inactive compounds—decarboxylated and deacetylated cefotaxime (~370 Da) and deacetyl cefotaxime (~414 Da), both with the hydrolyzed beta-lactam ring. Reaction products were not obtained when the experiment was performed with ceftriaxone or ceftazidime. From a clinical point of view, our study supports the idea that the efficacy of cefotaxime against OXA-48-producing Enterobacteriaceae is doubtful, in contrast to ceftazidime and ceftriaxone which could be valid choices for treating infections caused by these bacteria. However, further clinical studies confirming this hypothesis are required.

Comparative Proteomics of Commensal and Pathogenic Strains of Escherichia coli

2020 ◽  
Vol 27 (11) ◽  
pp. 1171-1177
Author(s):  
Neelja Singhal ◽  
Divakar Sharma ◽  
Manish Kumar ◽  
Deepa Bisht ◽  
Jugsharan Singh Virdi
Keyword(s):  
Escherichia Coli ◽  
Proteomic Analysis ◽  
Two Dimensional Gel Electrophoresis ◽  
Maldi Tof Ms ◽  
E Coli ◽  
Comparative Proteomic Analysis ◽  
Maldi Tof ◽  
Northern India ◽  
Flight Mass Spectrometry ◽  
Tof Ms

Background: Most of the proteomic studies in Escherichia coli have focussed on pathogenic strains, while very few studies have studied the commensal strains. It is important to study the commensal strains because under the selective pressure of their habitat, commensal strains might serve as reservoirs of virulent and pathogenic strains. Objective: In this study, we have performed a comparative proteomic analysis of commensal and pathogenic strains of E. coli isolated from a major river flowing through northern India. Methods: Proteins were resolved by two dimensional gel electrophoresis and the differentially expressed proteins were identified using matrix-assisted laser desorption ionization-time of flight mass-spectrometry (MALDI-TOF MS). Results: Many proteins of the commensal strain showed an increased expression compared to the pathogenic strain, of which seventeen proteins were identified by MALDI-TOF MS. Functional classification of these proteins revealed that they belonged to different functional pathways like energy metabolism, nucleotide and nucleoside conversions, translation, biosynthesis of amino acids and motility and energytaxis/chemotaxis. Conclusion: As per the best of our knowledge, this is the first report on comparative proteomic analysis of E. coli commensal and pathogenic strains of aquatic origin. Our results suggest that the increased production of these proteins might play an important role in adaptation of E. coli to a commensal/pathogenic lifestyle. However, further experiments are required to understand the precise role of these proteins in regulating the pathogenicity/commensalism of E. coli.

scholarly journals Rapid, Sensitive, and Specific Escherichia coli H Antigen Typing by Matrix-Assisted Laser Desorption Ionization–Time of Flight-Based Peptide Mass Fingerprinting

2015 ◽  
Vol 53 (8) ◽  
pp. 2480-2485 ◽  
Author(s):  
Huixia Chui ◽  
Michael Chan ◽  
Drexler Hernandez ◽  
Patrick Chong ◽  
Stuart McCorrister ◽  
...  
Keyword(s):  
Peptide Mass Fingerprinting ◽  
Laser Desorption Ionization ◽  
Ionization Time ◽  
Content Type ◽  
Maldi Tof Ms ◽  
E Coli ◽  
Maldi Tof ◽  
Peptide Mass ◽  
Reference Strains ◽  
Tof Ms

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has gained popularity in recent years for rapid bacterial identification, mostly at the genus or species level. In this study, a rapid method to identify the Escherichia coli flagellar antigen (H antigen) at the subspecies level was developed using a MALDI-TOF MS platform with high specificity and sensitivity. Flagella were trapped on a filter membrane, and on-filter trypsin digestion was performed. The tryptic digests of each flagellin then were collected and analyzed by MALDI-TOF MS through peptide mass fingerprinting. Sixty-one reference strains containing all 53 H types and 85 clinical strains were tested and compared to serotyping designations. Whole-genome sequencing was used to resolve conflicting results between the two methods. It was found that DHB (2,5-dihydroxybenzoic acid) worked better than CHCA (α-cyano-4-hydroxycinnamic acid) as the matrix for MALDI-TOF MS, with higher confidence during protein identification. After method optimization, reference strains representing all 53 E. coli H types were identified correctly by MALDI-TOF MS. A custom E. coli flagellar/H antigen database was crucial for clearly identifying the E. coli H antigens. Of 85 clinical isolates tested by MALDI-TOF MS-H, 75 identified MS-H types (88.2%) matched results obtained from traditional serotyping. Among 10 isolates where the results of MALDI-TOF MS-H and serotyping did not agree, 60% of H types characterized by whole-genome sequencing agreed with those identified by MALDI-TOF MS-H, compared to only 20% by serotyping. This MALDI-TOF MS-H platform can be used for rapid and cost-effective E. coli H antigen identification, especially during E. coli outbreaks.

scholarly journals MALDI-TOF MS: the proteomic approach, the future of fungal identification in India

2020 ◽  
Vol 8 (7) ◽  
pp. 2575
Author(s):  
Mallika Fonseca ◽  
Sarala Menon ◽  
Praveen Rahi ◽  
Prachi Patekar ◽  
Abhay S. Chowdhary
Keyword(s):  
Yeast Species ◽  
Fungal Infections ◽  
Tertiary Care ◽  
Turnaround Time ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Flight Mass Spectrometry ◽  
Proteomic Approach ◽  
Conventional Methods ◽  
Tof Ms

Background: India, being a country where fungal infections are rampant, is urgently in need of effective tools for early and accurate diagnosis of fungal infections. Matrix-assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) is a recent method which has shown potential in identifying clinically important bacterial pathogens as well as clinically important fungi. The main objective of this study was to compare the utility of MALDI-TOF MS for the identification of fungi against that of conventional methods.Methods: The project was carried out in a tertiary care government hospital in India. Fifty clinical isolates comprising mainly various yeast species were subjected to conventional identification (Phenotypic) as well as MALDI-TOF-MS. Their results were further compared.Results: MALDI-TOF MS showed a high concordance with conventional methods while identifying species like C. albicans, C. tropicalis, C. parapsilosis and C. neoformans, although the concordance for species such as Rhodotorula and Trichosporon could only be matched up to genus level.Conclusions: MALDI-TOF MS-based identification is both a rapid and a viable tool for identification of clinically relevant yeast species with good correlation to conventional methods and a quick turnaround time.

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

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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