Development of XRM-MacConkey agar selective medium for the isolation of Escherichia albertii

Journal Pre-proof Development of XRM-MacConkey agar selective medium for the isolation of Escherichia albertii

Atsushi Hinenoya, Keigo Nagano, Kentaro Okuno, Akira Nagita, Noritoshi Hatanaka, Sharda Prasad Awasthi, Shinji Yamasaki PII:

S0732-8893(19)31010-7

DOI:

https://doi.org/10.1016/j.diagmicrobio.2020.115006

Reference:

DMB 115006

To appear in:

Diagnostic Microbiology & Infectious Disease

Received date:

5 October 2019

Revised date:

25 January 2020

Accepted date:

26 January 2020

Please cite this article as: A. Hinenoya, K. Nagano, K. Okuno, et al., Development of XRM-MacConkey agar selective medium for the isolation of Escherichia albertii, Diagnostic Microbiology & Infectious Disease(2020), https://doi.org/10.1016/ j.diagmicrobio.2020.115006

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© 2020 Published by Elsevier.

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Development of XRM-MacConkey agar selective medium for the isolation of Escherichia albertii

Atsushi Hinenoyaa,b,c, Keigo Naganoa, Kentaro Okunob, Akira Nagitad, Noritoshi

b

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School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan Graduate School of Life and Environmental Sciences, Osaka Prefecture University,

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a

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Hatanakab, Sharda Prasad Awasthib and Shinji Yamasakia,b,c

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Osaka, Japan

Asian Health Science Institute, Osaka Prefecture University, Osaka, Japan Department of Pediatrics, Mizushima General Hospital, Okayama, Japan

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d

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Running title: Escherichia albertii selective medium

Word count of abstract: 150/150 words Word count of text: 2480/3500 words excluding reference section

#

Corresponding author: Shinji Yamasaki, PhD

Mailing address: Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58, Rinku ourai-kita, Izumisano, Osaka 598-8531, Japan Tel/Fax: +81-72-463-5653 E-mail address: [email protected] 1

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Abstract (150/150) Escherichia albertii has increasingly been recognized as an emerging pathogen. However, lack of selective medium for E. albertii is the bottleneck for clinical and epidemiological investigations. In this study, a selective medium for E. albertii named XRM-MacConkey agar, which is modified MacConkey agar supplemented with xylose

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(X), rhamnose (R) and melibiose (M) instead of lactose was developed and evaluated. All 49 E. albertii and 6 different species out of 23 grew as colorless colonies whereas

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17 remaining species grew as red colonies. Detection limit of E. albertii by this medium

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was 105 CFU/g stool when examined with spiked healthy human stool. Furthermore,

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colorless colonies on XRM-MacConkey agar obtained from 7 E. albertii-positive

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diarrheal stools were consistently E. albertii. In contrast, 57, 18 and 36% colorless colonies on MacConkey, DHL and mEA agars, respectively, were non-E. albertii. We

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concluded that XRM-MacConkey agar could specifically be used for isolation of E.

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albertii.

Keyword: Escherichia albertii, selective medium, sugar fermentation, MacConkey agar, XRM-MacConkey agar

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Introduction Escherichia albertii is a human enteropathogen and an avian pathogen with epidemic mortality (Hinenoya et al., 2017a, 2017b; Huys et al., 2003; Murakami et al., 2014; Oaks et al., 2010; Ooka et al., 2012). E. albertii is an attaching and effacing pathogen like enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and

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EHEC, respectively) and Citrobacter rodentium, which causes characteristic lesions of pedestal structure on intestinal epithelial cells by using type 3 secretion system encoded

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by the locus of enterocyte effacement (LEE), a pathogenicity island. Furthermore, E.

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albertii genome carries cytolethal distending toxin (cdt) genes and generally produces

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CDT consisting of 3 subunits, CdtA, CdtB and CdtC, which could be associated with

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virulence and persistent colonization of host (Scuron et al., 2016). Certain strains additionally produce Shiga toxin 2 (Stx2a, Stx2f) or carry those genes (Brandal et al.,

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2005; Hinenoya et al., 2017a, 2019a; Murakami et al., 2014; Ooka et al., 2012), which is one of the most important virulence factors in Shiga toxin-producing E. coli including

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serotype O157:H7, causing severe diseases such as hemorrhagic colitis, hemolytic uremic syndrome and neurological disorder. E. albertii has increasingly been recognized as an important emerging zoonotic pathogen worldwide, since it has been isolated from patients (Hinenoya et al., 2017a, 2017b; Huys et al., 2003; Murakami et al., 2014; Ooka et al., 2012) as well as from animals (Hinenoya et al., 2014; Oaks et al., 2010). However, except for a few outbreaks that occurred in Kumamoto (2012), Okinawa (2016) and Shizuoka (2016) in Japan (Ooka et al., 2013; National Institute of Infectious Diseases 2016), most E. albertii strains were re-identified from isolates which were initially identified as Hafnia alvei, EPEC, EHEC, EcCDT-II-producing E. coli or Shigella boydii due to similar 3

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biochemical properties of these pathogens. These misidentifications were due to conventional

biochemical

Enterobacteriaceae-selective

tests media

performed such

as

after

isolation

by

using

DHL

(deoxycholate-hydrogen

sulfide-lactose), XLD (xylose lysine deoxycholate) and MacConkey agars. Thereafter, they were re-identified as E. albertii through their extensive characterizations by

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DNA-DNA hybridization or multilocus sequence analysis (Hinenoya et al., 2014, 2017b, 2019a; Huys et al., 2003; Oaks et al., 2010; Ooka et al., 2012). Genomic

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characterization of those E. albertii strains has led to the development of multiple E.

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albertii-specific PCRs by which this bacterium could be identified accurately, in a

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simple and rapid manner (Hinenoya et al., 2019b; Hyma et al., 2005; Lindsey et al.,

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2017; Ooka et al., 2015). However, in addition to E. albertii-specific PCR, E. albertii-selective medium is required to isolate and further characterize E. alberti strains

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for examining their virulence properties, clonality and antimicrobial resistance. Recently, mEA agar has been developed as an E. albertii isolation medium but this medium itself

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was not sufficient for specific isolation of E. albertii (Maheux et al., 2018). It has been reported that E. albertii cannot utilize xylose, rhamnose and melibiose whereas most E. coli strains can utilize these sugar (Hinenoya et al., 2014, 2017a, 2017b, 2019a; Maheux et al., 2014; Ooka et al., 2012). In this study, therefore, we have attempted to develop a selective medium for E. albertii based on the utilization of different sugars between E. coli and E. albertii, and evaluated its sensitivity and specificity for the isolation of E. albertii by comparing with various selective media. The medium developed in this study showed much better isolation efficiency of E. albertii than DHL, MacConkey and mEA agars.

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Materials and Methods Bacterial strains Bacterial strains used in this study are listed in Table S1 and they were grown in Luria-Bertani (LB) broth (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) at 37°C for 14-16 h with vigorous shaking. Providencia spp. GTC strains, and E. coli

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strain JCM1649T and H. alvei strain JCM1666T were purchased from Gifu University Center for Conservation of Microbial Genetic Resource Organization for Research and

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Community Development (GCMR), and RIKEN BioResource Research Center,

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respectively.

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Growth on MacConkey agar supplemented with various sugars instead of lactose

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Forty gram per liter of MacConkey agar base powder (Becton, Dickinson and Company) was dissolved in appropriate volume of distilled water, and the mixture was

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autoclaved. Then, D-(+)-xylose, L-(+)-rhamnose and α-D-(+)-melibiose solutions separately sterilized by autoclaving were added to the medium at the final concentration

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of 1% (w/v) each, and the medium was poured into petri dishes (90 mm in diameter). A loopful of enrichment culture of LB broth was streaked onto the agar plates, and the agar plates were incubated at 37°C for 18-24 h. D-(+)-xylose and α-D-(+)-melibiose, and L-(+)-rhamnose were purchased from Nacalai Tesque, Inc. (Kyoto, Japan) and FUJIFILM Wako Pure Chemical Corp. (Osaka, Japan), respectively. Spiking experiment A fresh stool sample was obtained from a healthy person. A stool culture was confirmed negative for enteric bacterial pathogens including cdt- and eae-gene positive bacteria by specific PCR assays for both genes (Hinenoya et al, 2014). A portion of the stool (0.2 g) was mixed with 10 μL of early log-phase E. albertii strain 19982T, which 5

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was serially diluted to 108 to 103 CFU/mL. The spiked stools were homogenized with 9 volume of sterilized PBS (-) yielding 10% (w/v) stool suspension and then, a loopful of spiked suspension was streaked onto XRM-MacConkey agar, and the media were incubated at 37°C for 18-24 h. Simultaneously, another set of spiked stools were prepared, from which DNA was extracted by QIAamp® DNA Stool Mini Kit (QIAGEN,

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Venlo, Netherlands) following a protocol for pathogen detection. DNA extracts from

targeting Eacdt genes (Hinenoya et al., 2019b).

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spiked stools and colorless colonies were analyzed by an E. albertii-specific PCR assay

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isolation from clinical specimens

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Comparison between XRM-MacConkey and other selective agars for E. albertii

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Seven diarrheal stool specimens kept at -80C as a glycerol stock of enrichment culture of trypticase soy broth (TSB; Becton, Dickinson and Company) from which E.

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albertii was previously isolated (Hinenoya et al., 2017b) were used for evaluating the specificity and efficiency of XRM-MacConkey agar. Aliquot of the glycerol stock was

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inoculated into 3 mL of TSB (Becton, Dickinson and Company) and incubated at 37C for about 12 h. The enrichment culture was serially diluted and the diluents were spread onto XRM-MacConkey, MacConkey (Eiken Chemical Co., Ltd., Tokyo, Japan), DHL (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) and mEA (Maheux et al., 2018) agars, respectively, and the media were incubated at 37°C for 18-24 h. Red, blue and colorless colonies obtained from each medium was inoculated on Hybond-N+ membrane (GE Healthcare UK Ltd., Buckinghamshire, UK) overlaid on MacConkey agar and grown at 37°C. The membranes were subjected to colony hybridization assay using

32

P-labeled

EacdtB (former Eccdt-IIB) gene-probe, which is E. albertii-specific, as described previously (Hinenoya et al., 2014). EacdtB gene-positive colonies were judged to be E. 6

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albertii. Since majority of E. albertii strains cannot ferment lactose and sucrose (Hinenoya et al., 2017b; Maheux et al., 2014; Ooka et al., 2012), most of E. albertii form colorless colonies and can be differentiated from lactose and sucrose fermenting bacteria based on the color of colonies by MacConkey and DHL agars, which contain lactose and lactose/sucrose, respectively. E. albertii also form colorless colonies on

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mEA agar (Maheux et al., 2018).

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Results

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Selection of sugar combination supplemented into MacConkey agar base

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All the 49 E. albertii strains grew as colorless colonies on MacConkey agar base

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supplemented either with xylose (X), rhamnose (R) and/or melibiose (M). When supplemented with single sugar (X, R or M), out of 23 different species, 11 (48%), 12

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(52%) and 16 (70%) non-E. albertii species formed colorless colonies, respectively. When supplemented with two sugars (XR, XM or RM), 7 (30%), 9 (39%) and 10 (43%)

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non-E. albertii species formed colorless colonies, respectively. Furthermore, when supplemented with all 3 sugars (XRM), 6 (26%) non-E. albertii species, namely Morganella morganii, Plesiomonas shigelloides, Providencia alcalifaciens, P. rustigianii, P. stuartii and Pseudomonas aeruginosa formed colorless colonies (Table 1). Representative pictures of E. coli and/or E. albertii colonies are shown in Fig. 1. These data suggest that MacConkey agar base medium supplemented with 3 sugars (XRM) was the best to differentiate E. albertii from other bacterial species and thus, the medium developed was named XRM-MacConkey agar. XRM-MacConkey agar was further evaluated with several strains of Shigella spp., Salmonella enterica and E. coli including intestinal and extra-intestinal pathotypes. 7

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While E. coli and S. enterica grew as red colonies on the XRM-MacConkey agar, most of the Shigella strains formed colorless colonies. Detection limit of XRM-MacConkey agar for E. albertii Detection limit of XRM-MacConkey agar for E. albertii was evaluated in healthy human stool spiked with various number of E. albertii (107-102 CFU/g stool). As shown

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in Table 2, E. albertii-like colorless colonies were obtained from the samples spiked with 107 to 105 CFU/g stool, and confirmed to be E. albertii by an E. albertii-specific

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PCR (Hinenoya et al, 2019b). However, the samples less than 104 CFU/g stool

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including the non-spiked stool yielded only red colonies. These data suggested that

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detection limit of this selective medium is 105 CFU/g stool (Table 2).

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Efficiency of XRM-MacConkey agar to isolate E. albertii from clinical specimens Enrichment culture of 7 clinical human fecal specimens, from which E. albertii

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was previously isolated, was spread onto XRM-MacConkey, MacConkey, DHL and mEA agars, respectively. As shown in Table 3, when 7 samples were cultured by

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XRM-MacConkey agar, all the colorless colonies were positive for E. albertii by colony hybridization assay but all red colonies were negative. Similarly, when cultured by MacConkey agar, all colorless colonies obtained from P3321 and P4839 were E. albertii as described above. However, E. albertii-positive rates of P2543, P3502 and P2855 were 76, 15 and 12%, respectively. None of the colorless colonies from P3662 and P8790 were positive for E. albertii. When cultured by DHL agar, 6 samples showed E. albertii-positive rate of 88 to 100%. However, in P4839, no colorless colonies were obtained and E. albertii-specific genes were detected in 14 out of 50 red colonies by colony hybridization assay. When the same samples were cultured by mEA agar, E. albertii-positive rate was 81 to 100% in 5 samples and only 2.3 and 9.3% positive rates 8

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were obtained from P3662 and P3502, respectively. These data indicate that XRM-MacConkey agar is the most efficient medium for the isolation of E. albertii from fecal specimens of diarrheal patients.

Discussion

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Since there was no selective medium for E. albertii, which can differentiate from other bacteria including E. coli, real picture of E. albertii infection was not clear.

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Therefore, a selective medium for differential isolation of E. albertii has been desired.

for

various

pathogens.

For

example,

sorbitol-MacConkey

and

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developed

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Previously based on differential utilization of sugars several selective media have been

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rhamnose-MacConkey agars have been widely used for the isolation of EHEC serogroups O157 and O26, respectively (Hiramatsu et al., 2002; March and Ratnam,

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1986). Recent studies revealed that E. albertii is unable to ferment certain carbohydrates such as cellobiose, dulcitol, melibiose, rhamnose and xylose (Hinenoya et al., 2017b,

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2019a; Ooka et al., 2012). Among them, we selected 3 sugars, xylose, rhamnose and melibiose, which can be utilized by most E. coli but not by E. albertii, and developed an E. albertii-selective medium named XRM-MacConkey agar in this study. This newly developed selective medium has a detection limit of 105 CFU/g stool for E. albertii and could detect E. albertii from E. albertii-positive clinical stool specimens as colorless colonies with 100% sensitivity and specificity by a retrospective analysis. Thus, XRM-MacConkey agar could be an efficient and perhaps reliable E. albertii-selective medium for E. albertii. Recently, Maheux et al. (2018) have developed an E. albertii-specific isolation medium, called mEA agar. This medium differentiated E. albertii from other bacteria 9

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based on the capabilities of fermentation of cellobiose and -glucuronidase production. However, similar to E. albertii (0%) fermentation of cellobiose in E. coli is also quite low (2%) and certain E. coli strains, such as EHEC O157:H7, cannot produce -glucuronidase. Indeed, E. albertii and EHEC O157:H7 could not be differentiated by mEA agar (Maheux et al., 2018). In addition, specificity of the mEA was not high

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enough for the isolation of E. albertii from clinical specimens. When mEA agar was used for the isolation of E. albertii with the 7 clinical specimens, although isolation rate

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was high (81 to 100%) in 4 samples but was very low (0 to 9%) in 3 samples (Table 3).

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On the other hand, XRM-MacConkey agar could differentiate E. albertii from E. coli

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including EHEC O157:H7. Furthermore, XRM-MacConkey agar could also distinguish

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E. albertii from Citrobacter freundii, S. enterica and Proteus mirabilis, which formed E. albertii-like colonies on mEA agar. However, similar to mEA agar, XRM-MacConkey

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agar could not distinguish E. albertii clearly from M. morganii, P. shigelloides, Providencia spp. and Shigella spp., suggesting that there is also a limitation of

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XRM-MacConkey agar. Therefore, E. albertii-specific PCR is recommended to apply for species confirmation after isolation of suspicious E. albertii colonies on XRM-MacConkey agar.

Previously, E. albertii has been isolated by using Enterobacteriaceae-selective media such as MacConkey and sorbitol MacConkey agars on which E. albertii grew as colorless colonies due to their inability to ferment lactose and sorbitol, respectively (Hinenoya et al., 2014, 2017a, 2017b; National Institute of Infectious Diseases 2016). However, isolation of E. albertii by using MacConkey agar was not efficient owing to non-specific colorless colony formation by non-E. albertii bacteria (Table 3). In contrast, DHL agar could detect and differentiate E. albertii in 6 out of the 7 clinical specimens, 10

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where E. albertii was detected as colorless colonies due to non-fermentation of both lactose and sucrose. But E. albertii in P4839 was detected as red colony due to its sucrose fermentation. Further, given that E. albertii strains fermenting lactose and sorbitol in addition to sucrose have also been isolated (Hinenoya et al., 2017b, 2019a; Maheux et al., 2014; Ooka et al., 2012). Therefore, DHL, XLD and Hektoen enteric

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agars, which contain lactose and sucrose, are not sufficient for selective isolation of E. albertii.

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In summary, in this study a novel, simple and efficient E. albertii-specific

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selective medium, named XRM-MacConkey agar, has been developed, which could be

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useful for the isolation of E. albertii from clinical samples and unveil the real burden of

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human and animal E. albertii infections, its animal reservoir, infection source and route. However, this study indicated that XRM-MacConkey agar was not 100% specific. Thus,

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it is recommended to use both XRM-MacConkey agar and E. albertii-specific PCR for

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isolation and accurate identification of E. albertii.

Acknowledgements

We thank Dr. Rupak K. Bhadra, CSIR-Indian Institute of Chemical Biology, Kolkata, India for critically reading the manuscript. We also thank Dr. Eric Oswald, Inserm-INRA-ENVT-Université de Toulouse, Drs. T. Ramamurthy and G.B. Nair, National Institute of Cholera and Enteric Diseases, and Dr. Wanpen Chaicumpa, Mahidol University for providing E. coli, Shigella spp., Vibrio cholerae and Salmonella strains. This work was supported in part by JSPS KAKENHI Grant Numbers 17659131 to S.Y.

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Conflict of interest None to declare. Ethical statement None to declare.

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References Brandal LT, Tunsjø HS, Ranheim TE, Løbersli I, Lange H, Wester AL. 2005. Shiga

ro

toxin 2a in Escherichia albertii. J Clin Microbiol 53:1454-1455.

-p

Hinenoya A, Shima K, Asakura M, Nishimura K, Tsukamoto T, Ooka T, Hayashi T,

re

Ramamurthy T, Faruque SM, Yamasaki S. 2014. Molecular characterization of

lP

cytolethal distending toxin gene-positive Escherichia coli from healthy cattle and swine in Nara, Japan. BMC Microbiol 14:97.

na

Hinenoya A, Yasuda N, Hibino T, Shima A, Nagita A, Tsukamoto T, Yamasaki S. 2017a. Isolation and characterization of an Escherichia albertii producing three

Jo ur

different toxins isolated from a child with diarrhea. Jpn J Infect Dis 70:252-257. Hinenoya A, Yasuda N, Mukaizawa N, Sheikh S, Niwa Y, Awasthi SP, Asakura M, Tsukamoto T, Nagita A, Albert MJ, Yamasaki S. 2017b. Association of cytolethal distending toxin-II gene-positive Escherichia coli with Escherichia albertii, an emerging zoonotic pathogen. Int J Med Microbiol 307:564-571. Hinenoya A, Ichimura H, Awasthi SP, Yasuda N, Yatsuyanagi J, Yamasaki S. 2019a. Phenotypic and molecular characterization of Escherichia albertii: Further surrogates to avoid potential laboratory misidentification. Int J Med Microbiol 309:108-115. Hinenoya A, Ichimura H, Yasuda N, Harada S, Suzuki M, Iijima Y, Hatanaka N, Awasthi SP, Yamasaki S. 2019b. Development of a cytolethal distending toxin (cdt) 12

Journal Pre-proof

gene-based PCR for the detection and identification of Escherichia albertii. Diagn Microbiol Infect Dis 95:119-124. Hiramatsu R, Matsumoto M, Miwa Y, Suzuki Y, Saito M, Miyazaki Y. 2002. Characterization of Shiga toxin-producing Escherichia coli O26 strains and establishment of selective isolation media for these strains. J Clin Microbiol

of

40:922-925. Huys G, Cnockaert M, Janda JM, Swings J. 2003. Escherichia albertii sp. Nov., a

ro

diarrhoeagenic species isolated from stool specimens of Bangladeshi children. Int J Syst

-p

Evol Microbiol 53:807-810.

re

Hyma KE, Lacher DW, Nelson AM, Bumbaugh AC, Janda JM, Strockbine NA,

lP

Young VB, Whittam TS. 2005. Evolutionary genetics of a new pathogenic Escherichia species: Escherichia albertii and related Shigella boydii strains. J Bacteriol

na

187:619-628.

Lindsey RL, Garcia-Toledo L, Fasulo D, Gladney LM, Strockbine N. 2017.

Jo ur

Multiplex polymerase chain reaction for identification of Escherichia coli, Escherichia albertii and Escherichia fergusonii. J Microbiol Methods 140:1-4. Maheux AF, Boudreau DK, Bergeron MG, Rodriguez MJ. 2014. Characterization of Escherichia fergusonii and Escherichia albertii isolated from water. J Appl Microbiol 117:597-609. Maheux AF, Brodeur S, Bérubé È, Boudreau DK, Abed JY, Boissinot M, Bissonnette L, Bergeron MG. 2018. Method for isolation of both lactose-fermenting and - non-fermenting Escherichia albertii strains from stool samples. J Microbiol Methods 154:134-140. March SB, Ratnam S. 1986. Sorbitol-MacConkey medium for detection of 13

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Escherichia coli O157:H7 associated with hemorrhagic colitis. J Clin Microbiol 23:869-872. Murakami K, Etoh Y, Tanaka E, Ichihara S, Horikawa K, Kawano K, Ooka T, Kawamura Y, Ito K. 2014. Shiga toxin 2f-producing Escherichia albertii from a symptomatic human. Jpn J Infect Dis 67:204-208.

of

National Institute of Infectious Diseases. 2016. Infect Agents Surveillance Rep 37:252-255.

ro

Oaks JL, Besser TE, Walk ST, Gordon DM, Beckmen KB, Burek KA, Haldorson

-p

GJ, Bradway DS, Ouellette L, Rurangirwa FR, Davis MA, Dobbin G, Whittam TS.

re

2010. Escherichia albertii in wild and domestic birds. Emerg Infect Dis 16:638-646.

lP

Ooka T, Seto K, Kawano K, Kobayashi H, Etoh Y, Ichihara S, Kaneko A, Isobe J, Yamaguchi K, Horikawa K, Gomes TA, Linden A, Bardiau M, Mainil JG, Beutin L,

Dis 18:488-492.

na

Ogura Y, Hayashi T. 2012. Clinical significance of Escherichia albertii. Emerg Infect

Jo ur

Ooka T, Tokuoka E, Furukawa M, Nagamura T, Ogura Y, Arisawa K, Harada S, Hayashi T. 2013. Human gastroenteritis outbreak associated with Escherichia albertii, Japan. Emerg Infect Dis 19:144-146. Ooka T, Ogura Y, Katsura K, Seto K, Kobayashi H, Kawano K, Tokuoka E, Furukawa M, Harada S, Yoshino S, Seto J, Ikeda T, Yamaguchi K, Murase K, Gotoh Y, Imuta N, Nishi J, Gomes TA, Beutin L, Hayashi T. 2015. Defining the genome features of Escherichia albertii, an emerging enteropathogen closely related to Escherichia coli. Genome Biol Evol 7:3170-3179. Scuron MD, Boesze-Battaglia K, Dlakić M, Shenker BJ. 2016. The cytolethal distending toxin contributes to microbial virulence and disease pathogenesis by acting 14

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as a tri-perditious toxin. Front Cell Infect Microbiol 6:168.

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Author statement

A. H. and S. Y. designed research, A. H., K. N., K. O., N. H. and S. P. A. performed research, A. H., K. N., A. N., N. H., S. P. A. and S. Y. analyzed data, and A. H. and S. Y.

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wrote the paper.

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T

Fig. 1. A) Colorless colonies of Escherichia albertii LMG20976 , B) Red colonies of T

Escherichia coli strain JCM1649 , C) Colonies from mixture of E. albertii LMG20976

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and E. coli JCM1649 .

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Table 1. Growth of E. albertii and other bacteria on MacConkey agar plates supplemented with sugars. Strain

n=

E. albertii

Supplemented sugars XRM

XR

XM

RM

X

R

M

49

Colorless

Colorless

Colorless

Colorless

Colorless

Colorless

Colorless

E. coli (EHEC O157:H7)

1

Red

Red

Red

Red

Red

Red

Red

E. vulneris

1

Red

Red

Red

Red

Red

Red

Red

Citrobacter freundii

1

Red

Red

Red

Red

Red

Red

Red

C. koseri

1

Red

Red

Red

Red

Red

Red

Colorless

Hafnia alvei

1

+/-

+/-

+/-

+/-

+/-

+/-

Klebsiella oxytoca

1

Red

Red

K. pneumoniae

1

Red

Red

Morganella morganii

1

Colorless

Colorless

r P

Red

Red

Red

Red

Red

Red

Red

Red

Red

Colorless

Colorless

Colorless

Colorless

Colorless

Proteus mirabilis

3

Red

Red

Red

Colorless

Red

Colorless

Colorless

P. penneri

2

Red

Red

Red

Colorless

Red

Colorless

Colorless

P. vulgaris

2

Red

Red

Red

Colorless

Red

Colorless

Colorless

Plesiomonas shigelloides

1

Colorless

Colorless

Colorless

Colorless

Colorless

Colorless

Colorless

Providencia alcalifaciens

1

Colorless

Colorless

Colorless

Colorless

Colorless

Colorless

Colorless

P. heimbachae

1

Red

Red

Colorless

Red

Colorless

Red

Colorless

P. rettgeri

1

Red

Red

Colorless

Red

Colorless

Red

Colorless

P. rustigianii

1

Colorless

Colorless

Colorless

Colorless

Colorless

Colorless

Colorless

P. stuartii

1

Colorless

Colorless

Colorless

Colorless

Colorless

Colorless

Colorless

Pseudomonas aeruginosa

1

Colorless

Colorless

Colorless

Colorless

Colorless

Colorless

Colorless

l a

o J

n r u

+/-

Red

f o

o r p

e

18

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Salmonella Enteritidis

1

Red

Red

Red

Red

Red

Red

Red

S. Typhimurium

1

Red

Colorless

Red

Red

Colorless

Colorless

Red

S. Brunei/Krefeld

1 each

Red

Red

Red

Red

Red

Colorless

Red

1

Red

Red

Red

Red

Colorless

Red

Red

15

Red

Red

Red

Red

Red

Red

Shigella dysenteriae

1

Red

Red

Colorless

Red

Colorless

Red

Colorless

S. boydii

1

Red

Red

Red

Colorless

Red

Colorless

Colorless

S. sonnei

1

Red

Red

Red

Red

f o

Red

Red

Red

Colorless

S. flexneri

1

Red

Red

Red

Red

Red

Red

Colorless

Other E. coli

41

Red

ND

ND

ND

ND

ND

S. Enteritidis

5

Red

ND

ND

ND

ND

ND

ND

S. Typhimurium

3

Red

Red

r P Red

Red

ND

ND

ND

S. dysenteriae

9

Colorless

ND

ND

ND

ND

ND

ND

S. boydii

1

Red

ND

ND

ND

ND

ND

ND

Colorless

ND

ND

ND

ND

ND

ND

Red

ND

ND

ND

ND

ND

ND

14

Colorless

ND

ND

ND

ND

ND

ND

9

Colorless

ND

ND

ND

ND

ND

ND

S. Anatum Other Salmonella enterica

*

Additionally analyzed

S. sonnei S. flexneri

1

n r u

o J

18

l a

ND

o r p

e

+/-, almost no growth; ND, not done; X, xylose; R, rhamnose, M, Melibiose; XR, xylose and rhamnose; XM, xylose and rhamnose; RM, rhamnose and melibiose; XRM, xylose, rhamnose and rhamnose *One of each species of S. Agona, Derby, Eastboune, Hadar, Huittinfoss, Infantis, Mueachen, Newport, Poona, Saintpaul, Stanley, Welikade, Weltevreden, Virchow, and i,8,20:y-

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Table 2. Detection limit of Escherichia albertii in spiked healthy human stools. No. of bacteria spiked

XRM-MacConkey Eacdt-PCR

(CFU/g stool)

Colorless +

+

2.5×106

+

+

2.5×105

+

+

2.5×104

-

2.5×103

-

2.5×102

-

0

-

of

2.5×107

ro

-

-

Jo ur

na

lP

re

-p

-

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Table 3. Detection of Escherichia albertii in human diarrheal stool specimens by using E. albertii-selective and other Enterobacteriaceae -selective agar plates. XRM-MacConkey agar

MacConkey agar

DHL agar

Specimen

Colorless

Red

Colorless

Red

Colorless

Red

P2543

50a/50b (100%c)

0/50 (0%)

38/50 (76%)

0/4 (0%)

46/50 (92%)

P2855

99/99 (100%)

0/101 (0%)

6/50 (12%)

0/50 (0%)

50/50 (100%)

P3321

50/50 (100%)

0/50 (0%)

50/50 (100%)

0/50 (0%)

50/50 (100%)

P3662

8/8 (100%)

0/50 (0%)

0/50 (0%)

0/50 (0%)

P3502

39/39 (100%)

0/50 (0%)

8/54 (15%)

0/50 (0%)

P8790

4/4 (100%)

0/50 (0%)

0/50 (0%)

P4839

50/50 (100%)

0/50 (0%)

49/49 (100%)

a

No. of tested colonies

b c

Colorless

Red/Pink

Blue

0/12 (0%)

42/43 (98%)

0/38 (0%)

0/5 (0%)

0/50 (0%)

38/43 (88%)

0/43 (0%)

No colony

0/40 (0%)

43/43 (100%)

0/12 (0%)

0/31 (0%)

4/4 (100%)

0/50 (0%)

4/43 (9.3%)

0/33 (0%)

0/10 (0%)

31/32 (97%)

0/50 (0%)

1/43 (2.3%)

0/42 (0%)

0/43 (0%)

0/50 (0%)

7/8 (88%)

0/50 (0%)

6/6 (100%)

0/37 (0%)

No colony

0/50 (0%)

No colony

14/50 (28%)

35/43 (81%)

0/43 (0%)

No colony

l a

r P

J

No. of E. albertii-specific gene (Eacdt)-positive colonies

E. albertii-positive rate

21

f o

o r p

e

rn

u o

mEA agar

Figure 1