Evaluating the Occurrence of Escherichia albertii in Chicken Carcass Rinses by PCR, Vitek Analysis, and Sequencing of the rpoB Gene

Abstract

Escherichia albertii is a recently described species that has been associated with gastroenteritis in humans and with healthy and ill birds. Most recently, it has been identified as the causative agent in a food-borne outbreak in Japan. The distribution and clinical importance of E. albertii are not well studied because its importance is unclear. Culture methods for clinical isolation frequently miss E. albertii or incorrectly identify it as Shigella spp., Escherichia coli, or Hafnia alvei. This study was designed to determine if E. albertii could be recovered from chicken carcass rinses collected at slaughter during a 1-year period from November 2009 until October 2010. Colonies were isolated from chicken carcass rinses and tested by PCR for the presence or absence of clpX, lysP, mdh, intimin (eae), Shiga toxins 1 and 2 (stx 1 , stx 2 , and stx 2f ), heat-stable enterotoxin A (staA), and cytolethal distending toxins 1 and 2 (cdtB) genes. Sixty-five isolates were analyzed by sequencing a section of the rpoB gene. Analysis of the rpoB gene sequences revealed 14 fixed differences between E. albertii and other, closely related organisms. The fixed differences found in the rpoB gene could aid in future discrimination of E. albertii from closely related bacteria. Escherichia albertii is an emerging enteric pathogen first described by Albert and associates, Huys et al., and Hyma et al.(1-4). Isolates initially recovered from stool specimens from sick Bangladeshi children (1, 2) were preliminarily identified as atypical eae-positive Hafnia alvei and later were determined to be a new species, named E. albertii. From 1992 until now, E. albertii has been recovered on multiple continents from healthy and diseased birds, humans, and a cat (5-9). Most recently, E. albertii has been detected in sick humans in Japan, where Ooka et al. and Konno et al. found it to be the major causative agent in a restaurant-associated outbreak (9, 10). Additional studies have shown that E. albertii can be transmitted via food-borne routes, including lettuce, ground beef, and turkey (11-13).The true clinical importance and prevalence of E. albertii are unknown, which is in part attributable to the inability to identify isolates using commercially available biochemical identification systems, such as Vitek (bioMérieux, Inc., Durham, NC) and API biochemical test strips (bioMérieux). E. albertii is not in the VItek or API database, so currently, isolates may be incorrectly identified as Shigella spp., Escherichia coli, or H. alvei (3, 14-17). Molecular genetic approaches, such as PCR, for identification and sequencing for phylogenetic studies of specific genes have been used to distinguish E. albertii (4,6,8,16,18). Sequence analysis of the 16S rRNA gene has been used for E. albertii identification (3, 4, 6), but it is not sufficiently discriminative (19). Multilocus sequence typing (MLST), diagnostic multiplex PCR, and sequencing of the 16S rRNA, eae, and cdtB genes was used to show that atypical Shigella sp. strain B13 isolates were in fact E. a...

Whole-Genome Characterization and Strain Comparison of VT2f-ProducingEscherichiacoliCausing Hemolytic Uremic Syndrome

“…All the VT2f-producing isolates had an E. coli- related rpoB sequence ( 23 ). This finding verified that all the strains investigated were E. coli .…”

“…Amplification and sequencing of the rpoB gene were conducted to discriminate between E. coli and E. albertii species, as previously described ( 23 ). The amplicons were purified with the SureClean Plus kit (Bioline, London, UK) and sequenced using the BigDye Terminator v1.1 kit on a Genetic Analyzer 3130 (Thermo Fisher Scientific).…”

“…The amplicons were purified with the SureClean Plus kit (Bioline, London, UK) and sequenced using the BigDye Terminator v1.1 kit on a Genetic Analyzer 3130 (Thermo Fisher Scientific). The obtained sequences were trimmed and aligned to the reference sequences as indicated ( 23 ), using the Clustal Omega free software (http://www.ebi.ac.uk/Tools/msa/clustalo/). …”

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

“…A PCR that detects the uidA gene (encoding the b–glucuronidase enzyme) is frequently used to identify E. coli (Bej et al, 1991, (Frahm and Obst, 2003), Pavlovic et al, 2010). Traditionally, E. albertii was detected with a multiplex PCR for the presence of clpX, lysP , and mdh genes but this does not detect all E. albertii (Hyma et al, 2005, Murakami et al, 2014, Lindsey et al, 2015). Recently, Ooka et al developed a nested PCR to detect E. albertii (Ooka et al, 2015); however, this assay does not detect additional Escherichia species.…”

“…Recently, Ooka et al developed a nested PCR to detect E. albertii (Ooka et al, 2015); however, this assay does not detect additional Escherichia species. There are also rpoB gene sequencing based methods or multi locus sequencing of whole genome sequence (WGS), but these methods are time consuming and take days to complete (Hyma et al, 2005, Murakami et al, 2014, Lindsey et al, 2015). The wealth of WGS data provides an opportunity to develop a much needed multiplex polymerase chain reaction (PCR) assay to quickly speciate the common Escherichia species in a single reaction.…”