Faculty Opinions recommendation of A culture-independent sequence-based metagenomics approach to the investigation of an outbreak of Shiga-toxigenic Escherichia coli O104:H4.

Shiga toxin-producing Escherichia coli (STEC) are associated with acute gastroenteritis worldwide, which induces a high economic burden on both healthcare and individuals. Culture-independent diagnostic tests (CIDT) in frontline microbiology laboratories have been implemented in Alberta since 2019. The objectives of this study were to determine the association between gene detection and culture positivity over time using STEC microbiological clearance samples and also to establish the frequency of specimen submission. Both stx genes’ amplification by real-time PCR was performed with DNA extracted from stool samples using the easyMAG system. Stools were inoculated onto chromogenic agar for culture. An association between gene detection and culture positivity was found to be independent of which stx gene was present. CIDT can provide rapid reporting with less hands-on time and technical expertise. However, culture is still important for surveillance and early cluster detection. In addition, stool submissions could be reduced from daily to every 3–5 days until a sample is negative by culture.

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Comparison of Common Enrichment Broths Used in Diagnostic Laboratories for Shiga Toxin—Producing Escherichia coli

Microorganisms ◽

10.3390/microorganisms9030503 ◽

2021 ◽

Vol 9 (3) ◽

pp. 503

Author(s):

Michael Bording-Jorgensen ◽

Hannah Tyrrell ◽

Colin Lloyd ◽

Linda Chui

Keyword(s):

Escherichia Coli ◽

Shiga Toxin ◽

Real Time Pcr ◽

Acute Gastroenteritis ◽

Diagnostic Testing ◽

First Line ◽

Gram Negative ◽

Culture Independent ◽

Surveillance Programs ◽

Selection Of

Acute gastroenteritis caused by Shiga toxin-producing Escherichia coli (STEC) affects more than 4 million individuals in Canada. Diagnostic laboratories are shifting towards culture-independent diagnostic testing; however, recovery of STEC remains an important aspect of surveillance programs. The objective of this study was to compare common broth media used for the enrichment of STEC. Clinical isolates including O157:H7 as well as non-O157 serotypes were cultured in tryptic soy (TSB), MacConkey (Mac), and Gram-negative (GN) broths and growth was compared using culture on sheep’s blood agar and real-time PCR (qPCR). In addition, a selection of the same isolates was spiked into negative stool and enriched in the same three broths, which were then evaluated using culture on CHROMagarTM STEC agar and qPCR. TSB was found to provide the optimal enrichment for growth of isolates with and without stool. The results from this study suggest that diagnostic laboratories may benefit from enriching STEC samples in TSB as a first line enrichment instead of GN or Mac.

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Single Clinical Isolates from Acute Uncomplicated Urinary Tract Infections Are Representative of Dominant In Situ Populations

mBio ◽

10.1128/mbio.01064-13 ◽

2014 ◽

Vol 5 (2) ◽

Cited By ~ 18

Author(s):

Dana Willner ◽

Serene Low ◽

Jason A. Steen ◽

Narelle George ◽

Graeme R. Nimmo ◽

...

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Urinary Tract Infections ◽

Clinical Isolates ◽

Content Type ◽

E Coli ◽

Strain Diversity ◽

Amplicon Pyrosequencing ◽

Culture Independent ◽

Tract Infections

ABSTRACTUrinary tract infections (UTIs) are one of the most commonly acquired bacterial infections in humans, and uropathogenicEscherichia colistrains are responsible for over 80% of all cases. The standard method for identification of uropathogens in clinical laboratories is cultivation, primarily using solid growth media under aerobic conditions, coupled with morphological and biochemical tests of typically a single isolate colony. However, these methods detect only culturable microorganisms, and characterization is phenotypic in nature. Here, we explored the genotypic identity of communities in acute uncomplicated UTIs from 50 individuals by using culture-independent amplicon pyrosequencing and whole-genome and metagenomic shotgun sequencing. Genus-level characterization of the UTI communities was achieved using the 16S rRNA gene (V8 region). Overall UTI community richness was very low in comparison to other human microbiomes. We strain-typedEscherichia-dominated UTIs using amplicon pyrosequencing of the fimbrial adhesin gene,fimH. There were nine highly abundantfimHtypes, and each UTI sample was dominated by a single type. Molecular analysis of the corresponding clinical isolates revealed that in the majority of cases the isolate was representative of the dominant taxon in the community at both the genus and the strain level. Shotgun sequencing was performed on a subset of eightE. coliurine UTI and isolate pairs. The majority of UTI microbial metagenomic sequences mapped to isolate genomes, confirming the results obtained using phylogenetic markers. We conclude that for the majority of acute uncomplicatedE. coli-mediated UTIs, single cultured isolates are diagnostic of the infection.IMPORTANCEIn clinical practice, the diagnosis and treatment of acute uncomplicated urinary tract infection (UTI) are based on analysis of a single bacterial isolate cultured from urine, and it is assumed that this isolate represents the dominant UTI pathogen. However, these methods detect only culturable bacteria, and the existence of multiple pathogens as well as strain diversity within a single infection is not examined. Here, we explored bacteria present in acute uncomplicated UTIs using culture-independent sequence-based methods.Escherichia coliwas the most common organism identified, and analysis ofE. colidominant UTI samples and their paired clinical isolates revealed that in the majority of infections the cultured isolate was representative of the dominant taxon at both the genus and the strain level. Our data demonstrate that in most cases single cultured isolates are diagnostic of UTI and are consistent with the notion of bottlenecks that limit strain diversity during UTI pathogenesis.

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Culture-independent Sequence-based Approaches for Diagnostics and Food Safety Testing

Pathogenic Escherichia coli: Evolution, Omics, Detection and Control ◽

10.21775/9781910190777.10 ◽

2018 ◽

Author(s):

Susan R. Leonard ◽

Christopher A. Elkins

Keyword(s):

Food Safety ◽

Independent Sequence ◽

Safety Testing ◽

Culture Independent

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A culture-independent method for studying transfer of IncI1 plasmids from wild-type Escherichia coli in complex microbial communities

Journal of Microbiological Methods ◽

10.1016/j.mimet.2018.07.009 ◽

2018 ◽

Vol 152 ◽

pp. 18-26 ◽

Cited By ~ 1

Author(s):

Mehreen Anjum ◽

Jonas Stenløkke Madsen ◽

Carmen Espinosa-Gongora ◽

Bimal Jana ◽

Maria Wiese ◽

...

Keyword(s):

Escherichia Coli ◽

Microbial Communities ◽

Independent Method ◽

Wild Type ◽

Culture Independent

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Crosstalk Between Culturomics and Microbial Profiling of Egyptian Mongoose (Herpestes ichneumon) Gut Microbiome

Microorganisms ◽

10.3390/microorganisms8060808 ◽

2020 ◽

Vol 8 (6) ◽

pp. 808 ◽

Cited By ~ 1

Author(s):

André C. Pereira ◽

Victor Bandeira ◽

Carlos Fonseca ◽

Mónica V. Cunha

Keyword(s):

Microbial Community ◽

Gut Microbiota ◽

Microbial Community Composition ◽

Shannon Index ◽

Data Series ◽

Independent Sequence ◽

Microbial Profiling ◽

Culture Independent ◽

Egyptian Mongoose ◽

Herpestes Ichneumon

Recently, we unveiled taxonomical and functional differences in Egyptian mongoose (Herpestes ichneumon) gut microbiota across sex and age classes by microbial profiling. In this study, we generate, through culturomics, extended baseline information on the culturable bacterial and fungal microbiome of the species using the same specimens as models. Firstly, this strategy enabled us to explore cultivable microbial community differences across sexes and to ascertain the influence exerted by biological and environmental contexts of each host in its microbiota signature. Secondly, it permitted us to compare the culturomics and microbial profiling approaches and their ability to provide information on mongoose gut microbiota. In agreement with microbial profiling, culturomics showed that the core gut cultivable microbiota of the mongoose is dominated by Firmicutes and, as previously found, is able to distinguish sex- and age class-specific genera. Additional information could be obtained by culturomics, with six new genera unveiled. Richness indices and the Shannon index were concordant between culture-dependent and culture-independent approaches, highlighting significantly higher values when using microbial profiling. However, the Simpson index underlined higher values for the culturomics-generated data. These contrasting results were due to a differential influence of dominant and rare taxa on those indices. Beta diversity analyses of culturable microbiota showed similarities between adults and juveniles, but not in the data series originated from microbial profiling. Additionally, whereas the microbial profiling indicated that there were several bioenvironmental features related to the bacterial gut microbiota of the Egyptian mongoose, a clear association between microbiota and bioenvironmental features could not be established through culturomics. The discrepancies found between the data generated by the two methodologies and the underlying inferences, both in terms of β-diversity and role of bioenvironmental features, confirm that culture-independent, sequence-based methods have a higher ability to assess, at a fine scale, the influence of abiotic and biotic factors on the microbial community composition of mongoose’ gut. However, when used in a complementary perspective, this knowledge can be expanded by culturomics.

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Epidemiology of Enterotoxigenic Escherichia coli infection in Minnesota, 2016–2017

Epidemiology and Infection ◽

10.1017/s0950268820001934 ◽

2020 ◽

Vol 148 ◽

Author(s):

S. Buuck ◽

K. Smith ◽

R. C. Fowler ◽

E. Cebelinski ◽

V. Lappi ◽

...

Keyword(s):

Escherichia Coli ◽

Stool Culture ◽

Enterotoxigenic Escherichia Coli ◽

Culture Methods ◽

E Coli ◽

Case Comparison ◽

Culture Independent ◽

The Usa ◽

Escherichia Coli Infection ◽

Traveller’S Diarrhoea

Abstract Enterotoxigenic Escherichia coli (ETEC) is a well-established cause of traveller's diarrhoea and occasional domestic foodborne illness outbreaks in the USA. Although ETEC are not detected by conventional stool culture methods used in clinical laboratories, syndromic culture-independent diagnostic tests (CIDTs) capable of detecting ETEC have become increasingly prevalent in the last decade. This study describes the epidemiology of ETEC infections reported to the Minnesota Department of Health (MDH) during 2016–2017. ETEC-positive stool specimens were submitted to MDH to confirm the presence of ETEC DNA by polymerase chain reaction (PCR). Cases were interviewed to ascertain illness and exposures. Contemporaneous Salmonella cases were used as a comparison group in a case-case comparison analysis of risk factors. Of 222 ETEC-positive specimens received by MDH, 108 (49%) were concordant by PCR. ETEC was the sixth most frequently reported bacterial enteric pathogen among a subset of CIDT-positive specimens. Sixty-nine (64%) laboratory-confirmed cases had an additional pathogen codetected with ETEC, including enteroaggregative E. coli (n = 40) and enteropathogenic E. coli (n = 39). Although travel is a risk factor for ETEC infection, only 43% of cases travelled internationally, providing evidence for ETEC as an underestimated source of domestically acquired enteric illness in the USA.

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Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli of novel phylogeny relative to depletion of Clostridiales in Crohn's disease involving the ileum

The ISME Journal ◽

10.1038/ismej.2007.52 ◽

2007 ◽

Vol 1 (5) ◽

pp. 403-418 ◽

Cited By ~ 388

Author(s):

Martin Baumgart ◽

Belgin Dogan ◽

Mark Rishniw ◽

Gil Weitzman ◽

Brian Bosworth ◽

...

Keyword(s):

Escherichia Coli ◽

Crohn’S Disease ◽

Crohn's Disease ◽

Ileal Mucosa ◽

Independent Analysis ◽

Culture Independent ◽

Selective Increase

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High Relative Frequency of Enteroaggregative Escherichia coli Among Patients With Reportable Enteric Pathogens, Minnesota, 2016–2017

Clinical Infectious Diseases ◽

10.1093/cid/ciy890 ◽

2018 ◽

Vol 69 (3) ◽

pp. 473-479 ◽

Cited By ~ 1

Author(s):

Aaron Beczkiewicz ◽

Elizabeth Cebelinski ◽

Marijke Decuir ◽

Victoria Lappi ◽

Xiong Wang ◽

...

Keyword(s):

Escherichia Coli ◽

Disease Surveillance ◽

Clinical Characteristics ◽

The United States ◽

Epidemiological Studies ◽

International Travel ◽

Enteric Pathogen ◽

Enteric Pathogens ◽

Entire Study ◽

Culture Independent

AbstractBackgroundEnteroaggregative Escherichia coli (EAEC) is increasingly recognized as an enteric pathogen as clinical laboratories transition to culture-independent diagnostic tests that detect EAEC. To date, epidemiological studies have focused on children aged <5 years, and information on EAEC incidence, illness outcomes, and transmission avenues is limited.MethodsEnteric disease surveillance data in Minnesota were used to describe EAEC illnesses reported to the Minnesota Department of Health from September 2016 through August 2017. We determined laboratory characteristics of EAEC using pulsed-field gel electrophoresis and next-generation sequencing. Frequency of EAEC illness, demographic profile of cases, clinical characteristics of illness, and plausible food or environmental exposures leading to EAEC transmission were assessed.ResultsDuring the study period, 329 EAEC cases were reported. Among a subset of health systems able to detect EAEC over the entire study, EAEC was the second most common reportable enteric pathogen detected after Campylobacter and the most detected diarrheagenic E. coli pathotype. No other reportable enteric pathogens were detected among 75.3% of EAEC cases, and 68% of cases reported no international travel before onset. Several virulence genes were associated with clinical characteristics.ConclusionsWe provide evidence that EAEC is a likely causative agent of diarrheal illness in the United States. Our study contributes to criteria development for identification of pathogenic EAEC and proposes potential exposure avenues.

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