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

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.

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Detection of Colistin Resistance in Escherichia coli by Use of the MALDI Biotyper Sirius Mass Spectrometry System

Journal of Clinical Microbiology ◽

10.1128/jcm.01427-19 ◽

2019 ◽

Vol 57 (12) ◽

Cited By ~ 3

Author(s):

R. Christopher D. Furniss ◽

Laurent Dortet ◽

William Bolland ◽

Oliver Drews ◽

Katrin Sparbier ◽

...

Keyword(s):

Mass Spectrometry ◽

Escherichia Coli ◽

Cost Effective ◽

Colistin Resistance ◽

Content Type ◽

Maldi Tof Ms ◽

E Coli ◽

Maldi Tof ◽

Maldi Biotyper ◽

Tof Ms

ABSTRACT Polymyxin antibiotics are a last-line treatment for multidrug-resistant Gram-negative bacteria. However, the emergence of colistin resistance, including the spread of mobile mcr genes, necessitates the development of improved diagnostics for the detection of colistin-resistant organisms in hospital settings. The recently developed MALDIxin test enables detection of colistin resistance by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) in less than 15 min but is not optimized for the mass spectrometers commonly found in clinical microbiology laboratories. In this study, we adapted the MALDIxin test for the MALDI Biotyper Sirius MALDI-TOF MS system (Bruker Daltonics). We optimized the sample preparation protocol by using a set of 6 mobile colistin resistance (MCR) protein-expressing Escherichia coli clones and validated the assay with a collection of 40 E. coli clinical isolates, including 19 confirmed MCR protein producers, 12 colistin-resistant isolates that tested negative for commonly encountered mcr genes (i.e., likely chromosomally resistant isolates), and 9 polymyxin-susceptible isolates. We calculated polymyxin resistance ratio (PRR) values from the acquired spectra; PRR values of 0, indicating polymyxin susceptibility, were obtained for all colistin-susceptible E. coli isolates, whereas positive PRR values, indicating resistance to polymyxins, were obtained for all resistant strains, independent of the genetic basis of resistance. Thus, we report a preliminary feasibility study showing that an optimized version of the MALDIxin test adapted for the routine MALDI Biotyper Sirius system provides an unbiased, fast, reliable, cost-effective, and high-throughput way of detecting colistin resistance in clinical E. coli isolates.

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

Journal of Pediatric Infectious Diseases ◽

10.1055/s-0041-1730995 ◽

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.

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

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.634215 ◽

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.

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Comparative Proteomics of Commensal and Pathogenic Strains of Escherichia coli

Protein and Peptide Letters ◽

10.2174/0929866527666200517104154 ◽

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.

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Rapid susceptibility testing of multi-drug resistant Escherichia coli and Klebsiella by glucose metabolization monitoring

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2018-1178 ◽

2019 ◽

Vol 57 (8) ◽

pp. 1271-1279 ◽

Cited By ~ 2

Author(s):

Maximilian Kittel ◽

Peter Findeisen ◽

Beniam Ghebremedhin ◽

Thomas Miethke ◽

Alexander Grundt ◽

...

Keyword(s):

Escherichia Coli ◽

Antibiotic Therapy ◽

Susceptibility Testing ◽

Gram Negative Bacteria ◽

Drug Resistant ◽

Gram Negative ◽

Maldi Tof Ms ◽

E Coli ◽

Maldi Tof ◽

Tof Ms

Abstract Background The increasing number of multi-drug resistant (MDR) bacteria provides enormous challenges for choosing an appropriate antibiotic therapy in the early phase of sepsis. While bacterial identification has been greatly accelerated by the introduction of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), the antibiotic susceptibility testing (AST) remains time-consuming. Here, we present a rapid susceptibility testing method for testing Gram-negative bacteria, exemplarily validated for Escherichia coli and Klebsiella spp. Methods Gram-negative isolates (E. coli and Klebsiella spp.) were either taken as single colonies from agar plates (n=136) or directly extracted and identified from positive blood cultures (n=42) using MALDI-TOF MS. Bacteria were incubated in glucose-supplemented Luria broths (LBs) each containing one antibiotic (ceftazidime, piperacillin, imipenem and ciprofloxacin), routinely used to classify Gram-negative bacteria in Germany. To determine susceptibility the dynamics of glucose utilization in bacterial suspensions were quantitatively measured in the presence or absence of antibiotics designated liquid-AST (L-AST). Results The L-AST can be run on clinical-chemistry analyzers and integrated into laboratory routines. It yields critical resistance information within 90–150 min downstream of a MS-based identification. The results showed a high concordance with routine susceptibility testing, with less than 1% very major errors (VME) and 3.51% major errors (ME) for 178 assessed isolates. Analysis of turnaround time (TAT) for 42 clinical samples indicated that L-AST results could be obtained 34 h earlier than the routine results. Conclusions As exemplified for E. coli and Klebsiella spp., L-AST provides substantial acceleration of susceptibility testing following MALDI-TOF MS identification. The assay is a simple and low-cost method that can be integrated into clinical laboratory to allow for 24/7 AST. This approach could improve antibiotic therapy.

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Proteomic Study of Ribosomal Proteins from Escherichia coli, Saccharomyces cerevisiae, Bos taurus, Gallus gallus, and Oncorhynchus tshawytscha: Application in a Teaching Laboratory Setting

American Journal of Undergraduate Research ◽

10.33697/ajur.2014.004 ◽

2014 ◽

Vol 12 (1) ◽

Cited By ~ 1

Author(s):

Yoshihiro Miura ◽

Eric Yeager ◽

James MacKenzie ◽

Kestutis Bendinskas

Keyword(s):

Escherichia Coli ◽

Ribosomal Proteins ◽

Oncorhynchus Tshawytscha ◽

Bos Taurus ◽

Laboratory Setting ◽

Maldi Tof Ms ◽

Teaching Laboratory ◽

Maldi Tof ◽

Proteomic Study ◽

Tof Ms

Ribosomes are central to protein synthesis and our understanding of ribosomes has advanced antibiotics research. The proteomic study of ribosomes presented here utilizes a combination of differential centrifugation and matrix assisted laser desorption/ionization – time of flight mass spectrometry (MALDI-TOF MS) to analyze ribosomes from various species in a teaching laboratory setting. Five biologically varied species were used: Escherichia coli (bacteria), Saccharomyces cerevisiae (yeast), Bos taurus (cow), Gallus gallus (chicken), and Oncorhynchus tshawytscha (Chinook salmon). Samples were lysed, ribosomes were isolated via ultracentrifugation using a discontinuous sucrose gradient and the individual protein subunits were separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis. Tryptic digest and MALDI-TOF MS were then conducted on fifteen bands excised from the gel, and the mass spectra of both the whole protein sample and peptides were analyzed. Five out of these fifteen bands were positively identified as various ribosomal proteins, with two uncertain identifications. Additionally, three of the five positively identified proteins that travelled the same distance on the gel were determined to be orthologous. Finally, a class of 14 Biochemistry II students utilized these protocols, identified 3 ribosomal proteins and provided their evaluations of the ultracentrifugation-proteomics teaching laboratory. Key Words: Proteomics, MALDI-TOF MS, ultracentrifugation, ribosomes, teaching laboratory

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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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Rapid Methods To Enumerate Escherichia coli in Foods Using 4-Methylumbelliferyl-ß-D-Glucuronide

Journal of AOAC International ◽

10.1093/jaoac/84.2.407 ◽

2001 ◽

Vol 84 (2) ◽

pp. 407-415

Author(s):

Diane F Ekholm ◽

Irvin N Hirshfield

Keyword(s):

Escherichia Coli ◽

Cost Effective ◽

Good Choice ◽

Biochemical Tests ◽

Rapid Methods ◽

E Coli ◽

Tree Nuts ◽

Cost Effective Method ◽

History Of ◽

Hard Cheese

Abstract Three methods to enumerate Escherichia coli in food were compared. They were based on AOAC methods using lauryl tryptose broth (LST) medium, LST-4-methylumbelliferyl-ß-D-glucuronide (MUG) medium, and a proposed method using regular LST in combination with E. coli (EC)–MUG medium. An efficacious and cost-effective method is needed that can detect E. Coli and does not produce false presumptive positives. We tested 170 cheeses, 40 frozen processed seafood samples, 210 tree nuts, and 40 other samples. The method of choice for enumerating E. Coli depends on the commodity itself. For a product, such as hard cheese or processed seafood, with a history of being negative for E. Coli and other lactose-fermenting organisms, the proposed method using regular LST/EC–MUG is a good choice. These samples were seldom presumptive positive in the primary LST medium. If gas was produced, EC–MUG was an effective secondary medium. No false positives (fluorescence) or negatives were detected in EC–MUG medium. For a product with a history of being positive for E. Coli and/or other lactose fermenting organisms, such as tree nutmeats or cheeses that are ripened by bacteria or mold, the method using LST–MUG is the method of choice. A presumptive positive in the LST–MUG medium was highly correlative with the biochemical tests that confirmed a sample contain E. Coli. For samples spiked with E. Coli, the results from each of these 3 methods were identical, and were consistent in enumerating E. Coli.

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

Microorganisms ◽

10.3390/microorganisms7120614 ◽

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.

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Rapid and cost-effective identification of Bartonella species using mass spectrometry

Journal of Medical Microbiology ◽

10.1099/jmm.0.009647-0 ◽

2009 ◽

Vol 58 (9) ◽

pp. 1154-1159 ◽

Cited By ~ 43

Author(s):

Pierre-Edouard Fournier ◽

Carine Couderc ◽

Sylvain Buffet ◽

Christophe Flaudrops ◽

Didier Raoult

Keyword(s):

Mass Spectrometry ◽

Species Identification ◽

Chemical Reactivity ◽

Bacterial Species ◽

Cost Effective ◽

Molecular Techniques ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Flight Mass Spectrometry ◽

Tof Ms

Bacteria of the genus Bartonella are emerging zoonotic bacteria recognized in a variety of human diseases. Due to their poor chemical reactivity, these fastidious bacteria are poorly characterized using routine phenotypic laboratory tests. Identification is usually achieved using molecular techniques that are time-consuming, expensive and technically demanding. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a new technique for bacterial species identification. This study evaluated the use of MALDI-TOF MS for rapid genus and species identification of Bartonella species. Reference strains representing 17 recognized Bartonella species were studied. For each species, MS spectra for four colonies were analysed. The consensus spectrum obtained for each species was unique among spectra obtained for 2843 bacteria within the Bruker database, including 109 alphaproteobacteria. Thirty-nine additional blind-coded Bartonella strains were correctly identified at the species level, including 36 with a significant score. Altogether, these data demonstrate that MS is an accurate and reproducible tool for rapid and inexpensive identification of Bartonella species.

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