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

Author(s):  
Mehmet E. Bulut ◽  
Gülen Hürkal ◽  
Nazan Dalgıç

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.

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Anastasia Pavelkovich ◽  
Arta Balode ◽  
Petra Edquist ◽  
Svetlana Egorova ◽  
Marina Ivanova ◽  
...  

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.


Author(s):  
Nahid Kondori ◽  
Amra Kurtovic ◽  
Beatriz Piñeiro-Iglesias ◽  
Francisco Salvà-Serra ◽  
Daniel Jaén-Luchoro ◽  
...  

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.


2020 ◽  
Vol 27 (11) ◽  
pp. 1171-1177
Author(s):  
Neelja Singhal ◽  
Divakar Sharma ◽  
Manish Kumar ◽  
Deepa Bisht ◽  
Jugsharan Singh Virdi

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.


2019 ◽  
Vol 57 (12) ◽  
Author(s):  
R. Christopher D. Furniss ◽  
Laurent Dortet ◽  
William Bolland ◽  
Oliver Drews ◽  
Katrin Sparbier ◽  
...  

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.


2019 ◽  
Vol 57 (8) ◽  
pp. 1271-1279 ◽  
Author(s):  
Maximilian Kittel ◽  
Peter Findeisen ◽  
Beniam Ghebremedhin ◽  
Thomas Miethke ◽  
Alexander Grundt ◽  
...  

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.


Author(s):  
Yoshihiro Miura ◽  
Eric Yeager ◽  
James MacKenzie ◽  
Kestutis Bendinskas

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


2019 ◽  
Vol 6 (1) ◽  
pp. e000307 ◽  
Author(s):  
Alexandra Royden ◽  
Emma Ormandy ◽  
Gina Pinchbeck ◽  
Ben Pascoe ◽  
Matthew D Hitchings ◽  
...  

Extended-spectrum β-lactamase (ESBL)-producing bacteria causing clinical infections are often also multidrug-resistant (MDR; resistance to ≥3 antimicrobial drug classes), therefore treatment options may be limited. High carriage rates of these potentially zoonotic bacteria have been found in livestock and companion animals. Therefore, people working in veterinary hospitals may be a high-risk population for carriage. This is the first study to determine the prevalence and longitudinal carriage of antimicrobial-resistant (AMR) and ESBL-producing faecal Escherichia coli in veterinary hospital staff and students. Prevalence of faecal AMR and ESBL-producing E coli was determined in 84 staff members and students in three UK veterinary hospitals. Twenty-seven participants were followed for six weeks to investigate longitudinal carriage. Antimicrobial susceptibility and phenotypic ESBL production were determined and selected isolates were whole genome sequenced. ESBL-producing E coli were isolated from five participants (5.95 per cent; 95 per cent CI 0.89 to 11.0 per cent); two participants carried ESBL-producing E coli resistant to all antimicrobials tested. Carriage of MDR E coli was common (32.1 per cent; 95per cent CI 22.2 to 42.1 per cent) and there was a high prevalence of ciprofloxacin resistance (11.9 per cent; 95 per cent CI 4.98 to 18.8 per cent). ESBL-producing E coli were isolated from seven longitudinal participants (25.9 per cent; 95 per cent CI 9.40 to 42.5 per cent); two participants carried ESBL-producing E coli for the entire study period. Twenty-six participants (96.3 per cent; 95 per cent CI 89.2 to 100) carried ≥1 MDR E coli isolate during the six-week period, with seven participants (25.9 per cent) carrying ≥1 MDR isolate for at least five out of six weeks. The prevalence of faecal ESBL-producing E coli in cross-sectional participants is similar to asymptomatic general populations. However, much higher levels of carriage were observed longitudinally in participants. It is vital that veterinary hospitals implement gold-standard biosecurity to prevent transmission of MDR and ESBL-producing bacteria between patients and staff. Healthcare providers should be made aware that people working in veterinary hospitals are a high-risk population for carriage of MDR and ESBL-producing bacteria, and that this poses a risk to the carrier and for transmission of resistance throughout the wider community.


mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Shivdeep Singh Hayer ◽  
Seunghyun Lim ◽  
Samuel Hong ◽  
Ehud Elnekave ◽  
Timothy Johnson ◽  
...  

ABSTRACT Fluoroquinolones and cephalosporins are critically important antimicrobial classes for both human and veterinary medicine. We previously found a drastic increase in enrofloxacin resistance in clinical Escherichia coli isolates collected from diseased pigs from the United States over 10 years (2006 to 2016). However, the genetic determinants responsible for this increase have yet to be determined. The aim of the present study was to identify and characterize the genetic basis of resistance against fluoroquinolones (enrofloxacin) and extended-spectrum cephalosporins (ceftiofur) in swine E. coli isolates using whole-genome sequencing (WGS). blaCMY-2 (carried by IncA/C2, IncI1, and IncI2 plasmids), blaCTX-M (carried by IncF, IncHI2, and IncN plasmids), and blaSHV-12 (carried by IncHI2 plasmids) genes were present in 87 (82.1%), 19 (17.9%), and 3 (2.83%) of the 106 ceftiofur-resistant isolates, respectively. Of the 110 enrofloxacin-resistant isolates, 90 (81.8%) had chromosomal mutations in gyrA, gyrB, parA, and parC genes. Plasmid-mediated quinolone resistance genes [qnrB77, qnrB2, qnrS1, qnrS2, and aac-(6)-lb′-cr] borne on ColE, IncQ2, IncN, IncF, and IncHI2 plasmids were present in 24 (21.8%) of the enrofloxacin-resistant isolates. Virulent IncF plasmids present in swine E. coli isolates were highly similar to epidemic plasmids identified globally. High-risk E. coli clones, such as ST744, ST457, ST131, ST69, ST10, ST73, ST410, ST12, ST127, ST167, ST58, ST88, ST617, ST23, etc., were also found in the U.S. swine population. Additionally, the colistin resistance gene (mcr-9) was present in several isolates. This study adds valuable information regarding resistance to critical antimicrobials with implications for both animal and human health. IMPORTANCE Understanding the genetic mechanisms conferring resistance is critical to design informed control and preventive measures, particularly when involving critically important antimicrobial classes such as extended-spectrum cephalosporins and fluoroquinolones. The genetic determinants of extended-spectrum cephalosporin and fluoroquinolone resistance were highly diverse, with multiple plasmids, insertion sequences, and genes playing key roles in mediating resistance in swine Escherichia coli. Plasmids assembled in this study are known to be disseminated globally in both human and animal populations and environmental samples, and E. coli in pigs might be part of a global reservoir of key antimicrobial resistance (AMR) elements. Virulent plasmids found in this study have been shown to confer fitness advantages to pathogenic E. coli strains. The presence of international, high-risk zoonotic clones provides worrisome evidence that resistance in swine isolates may have indirect public health implications, and the swine population as a reservoir for these high-risk clones should be continuously monitored.


Antibiotics ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 639 ◽  
Author(s):  
Wen-Jung Lu ◽  
Hsuan-Ju Lin ◽  
Pang-Hung Hsu ◽  
Hong-Ting Victor Lin

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.


2021 ◽  
Vol 12 ◽  
Author(s):  
Razib Mazumder ◽  
Arif Hussain ◽  
Ahmed Abdullah ◽  
Md. Nazrul Islam ◽  
Md. Tuhin Sadique ◽  
...  

Background:Escherichia coli is a major extended-spectrum β-lactamase (ESBL)–producing organism responsible for the rapid spread of antimicrobial resistance (AMR) that has compromised our ability to treat infections. Baseline data on population structure, virulence, and resistance mechanisms in E. coli lineages from developing countries such as Bangladesh are lacking.Methods: Whole-genome sequencing was performed for 46 ESBL–E. coli isolates cultured from patient samples at the International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b)-Dhaka. Sequence data were analyzed to glean details of AMR, virulence, and phylogenetic and molecular markers of E. coli lineages.Results: Genome comparison revealed presence of all major high-risk clones including sequence type 131 (ST131) (46%), ST405 (13%), ST648 (7%), ST410 (4.3%), ST38 (2%), ST73 (2%), and ST1193 (2%). The predominant ESBL gene and plasmid combination were blaCTX–M–15 and FII-FIA-FIB detected in diverse E. coli phylogroups and STs. The blaNDM–5 (9%) gene was present in prominent E. coli STs. One (2%) mcr-1–positive ST1011 E. coli, coharboring blaCTXM–55 gene, was detected. The extraintestinal pathogenic E. coli genotype was associated with specific E. coli lineages. The single nucleotide polymorphism (SNP)-based genome phylogeny largely showed correlation with phylogroups, serogroups, and fimH types. Majority of these isolates were susceptible to amikacin (93%), imipenem (93%), and nitrofurantoin (83%).Conclusion: Our study reveals a high diversity of E. coli lineages among ESBL-producing E. coli from Dhaka. This study suggests ongoing circulation of ST131 and all major non-ST131 high-risk clones that are strongly associated with cephalosporin resistance and virulence genes. These findings warrant prospective monitoring of high-risk clones, which would otherwise worsen the AMR crises.


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