Multiplex polymerase chain reaction for identification of Escherichia coli , Escherichia albertii and Escherichia fergusonii

Lindsey, Rebecca L.; Garcia-Toledo, Lisley; Fasulo, Daniel; Gladney, Lori M.; Strockbine, Nancy

doi:10.1016/j.mimet.2017.06.005

“…The detection parameters for gene detection using BLAST were set to 90% sequence identity and 60% sequence coverage. The E. coli genotyping plug-in also has an in silico PCR tool for the detection of virulence genes and Shiga toxin gene subtypes using previously published primers (27)(28)(29). The in silico PCR settings were set to allow for 1 mismatch in the primer sequence binding sites.…”

Section: Methodsmentioning

confidence: 99%

Validation of Whole-Genome Sequencing for Identification and Characterization of Shiga Toxin-Producing Escherichia coli To Produce Standardized Data To Enable Data Sharing

Holmes

¹

,

Dallman

²

,

Shabaan

³

et al. 2018

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Whole-genome sequencing (WGS) is rapidly becoming the method of choice for outbreak investigations and public health surveillance of microbial pathogens. The combination of improved cluster resolution and prediction of resistance and virulence phenotypes provided by a single tool is extremely advantageous. However, the data produced are complex, and standard bioinformatics pipelines are required to translate the output into easily interpreted epidemiologically relevant information for public health action. The main aim of this study was to validate the implementation of WGS at the Scottish O157/STEC Reference Laboratory (SERL) using the Public Health England (PHE) bioinformatics pipeline to produce standardized data to enable interlaboratory comparison of results generated at two national reference laboratories. In addition, we evaluated the BioNumerics whole-genome multilocus sequence typing (wgMLST) and genotyping plug-in tools using the same data set. A panel of 150 well-characterized isolates of Shiga toxin-producing (STEC) that had been sequenced and analyzed at PHE using the PHE pipeline and database (SnapperDB) was assembled to provide identification and typing data, including serotype (O:H type), sequence type (ST), virulence genes ( and Shiga toxin [] subtype), and a single-nucleotide polymorphism (SNP) address. To validate the implementation of sequencing at the SERL, DNA was reextracted from the isolates and sequenced and analyzed using the PHE pipeline, which had been installed at the SERL; the output was then compared with the PHE data. The results showed a very high correlation between the data, ranging from 93% to 100%, suggesting that the standardization of WGS between our reference laboratories is possible. We also found excellent correlation between the results obtained using the PHE pipeline and BioNumerics, except for the detection of and when these subtypes are both carried by strains.

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“…It can be argued that although most studies have identi ed the two genes of lysP and mdh as speci c genes in the diagnosis of E. albertii, in several studies these two genes have not been able to identify all E. albertii. Therefore, efforts have been made to design more speci c areas of the genome (10,(13)(14)(15).…”

Section: Discussionmentioning

confidence: 99%

“…On the other hand, to identify E. albertii, tracking of two genes of mdh (Encoding Malate dehydrogenase) and lysP (Encoding Lysine Permease) as speci c and protected genes in E. albertii is performed in the form of a multiplex PCR method (8-10, 12, 13). Also rpoB gene sequencebased identi cation, Multilocus sequence typing (MLST), or Whole Genome Sequencing (WGS) methods have been proposed, one of the limitations of which is being time-consuming to complete the results (10,(13)(14)(15).…”

Section: Introductionmentioning

confidence: 99%

Detection of Escherichia albertii in urinary and gastrointestinal infections in Kermanshah, Iran

Namdari¹,

Rahimian-Zarif²,

Foroughi³

2020

Preprint

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Background Escherichia albertii (E. albertii) is a Gram-negative and facultative anaerobe bacterium. In recent years, the bacterium has been isolated from the feces of people with gastroenteritis as a pathogen that causes diarrhea. Due to insufficient information on the phenotypic and biochemical characteristics of E. albertii, it is difficult to distinguish it from other species of the Enterobacteriaceae family. This is especially prevalent in the pathotypes of Escherichia coli (E. coli). Moreover, in clinical laboratories, it is mistakenly identified as E. coli or even Hafnia alvei (H. alvei). This study was performed for the first time in Iran to identify E. albertii by PCR method from a sample of urinary and gastrointestinal infections obtained from clinical laboratories in Kermanshah, which were distinguished as E. coli. Methods In this study, 60 urinary samples and 40 fecal samples that were identified as E. coli by phenotypic and biochemical methods in clinical laboratories. The samples were re-evaluated in the first step in terms of specific phenotypic and biochemical characteristics of E. coli. Subsequently, DNA was extracted from the isolates by the phenol method. Then, two lysP and mdh genes were detected for E. albertii and the uidA gene for E. coli by PCR using specific primers pairs. Results The results obtained from phenotypic and biochemical tests indicated that all samples were consistent with E. coli characteristics. However, findings from PCR showed that out of a total of 100 samples, specific genes of E. coli were identified in 6 samples (6%) and uidA gene in 94 remaining samples (94%). Of these 6 samples, 5 samples were urinary tract infections, and only one was a gastrointestinal infection. Conclusion The findings of this study show that E. albertii can be considered as one of the causes of urinary and gastrointestinal infections that are mistakenly identified in clinical laboratories as E. coli.Therefore, the use of molecular methods for accurate and definitive diagnosis of bacteria can be useful.

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“…However, this method of analysis is insufficient for identifying the bacterial strains belonging to the various families in the order Enterobacterales. Reclassification of these families have been attempted using multilocus sequence analysis (MLSA) [1,5,6], conserved signature indels (CSIs) [1][2][3], housekeeping genes for phylogenetic analysis [7,8], and matrix-assisted laser desorption/ ionization-time of flight mass spectrometry (MALDI-TOF MS) [9]. A recent study by Adeolu [3], supports the existence of seven distinct monophyletic groups of genera or clades within the order Enterobacterales which can be distinguished from the members of this order by specific CSIs [10].…”

mentioning

confidence: 99%

“…This has prevented further confusion in the family-level taxonomy of the order Enterobacterales. Based on the existing method, the accuracy of MLSA is limited due to variations based on gene numbers and also since some genera such as Escherichia and Kluyvera, exhibit polyphyletic branching [7]. There is a need for a novel and efficient method capable of higher phylogenetic resolution so as to resolve these anomalies at the genus and species level.…”

mentioning

confidence: 99%

Reclassification of genus Izhakiella into the family Erwiniaceae based on phylogenetic and genomic analyses

Jiang

¹

,

Wang

²

,

Kim

³

et al. 2020

International Journal of Systematic and Evolutionary Microbiology

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The genus Izhakiella was established and designated as a member of the family Enterobacteriaceae in 2016. Although the taxonomical classification of most members in this family has been relatively resolved after two reclassifications in 2016 and 2017, the classification of the genus Izhakiella remains ambiguous. In this study, a polyphasic approach was used to provide evidence supporting the fact that the genus Izhakiella should no longer be considered a member of Enterobacteriaceae and proposes its reclassification into the family Erwiniaceae . The phylogenetic tree of type species in the families Enterobacteriaceae and Erwiniaceae based on the sequences of the 16S rRNA gene, rpoB housekeeping gene, and the whole-genome comprising the 92 core genes revealed that the genus Izhakiella forms a phylogenetic lineage within the family Erwiniaceae . The average nucleotide identity (ANI) value of the type species with genus Izhakiella was found to be higher for the family Erwiniaceae than that for the family Enterobacteriaceae . Notably, 12 conserved signature indels (CSIs) that are exclusively shared among the Erwiniaceae clade members were found in the type strains of the genus Izhakiella . Based on these analyses, this study suggests the reclassification of I. capsodis and I. australiensis into the family Erwiniaceae .

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Multiplex polymerase chain reaction for identification of Escherichia coli , Escherichia albertii and Escherichia fergusonii

Cited by 70 publications

References 18 publications

Validation of Whole-Genome Sequencing for Identification and Characterization of Shiga Toxin-Producing Escherichia coli To Produce Standardized Data To Enable Data Sharing

Validation of Whole-Genome Sequencing for Identification and Characterization of Shiga Toxin-Producing Escherichia coli To Produce Standardized Data To Enable Data Sharing

Detection of Escherichia albertii in urinary and gastrointestinal infections in Kermanshah, Iran

Reclassification of genus Izhakiella into the family Erwiniaceae based on phylogenetic and genomic analyses

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