Development of a Novel Real-Time Polymerase Chain Reaction Assay to Detect Escherichia albertii in Chicken Meat

Abstract

Escherichia albertii is an emerging enteropathogen. Several foodborne outbreaks of E. albertii have been reported in Japan; however, foods associated with most outbreaks remain unidentified. Therefore, polymerase chain reaction (PCR) assays detecting E. albertii specifically and sensitively are required. Primers and probe for real-time PCR assays targeting E. albertii-specific gene (EA-rtPCR) was designed. With 74 strains, including 43 E. albertii strains and several of its close relatives, EA-rtPCR specifically amplified E. albertii; therefore, the sensitivity of EA-rtPCR was then evaluated. The detection limits were 2.8 and 2.0–3.2 log colony-forming unit (CFU)/mL for E. albertii culture and enriched chicken culture inoculated with the pathogen, respectively. In addition, E. albertii was detected from 25 g of chicken meat inoculated with 0.1 log CFU of the pathogen by EA-rtPCR. The detection of E. albertii from chicken meat by EA-rtPCR was also evaluated by comparing with the nested-PCR assay, and 28 retail chicken meat and 193 dissected body parts from 21 chicken carcass were tested. One and three chicken meat were positive in the nested-PCR assay and EA-rtPCR, respectively. Fourteen carcasses had at least one body part that was positive for EA-rtPCR, and 36 and 48 samples were positive for the nested-PCR assay and EA-rtPCR, respectively. A total of 37 strains of E. albertii were isolated from seven PCR-positive samples obtained from six chicken carcass. All E. albertii isolates harbored eae gene, and were classified as E. albertii O-genotype (EAOg)3 or EAOg4 by EAO-genotyping. The EA-rtPCR developed in this study has potential to improve E. albertii detection in food and advance research on E. albertii infection.

Introduction

Escherichia albertii is an emerging enteropathogen that causes diarrhea and gastroenteritis in humans (Huys et al, 2003). The virulence factors of E. albertii, which have also been preserved in other pathogens, are as follows: eae that encodes intimin (Hinenoya et al, 2009; Oaks et al, 2010), an outer membrane protein associated with intestinal epithelial binding, and a cytolethal distending toxin, which is considered to be associated with increased persistent colonization, bacterial invasion, and disease severity (Ge et al, 2007; McAuley et al, 2007; Pandey et al, 2003; Young et al, 2004). Some E. albertii strains also produce Shiga toxin 2 (Stx2a and Stx2f) or carry genes encoding these toxins (Brandal et al, 2015; Hinenoya et al, 2017a; Murakami et al, 2014; Ooka et al, 2012).

Animals would be potential reservoir for E. albertii, either contaminating or being colonized/infected via water. Thus foods such as chicken meat or vegetables can be direct sources of E. albertii to humans (Muchaamba et al, 2022). Ten foodborne outbreaks of E. albertii have been reported in Japan between 2003 and 2017 (Etoh et al, 2009; Kashio et al, 2020; Ooka, 2017; Tokoi et al, 2018). However, foods associated with most outbreaks remain unidentified. Therefore, an efficient selective agar and polymerase chain reaction (PCR) assay detecting E. albertii specifically and sensitively is required to identify and isolate E. albertii from food residues to prevent foodborne outbreaks.

MacConkey agar (MAC) supplemented with melibiose, l-(+)-rhamnose, and d-(+)-xylose is effective for isolating E. albertii from stool samples (Hinenoya et al, 2020). In a previous study, we modified this agar plate and demonstrated that deoxycholate hydrogen sulfide lactose agar (DHL) and MAC supplemented with l-(+)-rhamnose and d-(+)-xylose are effective for isolating E. albertii from chicken samples (Arai et al, 2021). Several PCR assays specific for E. albertii (Hinenoya et al, 2019; Hyma et al, 2005; Lindsey et al, 2017; Maeda et al, 2014; Ooka et al, 2015; Takahashi et al, 2020) have also been reported. Among these PCR assays, a nested-PCR assay for detecting E. albertii in food has been evaluated (Arai et al, 2021); however, this assay is complicated and time-consuming because it repeats the PCR assay twice.

Real-time PCR assays, which are time- and labor-saving methods, are widely used in the field of microbiological food analyses (Güven and Azizoglu, 2022; Hara-Kudo et al, 2020). In this study, a novel real-time PCR assay for E. albertii, which is designated as real-time PCR assays targeting E. albertii-specific gene (EA-rtPCR), was developed. Furthermore, because E. albertii has been reportedly isolated from chicken meat (Asoshima et al, 2015; Maeda et al, 2014; Wang et al, 2016), we utilized chicken as sample to demonstrate the applicability of our novel EA-rtPCR by comparing it with the nested-PCR assay.

Materials and Methods

Bacterial strains and culture conditions

In total, 74 strains, including 43 E. albertii strains, other foodborne disease bacteria, and other bacteria isolated from food, were used in this study (Supplementary Table S1). Vibrio parahaemolyticus and Morganella morganii subsp. Morganii were cultured in tryptone soya broth (TSB; Oxoid, Hampshire, United Kingdom) supplemented with 2% sodium chloride. All the other strains were cultured in TSB. All strains were incubated at 37°C for 18 h, and genomic DNA was extracted using a NucleoSpin Tissue Kit (TaKaRa Bio, Shiga, Japan). DNA concentrations were measured using a NanoDrop^® 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). TSB culture of several E. albertii strains were also used for the latter examinations as described hereunder.

Retail chicken meat samples

A total of 28 samples of retail chicken meat, including 2 breast meats, 1 drum stick, 4 gizzards, and 21 thigh meats from 10 chicken brands (A–J) were purchased from retail shops in Japan between November 2018 and January 2019 (Table 1). Each 100 g of chicken meat or 25 g of gizzard was placed into a sterile stomacher bag and homogenized with 300 or 225 mL of modified Escherichia coli broth (mEC; Nissui, Tokyo, Japan), respectively, for 1 min. After 22 ± 2 h incubation at 42°C, DNA was extracted from each 100 μL of the enriched culture using alkaline DNA extraction (Hara-Kudo et al, 2016). In brief, enriched culture was centrifuged and supernatant was drained. The pellet was resuspended in 50 mM NaOH, and heated at 100°C for 10 min. Suspension was neutralized with 1 M Tris-HCl (pH 7.0), and was then centrifuged. The supernatant (100 μL) containing DNA was used for nested and duplex EA-rtPCR as described hereunder. In addition, for EA-rtPCR evaluation, retail chicken meat (brand K) was purchased from supermarkets in Japan in December 2019.

Table 1.

Escherichia albertii Detection in Retail Chicken Meat Sample by Polymerase Chain Reaction Assays

Chicken meat parts (subtotal of samples)	Chicken brand^a	Number of samples	Number of PCR-positive sample (%)
Chicken meat parts (subtotal of samples)	Chicken brand^a	Number of samples	Nested PCR	Duplex EA-rtPCR^b
Breast (2)	A	1	0	0
	B	1	0	0
Drum stick (1)	C	1	0	1 (100)
Gizzard (4)	A	1	0	0
	C	1	0	0
	D	1	0	0
	E	1	0	0
Thigh (21)	A	3	0	0
	B	3	0	0
	C	2	0	1 (50)
	D	1	0	0
	E	2	0	0
	F	5	0	0
	G	2	0	0
	H	1	0	0
	I	1	0	0
	J	1	1 (100)	1 (100)
	Total	28	1 (3.6)	3 (10.7)

The chicken brand was indicated using alphabets.

E. albertii-specific real-time PCR was performed with primers and probe of 16S rRNA-specific real-time PCR.

EA-rtPCR, real-time PCR assays targeting E. albertii-specific gene; PCR, polymerase chain reaction.

Chicken carcass

Twenty-one chicken carcasses were purchased directly from three chicken farms in Japan between December 2018 and March 2019 (Supplementary Table S2). The chickens raised in the same chicken coop at the same period were recognized for the same lot, and farms 1, 2, and 3 had one, three, and four lots, respectively (Supplementary Table S2). The carcasses were dissected and 193 body parts were obtained: 16 crops and esophagi, 21 gallbladders, 21 gizzards, 21 glandular stomachs, 17 hearts, 17 hip skins, 21 intestines, 21 livers, 17 neck skins, and 21 tracheas (Table 2). Because of the large volume of gizzard and liver, three times the amount of mEC or mEC supplemented with novobiocin (NmEC; Eiken Chemicals, Tokyo, Japan) was added. For the other samples, nine times the amount of mEC or NmEC was added and homogenized for 1 min. After 22 ± 2 h incubation at 42°C, DNA was extracted from each 100 μL of the enriched culture using alkaline DNA extraction and used as a template for nested and duplex EA-rtPCR as described hereunder.

Table 2.

Escherichia albertii Detection in Body Parts of Chicken Carcass Samples

Body part^a	Number of samples	Number of PCR-positive sample (%)^b		Number of E. albertii-isolated samples (%)
Body part^a	Number of samples	Nested PCR	Duplex EA-rtPCR^c	Number of E. albertii-isolated samples (%)
Crop and esophagus	16	4 (25.0)	4 (25.0)	0
Gallbladder	21	3 (14.3)	4 (19.0)	0
Gizzard	21	2 (9.5)	5 (23.8)	1 (4.8)
Glandular stomach	21	4 (19.0)	4 (19.0)	0
Heart	17	2 (11.8)	4 (23.5)	0
Hip skin	17	0	0	NT
Intestine	21	8 (38.1)	11 (52.4)	1 (4.8)
Liver	21	6 (28.6)	6 (28.6)	1 (4.8)
Neck skin	17	0	1 (5.9)	0
Trachea	21	7 (33.3)	9 (42.9)	4 (19.0)
Total	193	36 (18.7)	48 (24.9)	7 (3.6)

Brand K.

All nested PCR-positive samples were positive for real-time PCR assay.

E. albertii-specific real-time PCR was performed with primers and probe of 16S rRNA-specific real-time PCR.

EA-rtPCR, real-time PCR assays targeting E. albertii-specific gene; NT, not tested; PCR, polymerase chain reaction.

Primer and probe design for EA-rtPCR and PCR conditions

EACBF0500 (CDS numbers in strain CB9786) that shared >99% nucleotide sequence identity in the 55 sequences of E. albertii by blastn analysis (Ooka et al, 2015) was selected as target genes for designing primers and probes (Supplementary Table S3). A total of 43 E. albertii strains listed in Supplementary Table S1 were amplified using the primers EACBF0500F and EACBF0500R (Supplementary Table S3). Sequencing was performed using a Big Dye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific). Nucleotide sequences were determined using SeqStudio Genetic Analyzer (Thermo Fisher Scientific), and assembled and aligned with CLC Genomics Workbench version 12.0.3 (CLC bio, Aarhus, Denmark). The nucleotide sequences of EACBF0500 in the other 75 E. albertii strains were obtained by a blastn search, and those of 113 E. albertii strains were assembled and aligned as described above. Nucleotide sequences that did not have gaps between E. albertii strains were selected, and primers and probes were designed using Primer 3 (http://bioinfo.ut.ee/primer3-0.4.0/) (Supplementary Table S3).

Nested-PCR assay for E. albertii (Ooka et al, 2015) was performed as described previously (Arai et al, 2021). Two types of EA-rtPCR were performed: one targeting only E. albertii (single) and another targeting both E. albertii and 16S rRNA gene as an internal control for real-time PCR (duplex). Each EA-rtPCR was performed in a total reaction volume of 30 μL containing 5 μL of DNA template, 1 × TaqMan Environmental Master Mix 2.0 (Thermo Fisher Scientific), 0.3 μM of each primer for E. albertii, and 0.15 μM probe for E. albertii. Duplex EA-rtPCR also contained 0.16 μM of each primer and 0.1 μM probe that is specific for 16S rRNA gene (Supplementary Table S3). The EA-rtPCR was performed using Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific) with the cycling conditions given in Supplementary Table S3.

Evaluation of the specificity and sensitivity of EA-rtPCR

The specificity of the nested PCR and EA-rtPCR were evaluated using 10 ng of genomic DNA from the bacteria listed in Supplementary Table S1. E. coli, Escherichia fergusonii, Hafnia alvei, Salmonella enterica subsp. enterica serovar Typhimurium, Shigella boydii, and Shigella flexneri have been found to be closely related to E. albertii (Lukjancenko et al, 2010; Na et al, 2018; Oh et al, 2011). A single EA-rtPCR for E. albertii was performed to evaluate specificity.

The sensitivity of the EA-rtPCR was evaluated using E. albertii type strain. The TSB culture of JCM 17328^T was diluted from 10⁻¹ to 10⁻⁷ with phosphate-buffered saline (PBS). DNA was extracted from 100 μL of each undiluted culture and dilutions by alkaline DNA extraction. Both single and duplex EA-rtPCR were performed. The highest bacterial dilution positive for PCR in triplicate was considered as the detection limit. A 100 μL of the appropriate dilution was inoculated onto tryptic soy agar (TSA; BD, Franklin Lakes, NJ). The colonies were counted after incubation at 37°C for 18 h. Sensitivity of EA-rtPCR with chicken meat culture was determined as described previously (Arai et al, 2021). In brief, E. albertii EA12, EA21, EA24, and EA29, isolated from individuals with foodborne infections or diarrhea in Japan, were chosen as representative strains. DNA extracted from the dilutions (10⁻⁴ to 10⁻⁷) of each bacterial culture with chicken mEC or NmEC culture were used as a template for the duplex EA-rtPCR.

Detection of E. albertii from chicken meat using EA-rtPCR

TSB culture of E. albertii strains EA12, EA21, EA24, and EA29 was diluted with PBS. Each 100 μL of the dilution of 10⁻⁶ to 10⁻⁸ was inoculated into 25 g of chicken meat placed in a sterile stomacher bag, homogenized with 225 mL of mEC or NmEC for 1 min, and incubated at 42°C for 22 ± 2 h. DNA was extracted and duplex EA-rtPCR was performed as described above.

Isolation and characterization of E. albertii from chicken samples

Retail chicken meat and carcass samples that tested positive in the nested-PCR assay were used for E. albertii isolation. Ten microliters of the enriched culture was streaked on DHL and incubated at 37 ± 1°C for 18 h. Noncolored colonies were subjected to a colony PCR assay as described previously (Arai et al, 2021), and PCR-positive isolate was streaked on TSA for purification and incubated at 37°C for 18 h. DNA was extracted using the heat extraction method. In brief, the colony on TSA was suspended in 100 μL of sterilized Tris-ethylenediaminetetraacetic acid buffer. After heating at 100°C for 10 min, the suspension was cooled on ice. The suspension was then centrifuged at 10,000 × g for 10 min, and the 100 μL of DNA-containing supernatant was used for single EA-rtPCR.

To define the biochemical properties, PCR-positive colonies were transferred to triple sugar iron agar (Oxoid, Hampshire, United Kingdom), lysine indole motility medium (Nissui), and Andrade peptone water (Oxoid) with one of the following three carbohydrates (1% w/v): lactose (Lac-AND), sucrose (Suc-AND), or d-(+)-xylose (Xyl-AND). The cultures were incubated at 37 ± 1°C for 22 ± 2 h. PCR targeting eae (Schmidt et al, 1994), stx1, stx2 (Nielsen and Andersen, 2003), and stx2f (Scheutz et al, 2012) was performed as described previously. In addition, E. albertii-specific O-genotyping (EAO genotyping), which distinguish variety of O-antigen biosynthesis gene clusters of E. albertii, of these isolates was performed using multiplex PCR as described previously (Ooka et al, 2019).

Results

Specificity of PCR assays

Nested PCR and the developed EA-rtPCR specifically amplified E. albertii and not other species, including close relatives of E. albertii (Supplementary Table S1).

E. albertii detection sensitivity of EA-rtPCR

With E. albertii-enriched culture, the detection limit of EA-rtPCR was 0.5 log colony-forming unit (CFU) per reaction (2.8 log CFU/mL) for both single and duplex assays (Table 3). The detection limits of EA-rtPCR were also determined using enriched chicken meat culture. The detection limits of EA-rtPCR for DNA extracted from the enriched mEC and NmEC culture were −0.2 to 0.9 log CFU per reaction (2.0–3.2 log CFU/mL) and −0.3 to 0.9 log CFU per reaction (2.0–3.2 log CFU/mL), respectively (Table 4).

Table 3.

Detection Limits of Real-Time Polymerase Chain Reaction Assays Targeting Escherichia albertii-Specific Gene

Bacterial concentration (log CFU/mL)	Bacterial concentration (log CFU/reaction)	EA-rtPCR-positive number/tested number ^a (average Ct values)^b
Bacterial concentration (log CFU/mL)	Bacterial concentration (log CFU/reaction)	Single EA-rtPCR^c		Duplex EA-rtPCR^d
8.8	6.5	3/3^c	(16.8)	3/3	(16.9)
7.8	5.5	3/3	(19.7)	3/3	(19.8)
6.8	4.5	3/3	(23.2)	3/3	(23.4)
5.8	3.5	3/3	(26.6)	3/3	(26.7)
4.8	2.5	3/3	(30.5)	3/3	(30.6)
3.8	1.5	3/3	(33.9)	3/3	(34.1)
2.8	0.5	3/3	(37.5)	3/3	(37.8)
1.8	−0.5	2/3	(40.0)	1/3	(40.8)

PCR reactions were performed in triplicate.

Average Ct values for E. albertii were indicated.

E. albertii-specific real-time PCR was performed without primers and probe of 16S rRNA-specific real-time PCR.

E. albertii-specific real-time PCR was performed with primers and probe of 16S rRNA-specific real-time PCR.

CFU, colony-forming unit; Ct, threshold cycle; EA-rtPCR, real-time PCR assays targeting E. albertii-specific gene; PCR, polymerase chain reaction.

Table 4.

Sensitivity of Real-Time Polymerase Chain Reaction Assays Targeting Escherichia albertii-Specific Gene in Chicken Meat Culture with Two Enrichment Broths and Four Escherichia albertii Strains

Enrichment broth	Strain	Detection limit of duplex EA-rtPCR^a,b
Enrichment broth	Strain	Bacterial concentration (log CFU/mL)	Bacterial concentration (log CFU/reaction)
mEC	EA12	2.0	−0.2
	EA21	3.2	0.9
	EA24	3.1	0.8
	EA29	3.0	0.7
	Noninoculation	ND	ND
NmEC	EA12	2.0	−0.2
	EA21	3.2	0.9
	EA24	3.1	0.8
	EA29	2.0	−0.3
	Noninoculation	ND	ND

E. albertii-specific real-time PCR was performed with primers and probe of 16S rRNA-specific real-time PCR.

PCR reactions were performed in triplicate.

CFU, colony-forming unit; EA-rtPCR, real-time PCR assays targeting E. albertii-specific gene; mEC, modified Escherichia coli broth; ND, not detected; NmEC, novobiocin; PCR, polymerase chain reaction.

E. albertii detection in chicken meat using EA-rtPCR

When E. albertii was inoculated in chicken meat, the detection limits of EA-rtPCR were equivalent (0.1 log CFU/25 g) among the two enrichment broths and four E. albertii strains (Table 5).

Table 5.

Comparison of the Detection Limits of Real-Time Polymerase Chain Reaction Assays Targeting Escherichia albertii-Specific Gene in Enriched Cultures of Chicken Inoculated with Escherichia albertii

Enrichment broth	Strain	Detection limit of duplex EA-rtPCR^a,b
Enrichment broth	Strain	Bacterial concentration (log CFU/mL)
mEC	EA12	0.1
	EA21	0.1
	EA24	0.1
	EA29	0.1
	Noninoculation	ND
NmEC	EA12	0.1
	EA21	0.1
	EA24	0.1
	EA29	0.1
	Noninoculation	ND

E. albertii-specific real-time PCR was performed with primers and probe of 16S rRNA-specific real-time PCR.

PCR reactions were performed in triplicate.

E. albertii detection and isolation from retail chicken meat and carcass samples

Of the 28 retail chicken meat samples, 1 (thigh meat of brand J) and 3 (drum stick and thigh meats of brand C and thigh meat of brand J) samples were positive for nested-PCR assay and EA-rtPCR, respectively (Table 1). A thigh meat sample from brand J showed positive results for all three of the triplicates subjected to EA-rtPCR with cycle threshold (Ct) values of 35.7, 35.8, and 36.6. The drum stick and thigh meats of brand C showed positive results for 1/3 triplicates in EA-rtPCR with Ct values of 38.2 and 41.0, respectively. E. albertii was not isolated from these PCR-positive retail chicken meat samples.

Carcasses with at least one EA-rtPCR-positive body part were considered E. albertii positive. Of the 21 carcasses obtained from three farms, 14 carcasses (67%) were positive for E. albertii (Supplementary Table S2). Chickens from farms 1, 2, and 3 showed 100%, 100%, and 30% E. alberii-positivity rate, respectively. On farm 3, lots 3-1 and 3-4, 3-2, and 3-3 exhibited E. alberii-positivity rates of 0%, 50%, and 67%, respectively. Among 193 body parts from these 21 carcasses, 36 (18.7%) and 48 (24.9%) samples were positive for nested-PCR assay and EA-rtPCR, respectively (Table 2). All nested PCR-positive samples were positive for EA-rtPCR. A total of 37 strains of E. albertii were isolated from seven PCR-positive samples: a gizzard, a intestine, a liver, and four trachea samples (Supplementary Table S4).

Genetic characteristics of E. albertii isolated from chicken samples

All E. albertii isolates were positive for eae and negative for stx1, stx2, and stx2f (Supplementary Table S4). E. albertii isolated from chicken nos. 1, 2, 3, and 6 and 4 and 5 were EAOg3 and EAOg4, respectively. E. albertii isolated from each chicken body part sample displayed the same results.

Discussion

Takahashi et al (2020) have reported an E. albertii-specific real-time PCR assay utilizing primers reported by Maeda et al (2014) and the originally designed probe but did not evaluate its specificity and sensitivity. We developed a novel real-time PCR assay for the specific detection of E. albertii in chickens. The sensitivity of EA-rtPCR was <0.9 log CFU/reaction (<3.2 log CFU/mL) in both cases: only E. albertii strains and E. albertii strains with enriched chicken meat culture were used. In addition, the detection limits of EA-rtPCR in enriched culture of chicken inoculated with E. albertii were the same among mEC and NmEC and four E. albertii strains (0.1 log CFU/25 g), suggesting that EA-rtPCR is sensitive enough to detect E. albertii in chicken meat (25 g) contaminated with 1 CFU of E. albertii. The contamination level of E. albertii in food residues from foodborne outbreaks remains unknown, but our EA-rtPCR may be effective in detecting E. albertii in such food samples.

Previous studies involving the same four E. albertii strains used in this study have reported nested-PCR assay sensitivities of −0.2 to 1.1 log CFU/reaction and 0.8 to 1.2 log CFU/reaction in chicken meat cultured in mEC and NmEC, respectively (Arai et al, 2021). This suggests that the sensitivity of EA-rtPCR for chicken meat is almost the same or slightly superior to that of the nested-PCR assay. The difference in sensitivity between these two assays may depend on the volume of template DNA used for PCR (1 μL for nested PCR assay and 5 μL for EA-rtPCR). Two retail chicken meat samples showed positive and negative reactions in EA-rtPCR and nested-PCR assay, respectively. The Ct values of these two samples were 38.2 and 41.0, suggesting that the quantity of their PCR products might be too less to observe specific bands on agarose gel by electrophoresis.

Three retail chicken meat samples showed a positive reaction in EA-rtPCR, but E. albertii was not isolated from these samples. Considering the obscurity of the source of this pathogen in previously reported outbreaks in Japan and the difficulty in isolating it from food and water sources, the development of two-step enrichment and bead concentration methods would be effective in isolating E. albertii from food residues in foodborne outbreaks, similar to those caused by other pathogens (Hara-Kudo et al, 2020; Hara-Kudo et al, 2016). In addition, the Ct value of real-time PCR assay may be an indicator for the second enrichment or isolation step because the Ct value is useful for estimating the population of E. albertii in the enriched culture. Of the 28 retail chicken meat samples, 1 showed a positive reaction with the nested-PCR assay but not with 1st PCR (data not shown). Thus, performing two independent PCR assays is effective for sensitive E. albertii detection.

Chickens from all farms were positive for E. albertii. Moreover, many E. albertii strains were isolated from tracheal samples (Supplementary Table S4), suggesting that the rearing environment in the farms may be polluted by E. albertii. Because E. albertii has been reported in the feces of many wild bird species (Gordon, 2011; Grillová et al, 2018; Hinenoya et al, 2022; Ooka, 2017) and 4.5% of pigeon droppings (Murakami et al, 2019), chickens should be raised inside the chicken coop to avoid E. albertii infection from wild birds.

Because all the E. albertii isolates from foodborne infections possess eae (Ooka, 2017), eae presence in E. albertii isolates is important for evaluating its impact on humans. All the E. albertii isolates in this study were positive for eae; therefore, to prevent foodborne infection with E. albertii, it is important to avoid intake of uncooked or undercooked chicken meat and cross-contamination from chicken to other foods. Furthermore, all the isolates were grouped into EAOg3 and EAOg4 by EAO genotyping, possibly because almost all these isolates were obtained from farm 2. It was reported that several isolates from chicken and humans belonged these EAO genotypes (Ooka et al, 2019). Information on the prevalence of E. albertii for each EAOg type is required to elucidate the variation in EAOg type in each host animal.

In conclusion, we report a novel real-time PCR assay specific for E. albertii. Because this EA-rtPCR showed high sensitivity and specificity for E. albertii detection, it is anticipated that it will improve E. albertii detection in food and advance research on E. albertii infection.

Footnotes

Acknowledgment

The authors thank Naoaki Misawa for assistance with collection of samples.

Authors' Contributions

S.A. and Y.H.-K. designed the study. S.A., T.O. and Y.H.-K. developed real-time PCR assays for E. albertii. S.A., M.S., Y.N., Y.T., H.N., R.M., A.T., Y.K., K.O., N.K., and K.O. collected chicken samples and performed PCR assays. T.O. provided bacterial DNA. Y.H.-K. provided funding. All authors approved the article.

Disclosure Statement

The authors declare no conflicts of interest associated with this article.

Funding Information

This work was supported by the Ministry of Health, Labour and Welfare, Japan (Health Labour Sciences Research Grant No. H30-syokuhinippan-001).

Supplementary Material

Supplementary Table S1

Supplementary Table S2

Supplementary Table S3

Supplementary Table S4

References

Arai

, Ohtsuka

, Konishi

, et al. Evaluating methods for detecting Escherichia albertii in chicken meat. J Food Prot, 2021; 84(4):553–562; doi: 10.4315/jfp-20-206

Asoshima

, Matsuda

, Shigemura

, et al. Isolation of Escherichia albertii from raw chicken liver in Fukuoka City, Japan. Jpn J Infect Dis, 2015; 68(3):248–250; doi: 10.7883/yoken.JJID.2014.530

Brandal

, Tunsjo

, Ranheim

, et al. Shiga toxin 2a in Escherichia albertii . J Clin Microbiol, 2015; 53(4):1454–1455; doi: 10.1128/jcm.03378-14

Etoh

, Murakami

, Ichihara

, et al. Isolation of Shiga toxin 2f-producing Escherichia coli (O115:HNM) from an adult symptomatic patient in Fukuoka Prefecture, Japan. Jpn J Infect Dis, 2009; 62(4):315–317.

, Rogers

, Feng

, et al. Bacterial cytolethal distending toxin promotes the development of dysplasia in a model of microbially induced hepatocarcinogenesis. Cell Microbiol, 2007; 9(8):2070-2080; doi: 10.1111/j.1462-5822.2007.00939.x

Gordon

. Reservoirs of Infection: The Epidemiological Characteristics of an Emerging Pathogen, Escherichia albertii . Department of Agirculture, Fisheries and Forestry; Division of Ecology, Evolution and Genetics; Research School of Biology; The Australian National University Canberra: Canberra, Australia; 2011.

Grillová

, Sedláček

, Páchníková

, et al. Characterization of four Escherichia albertii isolates collected from animals living in Antarctica and Patagonia. J Vet Med Sci, 2018; 80(1):138–146; doi: 10.1292/jvms.17-0492

Güven

, Azizoglu

. The recent original perspectives on nonculture-based bacteria detection methods: A comprehensive review. Foodborne Pathog Dis, 2022; 19(7):425–440; doi: 10.1089/fpd.2021.0078

Hara-Kudo

, Konishi

, Ohtsuka

, et al. An interlaboratory study on efficient detection of Shiga toxin-producing Escherichia coli O26, O103, O111, O121, O145, and O157 in food using real-time PCR assay and chromogenic agar. Int J Food Microbiol, 2016; 230:81–88; doi: 10.1016/j.ijfoodmicro.2016.03.031

10.

Hara-Kudo

, Ohtsuka

, Konishi

, et al. An interlaboratory study on the detection methods for enterotoxigenic Escherichia coli in vegetables using enterotoxin gene screening and selective agars for ETEC-specific isolation. Int J Food Microbiol, 2020; 334:108832; doi: 10.1016/j.ijfoodmicro.2020.108832

11.

Hinenoya

, Awasthi

, Yasuda

, et al. Detection, isolation and molecular characterization of Escherichia albertii in wild birds in West Japan. Jpn J Infect Dis, 2022; 75(2):156–163; doi: 10.7883/yoken.JJID.2021.355

12.

Hinenoya

, Ichimura

, Yasuda

, et al. Development of a specific cytolethal distending toxin (cdt) gene (Eacdt)-based PCR assay for the detection of Escherichia albertii . Diagn Microbiol Infect Dis, 2019; 95(2):119–124; doi: 10.1016/j.diagmicrobio.2019.04.018

13.

Hinenoya

, Nagano

, Okuno

, et al. Development of XRM-MacConkey agar selective medium for the isolation of Escherichia albertii . Diagn Microbiol Infect Dis, 2020; 97(1):115006; doi: 10.1016/j.diagmicrobio.2020.115006

14.

Hinenoya

, Naigita

, Ninomiya

, et al. Prevalence and characteristics of cytolethal distending toxin-producing Escherichia coli from children with diarrhea in Japan. Microbiol Immunol, 2009; 53(4):206–215; doi: 10.1111/j.1348-0421.2009.00116.x

15.

Hinenoya

, Yasuda

, Hibino

, et al. Isolation and characterization of an Escherichia albertii strain producing three different toxins from a child with diarrhea. Jpn J Infect Dis, 2017a;70(3):252–257; doi: 10.7883/yoken.JJID.2016.186

16.

Huys

, Cnockaert

, Janda

, et al. Escherichia albertii sp. nov., a diarrhoeagenic species isolated from stool specimens of Bangladeshi children. Int J Syst Evol Microbiol, 2003; 53(Pt 3):807–810; doi: 10.1099/ijs.0.02475-0

17.

Hyma

, Lacher

, Nelson

, et al. Evolutionary genetics of a new pathogenic Escherichia species: Escherichia albertii and related Shigella boydii strains. J Bacteriol, 2005; 187(2):619–628; doi: 10.1128/JB.187.2.619-628.2005

18.

Kashio

, Konno

, Takahashi

, et al. Identification of multiple Escherichia albertii O-genotypes as etiological agents in a foodborne disease outbreak. Jpn J Food Microbiol, 2020; 37(4):183–187; doi: 10.5803/jsfm.37.183

19.

Lindsey

, Garcia-Toledo

, Fasulo

, et al. Multiplex polymerase chain reaction for identification of Escherichia coli, Escherichia albertii and Escherichia fergusonii . J Microbiol Methods, 2017; 140:1–4; doi: 10.1016/j.mimet.2017.06.005

20.

Lukjancenko

, Wassenaar

, Ussery

. Comparison of 61 sequenced Escherichia coli genomes. Microb Ecol, 2010; 60(4):708–720; doi: 10.1007/s00248-010-9717-3

21.

Maeda

, Murakami

, Okamoto

, et al. Nonspecificity of primers for Escherichia albertii detection. Jpn J Infect Dis, 2014; 67(6):503–505; doi: 10.7883/yoken.67.503

22.

McAuley

, Linden

, Png

, et al. MUC1 cell surface mucin is a critical element of the mucosal barrier to infection. J Clin Invest, 2007; 117(8):2313–2324; doi: 10.1172/jci26705

23.

Muchaamba

, Barmettler

, Treier

, et al. Microbiology and epidemiology of Escherichia albertii-an emerging elusive foodborne pathogen. Microorganisms, 2022; 10(5):875; doi: 10.3390/microorganisms10050875

24.

Murakami

, Etoh

, Tanaka

, et al. Shiga toxin 2f-producing Escherichia albertii from a symptomatic human. Jpn J Infect Dis, 2014; 67(3):204–208; doi: 10.7883/yoken.67.204

25.

Murakami

, Maeda-Mitani

, Kimura

, et al. Non-biogroup 1 or 2 strains of the emerging zoonotic pathogen Escherichia albertii, their proposed assignment to biogroup 3, and their commonly detected characteristics. Front Microbiol, 2019; 10:1543; doi: 10.3389/fmicb.2019.01543

26.

, Kim

, Yoon

, et al. UBCG: Up-to-date bacterial core gene set and pipeline for phylogenomic tree reconstruction. J Microbiol, 2018; 56(4):280–285; doi: 10.1007/s12275-018-8014-6

27.

Nielsen

, Andersen

. Detection and characterization of verocytotoxin-producing Escherichia coli by automated 5’ nuclease PCR assay. J Clin Microbiol, 2003; 41(7):2884–2893; doi: 10.1128/jcm.41.7.2884-2893.2003

28.

Oaks

, Besser

, Walk

, et al. Escherichia albertii in wild and domestic birds. Emerg Infect Dis, 2010; 16(4):638–646; doi: 10.3201/eid1604.090695

29.

, Kang

, Hwang

, et al. Epidemiological investigation of eaeA-positive Escherichia coli and Escherichia albertii strains isolated from healthy wild birds. J Microbiol, 2011; 49(5):747–752; doi: 10.1007/s12275-011-1133-y

30.

Ooka

. Emerging enteropathogen, Escherichia albertii . Jpn J Food Microbiol, 2017; 34(3):151–157.

31.

Ooka

, Ogura

, Katsura

, et al. Defining the genome features of Escherichia albertii, an emerging enteropathogen closely related to Escherichia coli . Genome Biol Evol, 2015; 7(12):3170–3179; doi: 10.1093/gbe/evv211

32.

Ooka

, Seto

, Kawano

, et al. Clinical significance of Escherichia albertii . Emerg Infect Dis, 2012; 18(3):488–492; doi: 10.3201/eid1803.111401

33.

Ooka

, Seto

, Ogura

, et al. O-antigen biosynthesis gene clusters of Escherichia albertii: Their diversity and similarity to Escherichia coli gene clusters and the development of an O-genotyping method. Microb Genom, 2019; 5:e000314; doi: 10.1099/mgen.0.000314

34.

Pandey

, Khan

, Das

, et al. Association of cytolethal distending toxin locus cdtB with enteropathogenic Escherichia coli isolated from patients with acute diarrhea in Calcutta, India. J Clin Microbiol, 2003; 41(11):5277–5281; doi: 10.1128/jcm.41.11.5277-5281.2003

35.

Scheutz

, Teel

, Beutin

, et al. Multicenter evaluation of a sequence-based protocol for subtyping Shiga toxins and standardizing Stx nomenclature. J Clin Microbiol, 2012; 50(9):2951–2963; doi: 10.1128/jcm.00860-12

36.

Schmidt

, Plaschke

, Franke

, et al. Differentiation in virulence patterns of Escherichia coli possessing eae genes. Med Microbiol Immunol, 1994; 183(1):23–31; doi: 10.1007/bf00193628

37.

Takahashi

, Konno

, Kashio

, et al. Isolation and characterization of Escherichia albertii from environmental water in Akita Prefecture, Japan. Jpn J Food Microbiol, 2020; 37(2):81–86.

38.

Tokoi

, Kataoka

, Wakatsuki

, et al. Case report of Escherichia albertii food poisoning in Utsunomiya City. Jpn J Food Microbiol, 2018; 35(3):159–162.

39.

Wang

, Li

, Bai

, et al. Prevalence of eae-positive, lactose non-fermenting Escherichia albertii from retail raw meat in China. Epidemiol Infect, 2016; 144(1):45–52; doi: 10.1017/S0950268815001120

40.

Young

, Knox

, Pratt

, et al. In vitro and in vivo characterization of Helicobacter hepaticus cytolethal distending toxin mutants. Infect Immun, 2004; 72(5):2521–2527; doi: 10.1128/iai.72.5.2521-2527.2004

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.01 MB