Frequency and Mechanisms of Spontaneous Fosfomycin Nonsusceptibility Observed upon Disk Diffusion Testing of Escherichia coli

ABSTRACTFosfomycin maintains activity against mostEscherichia coliclinical isolates, but the growth ofE. colicolonies within the zone of inhibition around the fosfomycin disk is occasionally observed upon susceptibility testing. We aimed to estimate the frequency of such nonsusceptible inner colony mutants and identify the underlying resistance mechanisms. Disk diffusion testing of fosfomycin was performed on 649 multidrug-resistantE. coliclinical isolates collected between 2011 and 2015. For those producing inner colonies inside the susceptible range, the parental strains and their representative inner colony mutants were subjected to MIC testing, whole-genome sequencing, reverse transcription-quantitative PCR (qRT-PCR), and carbohydrate utilization studies. Of the 649E. coliclinical isolates, 5 (0.8%) consistently produced nonsusceptible inner colonies. Whole-genome sequencing revealed the deletion ofuhpTencoding hexose-6-phosphate antiporter in 4 of theE. coliinner colony mutants, while the remaining mutant contained a nonsense mutation inuhpA. The expression ofuhpTwas absent in the mutant strains withuhpTdeletion and was not inducible in the strain with theuhpAmutation, unlike in its parental strain. All 5 inner colony mutants had reduced growth on minimal medium supplemented with glucose-6-phosphate. In conclusion, fosfomycin-nonsusceptible inner colony mutants can occur due to the loss of function or induction of UhpT but are rare among multidrug-resistantE. coliclinical strains. Considering that these mutants carry high biological costs, we suggest that fosfomycin susceptibility of strains that generate inner colony mutants can be interpreted on the basis of the zone of inhibition without accounting for the inner colonies.

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Whole-Genome Sequencing of Multidrug-Resistant Escherichia coli Strains Harboring the mcr-1 Gene, Isolated from Seawater of the Algiers Coast in Algeria

Microbiology Resource Announcements ◽

10.1128/mra.00638-19 ◽

2019 ◽

Vol 8 (34) ◽

Cited By ~ 2

Author(s):

M. Berrazeg ◽

A. Deriet ◽

S. C. J. De Keersmaecker ◽

B. Verhaegen ◽

K. Vanneste ◽

...

Keyword(s):

Escherichia Coli ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Treatment Efficacy ◽

Multidrug Resistant ◽

Whole Genome ◽

Colistin Resistance ◽

Content Type ◽

E Coli

Colistin resistance has emerged worldwide and is threatening the treatment efficacy of multiresistant Escherichia coli strains in humans and animals. Here, we communicate the whole-genome sequencing (WGS) of two colistin-resistant E. coli strains, M49 and M78, with genomes sizes of 4,947,168 and 5,178,716 bp, respectively, isolated from seawaters of the Algiers coast.

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Genomic surveillance of Escherichia coli and Klebsiella spp. in hospital sink drains and patients

Microbial Genomics ◽

10.1099/mgen.0.000391 ◽

2020 ◽

Vol 6 (7) ◽

Author(s):

Bede Constantinides ◽

Kevin K. Chau ◽

T. Phuong Quan ◽

Gillian Rodger ◽

Monique I. Andersson ◽

...

Keyword(s):

Escherichia Coli ◽

Antimicrobial Resistance ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Type Species ◽

Whole Genome ◽

Content Type ◽

E Coli ◽

Link Type ◽

Antimicrobial Resistance Genes

Escherichia coli and Klebsiella spp. are important human pathogens that cause a wide spectrum of clinical disease. In healthcare settings, sinks and other wastewater sites have been shown to be reservoirs of antimicrobial-resistant E. coli and Klebsiella spp., particularly in the context of outbreaks of resistant strains amongst patients. Without focusing exclusively on resistance markers or a clinical outbreak, we demonstrate that many hospital sink drains are abundantly and persistently colonized with diverse populations of E. coli , Klebsiella pneumoniae and Klebsiella oxytoca , including both antimicrobial-resistant and susceptible strains. Using whole-genome sequencing of 439 isolates, we show that environmental bacterial populations are largely structured by ward and sink, with only a handful of lineages, such as E. coli ST635, being widely distributed, suggesting different prevailing ecologies, which may vary as a result of different inputs and selection pressures. Whole-genome sequencing of 46 contemporaneous patient isolates identified one (2 %; 95 % CI 0.05–11 %) E. coli urine infection-associated isolate with high similarity to a prior sink isolate, suggesting that sinks may contribute to up to 10 % of infections caused by these organisms in patients on the ward over the same timeframe. Using metagenomics from 20 sink-timepoints, we show that sinks also harbour many clinically relevant antimicrobial resistance genes including bla CTX-M, bla SHV and mcr, and may act as niches for the exchange and amplification of these genes. Our study reinforces the potential role of sinks in contributing to Enterobacterales infection and antimicrobial resistance in hospital patients, something that could be amenable to intervention. This article contains data hosted by Microreact.

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Is Shiga Toxin-Negative Escherichia coli O157:H7 Enteropathogenic or Enterohemorrhagic Escherichia coli? Comprehensive Molecular Analysis Using Whole-Genome Sequencing

Journal of Clinical Microbiology ◽

10.1128/jcm.01899-15 ◽

2015 ◽

Vol 53 (11) ◽

pp. 3530-3538 ◽

Cited By ~ 30

Author(s):

Mithila Ferdous ◽

Kai Zhou ◽

Alexander Mellmann ◽

Stefano Morabito ◽

Peter D. Croughs ◽

...

Keyword(s):

Escherichia Coli ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Shiga Toxin ◽

Enterohemorrhagic Escherichia Coli ◽

Cellular Damage ◽

Whole Genome ◽

Content Type ◽

E Coli ◽

Treatment And Prevention

The ability ofEscherichia coliO157:H7 to induce cellular damage leading to disease in humans is related to numerous virulence factors, most notably thestxgene, encoding Shiga toxin (Stx) and carried by a bacteriophage. Loss of the Stx-encoding bacteriophage may occur during infection or culturing of the strain. Here, we collectedstx-positive andstx-negative variants ofE. coliO157:H7/NM (nonmotile) isolates from patients with gastrointestinal complaints. Isolates were characterized by whole-genome sequencing (WGS), and their virulence properties and phylogenetic relationship were determined. Because of the presence of theeaegene but lack of thebfpAgene, thestx-negative isolates were considered atypical enteropathogenicE. coli(aEPEC). However, they had phenotypic characteristics similar to those of the Shiga toxin-producingE. coli(STEC) isolates and belonged to the same sequence type, ST11. Furthermore, EPEC and STEC isolates shared similar virulence genes, the locus of enterocyte effacement region, and plasmids. Core genome phylogenetic analysis using a gene-by-gene typing approach showed that the sorbitol-fermenting (SF)stx-negative isolates clustered together with an SF STEC isolate and that one non-sorbitol-fermenting (NSF)stx-negative isolate clustered together with NSF STEC isolates. Therefore, thesestx-negative isolates were thought either to have lost the Stx phage or to be a progenitor of STEC O157:H7/NM. As detection of STEC infections is often based solely on the identification of the presence ofstxgenes, these may be misdiagnosed in routine laboratories. Therefore, an improved diagnostic approach is required to manage identification, strategies for treatment, and prevention of transmission of these potentially pathogenic strains.

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MCR-1 and OXA-48 In Vivo Acquisition in KPC-Producing Escherichia coli after Colistin Treatment

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02540-16 ◽

2017 ◽

Vol 61 (8) ◽

Cited By ~ 32

Author(s):

Racha Beyrouthy ◽

Frederic Robin ◽

Aude Lessene ◽

Igor Lacombat ◽

Laurent Dortet ◽

...

Keyword(s):

Escherichia Coli ◽

Klebsiella Pneumoniae ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Whole Genome ◽

Content Type ◽

First Report ◽

E Coli ◽

Carbapenem Resistant

ABSTRACT The spread of mcr-1-encoding plasmids into carbapenem-resistant Enterobacteriaceae raises concerns about the emergence of untreatable bacteria. We report the acquisition of mcr-1 in a carbapenem-resistant Escherichia coli strain after a 3-week course of colistin in a patient repatriated to France from Portugal. Whole-genome sequencing revealed that the Klebsiella pneumoniae carbapenemase-producing E. coli strain acquired two plasmids, an IncL OXA-48-encoding plasmid and an IncX4 mcr-1-encoding plasmid. This is the first report of mcr-1 in carbapenemase-encoding bacteria in France.

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Mass Spectrometry–Based Escherichia coli H Antigen/Flagella Typing: Validation and Comparison with Traditional Serotyping

Clinical Chemistry ◽

10.1373/clinchem.2015.244236 ◽

2016 ◽

Vol 62 (6) ◽

pp. 839-847 ◽

Cited By ~ 11

Author(s):

Keding Cheng ◽

Yi-Min She ◽

Huixia Chui ◽

Larissa Domish ◽

Angela Sloan ◽

...

Keyword(s):

Mass Spectrometry ◽

Escherichia Coli ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Close Correlation ◽

Clinical Isolates ◽

Whole Genome ◽

Sequence Coverage ◽

E Coli ◽

Reference Strains

Abstract BACKGROUND Escherichia coli H antigen typing with antisera, a useful method for flagella clinical identification and classification, is a time-consuming process because of the need to induce flagella growth and the occurrence of undetermined strains. We developed an alternative rapid and analytically sensitive mass spectrometry (MS) method, termed MS-based H antigen typing (MS-H), and applied it at the protein sequence level for H antigen typing. We also performed a comparison with traditional serotyping on reference strains and clinical isolates. METHODS On the basis of international guidelines, the analytical selectivity and sensitivity, imprecision, correlation, repeatability, and reproducibility of the MS-H platform was evaluated using reference strains. Comparison of MS-H typing and serotyping was performed using 302 clinical isolates from 5 Canadian provinces, and discrepant results between the 2 platforms were resolved through whole genome sequencing. RESULTS Repeated tests on reference strain EDL933 demonstrated a lower limit of the measuring interval at the subsingle colony (16.97 μg or 1.465 × 107 cells) level and close correlation (r2 > 0.99) between cell culture biomass and sequence coverage. The CV was <10.0% among multiple repeats with 4 reference strains. Intra- and interlaboratory tests demonstrated that the MS-H method was robust and reproducible under various sample preparation and instrumentation conditions. Using discrepancy analysis via whole genome sequencing, performed on isolates with discrepant results, MS-H accurately identified 12.3% more isolates than conventional serotyping. CONCLUSIONS MS-H typing of E. coli is useful for fast and accurate flagella typing and could be very useful during E. coli outbreaks.

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Molecular Characteristics of Extraintestinal Pathogenic E. coli (ExPEC), Uropathogenic E. coli (UPEC), and Multidrug Resistant E. coli Isolated from Healthy Dogs in Spain. Whole Genome Sequencing of Canine ST372 Isolates and Comparison with Human Isolates Causing Extraintestinal Infections

Microorganisms ◽

10.3390/microorganisms8111712 ◽

2020 ◽

Vol 8 (11) ◽

pp. 1712

Author(s):

Saskia-Camille Flament-Simon ◽

María de Toro ◽

Vanesa García ◽

Jesús E. Blanco ◽

Miguel Blanco ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

One Health ◽

Multidrug Resistant ◽

Clinical Isolates ◽

Molecular Characteristics ◽

Whole Genome ◽

E Coli ◽

Healthy Dogs ◽

The One

Under a one health perspective and the worldwide antimicrobial resistance concern, we investigated extraintestinal pathogenic Escherichia coli (ExPEC), uropathogenic E. coli (UPEC), and multidrug resistant (MDR) E. coli from 197 isolates recovered from healthy dogs in Spain between 2013 and 2017. A total of 91 (46.2%) isolates were molecularly classified as ExPEC and/or UPEC, including 50 clones, among which (i) four clones were dominant (B2-CH14-180-ST127, B2-CH52-14-ST141, B2-CH103-9-ST372 and F-CH4-58-ST648) and (ii) 15 had been identified among isolates causing extraintestinal infections in Spanish and French humans in 2015 and 2016. A total of 28 (14.2%) isolates were classified as MDR, associated with B1, D, and E phylogroups, and included 24 clones, of which eight had also been identified among the human clinical isolates. We selected 23 ST372 strains, 21 from healthy dogs, and two from human clinical isolates for whole genome sequencing and built an SNP-tree with these 23 genomes and 174 genomes (128 from canine strains and 46 from human strains) obtained from public databases. These 197 genomes were segregated into six clusters. Cluster 1 comprised 74.6% of the strain genomes, mostly composed of canine strain genomes (p < 0.00001). Clusters 4 and 6 also included canine strain genomes, while clusters 2, 3, and 5 were significantly associated with human strain genomes. Finding several common clones and clone-related serotypes in dogs and humans suggests a potentially bidirectional clone transfer that argues for the one health perspective.

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Clinical and Genetic Characteristics of Extended-Spectrum Beta-Lactamase–Producing Enterobacteriaceae Among Canadian Children

Infection Control and Hospital Epidemiology ◽

10.1017/ice.2020.693 ◽

2020 ◽

Vol 41 (S1) ◽

pp. s167-s168

Author(s):

Nisha Thampi ◽

Jennifer Bowes ◽

Roberto Melano ◽

Nathalie Tijet ◽

Robert Slinger

Keyword(s):

Public Health ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Multidrug Resistant ◽

Clinical Isolates ◽

First Episode ◽

Whole Genome ◽

Comorbid Conditions ◽

Genetic Characteristics ◽

E Coli

Background: Infections with extended-spectrum β-lactamase–producing Enterobacteriaceae (ESBL-E) in nonoutbreak settings have not demonstrated the presence of dominant strains. Our objective was to determine the incidence, clinical characteristics, and genetic characteristics of ESBL-E infections among a group of Canadian children. Methods: From 2012 through 2017, patients aged ≤18 years with first-episode ESBL-E infections who presented at a pediatric center were reviewed. All clinical isolates were phenotypically identified in the laboratory as ESBL-producers. Demographic and clinical data were collected, including comorbid conditions, presence of devices, and previous antibacterial exposure. Community-associated infection was defined as a positive culture from a sterile site within the first 48 hours of hospital admission and no healthcare exposure during the preceding year. Isolates were sent to the Public Health Ontario Laboratory for whole-genome sequencing. Multilocus sequence typing was used to determine clonal relationship. Results: During the study period, 102 patients were identified with first-episode ESBL-E infection, and the proportion of ESBL-E isolates among all clinical isolates of E. coli and Klebsiella spp increased from 0.6% to 2.6% between 2012 and 2017, respectively (P = .001). The median age was 1 year (interquartile range, 0.8–5 years). Women comprised 66% of cases. No comorbid conditions were noted among 58 patients (57%), and 24% had previous antibiotic exposure, most frequently a cephalosporin (16%). ESBL-E was most frequently isolated in the urine (91%) and least frequently in the blood (2.2%) and was predominantly Escherichia coli (90%). Infection was most frequently diagnosed in the outpatient setting (61%); there were 11 healthcare-associated infections. Whole-genome sequencing of ESBL-E isolates revealed predominance of blaCTX-M-15 (63 isolates, 62%) and blaCTX-M-27 (16%) genes, and sequence type (ST) 131 (41%). Mutations conferring fluoroquinolone nonsusceptibility were noted among 62 isolates (61%), most frequently associated with ST131 (38 of 62 isolates, 61%) and among all 5 isolates with ST1193, an emerging multidrug-resistant E. coli clone. In addition, 15 patients had recurrence of ESBL-E infection at median of 113 days (IQR, 26–208); blaCTX-M-27 was found in 33% of recurrent infections compared to 12% of primary infections (P = 0.045). Conclusions: This study is the first in Canada to provide whole-genome sequencing data regarding ESBL-E in a pediatric population. The gene blaCTX-M-15 and ST131 clone were predominant. More than 60% of infections were community associated and demonstrated cross resistance to fluoroquinolones. With 76% of infections in antibiotic-naïve children, ESBL-E is a public health concern, and a One Health approach is critical to understanding the epidemiology and curbing the spread of multidrug-resistant Enterobacteriaceae.Funding: NoneDisclosures: None

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Utility of Whole-Genome Sequencing of Escherichia coli O157 for Outbreak Detection and Epidemiological Surveillance

Journal of Clinical Microbiology ◽

10.1128/jcm.01066-15 ◽

2015 ◽

Vol 53 (11) ◽

pp. 3565-3573 ◽

Cited By ~ 48

Author(s):

Anne Holmes ◽

Lesley Allison ◽

Melissa Ward ◽

Timothy J. Dallman ◽

Richard Clark ◽

...

Keyword(s):

Escherichia Coli ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Outbreak Detection ◽

Epidemiological Surveillance ◽

Personal Genome ◽

Whole Genome ◽

Content Type ◽

E Coli ◽

Antimicrobial Resistance Genes

Detailed laboratory characterization ofEscherichia coliO157 is essential to inform epidemiological investigations. This study assessed the utility of whole-genome sequencing (WGS) for outbreak detection and epidemiological surveillance ofE. coliO157, and the data were used to identify discernible associations between genotypes and clinical outcomes. One hundred fiveE. coliO157 strains isolated over a 5-year period from human fecal samples in Lothian, Scotland, were sequenced with the Ion Torrent Personal Genome Machine. A total of 8,721 variable sites in the core genome were identified among the 105 isolates; 47% of the single nucleotide polymorphisms (SNPs) were attributable to six “atypical”E. coliO157 strains and included recombinant regions. Phylogenetic analyses showed that WGS correlated well with the epidemiological data. Epidemiological links existed between cases whose isolates differed by three or fewer SNPs. WGS also correlated well with multilocus variable-number tandem repeat analysis (MLVA) typing data, with only three discordant results observed, all among isolates from cases not known to be epidemiologically related. WGS produced a better-supported, higher-resolution phylogeny than MLVA, confirming that the method is more suitable for epidemiological surveillance ofE. coliO157. A combination ofinsilicoanalyses (VirulenceFinder, ResFinder, and local BLAST searches) were used to determinestxsubtypes, multilocus sequence types (15 loci), and the presence of virulence and acquired antimicrobial resistance genes. There was a high level of correlation between the WGS data and our routine typing methods, although some discordant results were observed, mostly related to the limitation of short sequence read assembly. The data were used to identify sublineages and clades ofE. coliO157, and when they were correlated with the clinical outcome data, they showed that one clade, Ic3, was significantly associated with severe disease. Together, the results show that WGS data can provide higher resolution of the relationships betweenE. coliO157 isolates than that provided by MLVA. The method has the potential to streamline the laboratory workflow and provide detailed information for the clinical management of patients and public health interventions.

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In Silico Genotyping of Escherichia coli Isolates for Extraintestinal Virulence Genes by Use of Whole-Genome Sequencing Data

Journal of Clinical Microbiology ◽

10.1128/jcm.01269-20 ◽

2020 ◽

Vol 58 (10) ◽

Author(s):

Anna Maria Malberg Tetzschner ◽

James R. Johnson ◽

Brian D. Johnston ◽

Ole Lund ◽

Flemming Scheutz

Keyword(s):

Escherichia Coli ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

In Silico ◽

Virulence Genes ◽

Whole Genome Sequencing Data ◽

Whole Genome ◽

Content Type ◽

E Coli

ABSTRACT Extraintestinal pathogenic Escherichia coli (ExPEC) is the leading cause in humans of urinary tract infection and bacteremia. The previously published web tool VirulenceFinder (http://cge.cbs.dtu.dk/services/VirulenceFinder/) uses whole-genome sequencing (WGS) data for in silico characterization of E. coli isolates and enables researchers and clinical health personnel to quickly extract and interpret virulence-relevant information from WGS data. In this study, 38 ExPEC-associated virulence genes were added to the existing E. coli VirulenceFinder database. In total, 14,441 alleles were downloaded. A total of 1,890 distinct alleles were added to the database after removal of redundant sequences and analysis of the remaining alleles for open reading frames (ORFs). The database now contains 139 genes—of which 44 are related to ExPEC—and 2,826 corresponding alleles. Construction of the database included validation against 27 primer pairs from previous studies, a search for serotype-specific P fimbriae papA alleles, and a BLASTn confirmation of seven genes (etsC, iucC, kpsE, neuC, sitA, tcpC, and terC) not covered by the primers. The augmented database was evaluated using (i) a panel of nine control strains and (ii) 288 human-source E. coli strains classified by PCR as ExPEC and non-ExPEC. We observed very high concordance (average, 93.4%) between PCR and WGS findings, but WGS identified more alleles. In conclusion, the addition of 38 ExPEC-associated genes and the associated alleles to the E. coli VirulenceFinder database allows for a more complete characterization of E. coli isolates based on WGS data, which has become increasingly important considering the plasticity of the E. coli genome.

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Rapid and EasyIn SilicoSerotyping of Escherichia coli Isolates by Use of Whole-Genome Sequencing Data

Journal of Clinical Microbiology ◽

10.1128/jcm.00008-15 ◽

2015 ◽

Vol 53 (8) ◽

pp. 2410-2426 ◽

Cited By ~ 309

Author(s):

Katrine G. Joensen ◽

Anna M. M. Tetzschner ◽

Atsushi Iguchi ◽

Frank M. Aarestrup ◽

Flemming Scheutz

Keyword(s):

Escherichia Coli ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Antigen Processing ◽

Processing System ◽

Outbreak Detection ◽

Whole Genome ◽

Content Type ◽

E Coli ◽

O Antigen

Accurate and rapid typing of pathogens is essential for effective surveillance and outbreak detection. Conventional serotyping ofEscherichia coliis a delicate, laborious, time-consuming, and expensive procedure. With whole-genome sequencing (WGS) becoming cheaper, it has vast potential in routine typing and surveillance. The aim of this study was to establish a valid and publicly available tool for WGS-basedin silicoserotyping ofE. coliapplicable for routine typing and surveillance. A FASTA database of specific O-antigen processing system genes for O typing and flagellin genes for H typing was created as a component of the publicly available Web tools hosted by the Center for Genomic Epidemiology (CGE) (www.genomicepidemiology.org). AllE. coliisolates available with WGS data and conventional serotype information were subjected to WGS-based serotyping employing this specific SerotypeFinder CGE tool. SerotypeFinder was evaluated on 682E. coligenomes, 108 of which were sequenced for this study, where both the whole genome and the serotype were available. In total, 601 and 509 isolates were included for O and H typing, respectively. The O-antigen geneswzx,wzy,wzm, andwztand the flagellin genesfliC,flkA,fllA,flmA, andflnAwere detected in 569 and 508 genome sequences, respectively. SerotypeFinder for WGS-based O and H typing predicted 560 of 569 O types and 504 of 508 H types, consistent with conventional serotyping. In combination with other available WGS typing tools,E. coliserotyping can be performed solely from WGS data, providing faster and cheaper typing than current routine procedures and making WGS typing a superior alternative to conventional typing strategies.

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