Abstract
It is challenging to isolate Escherichia albertii from clinical specimens. Therefore, a medium that can selectively grow E. albertii and differentiate it from E. coli is earnestly desired. Here, we describe the evaluation of a recently developed selective differential medium, called cefixime-tellurite-phosphate-xylose-rhamnose-MacConkey (CT-PS-XR-MacConkey) medium, which enables the specific growth of E. albertii and differentiation of E. albertii (colorless) from E. coli (red) based on colony color and thus, facilitating the efficient isolation of E. albertii from diarrheal stool. When three E. albertii negative diarrheal stools were inoculated onto CT-PS-XR-MacConkey and xylose-rhamnose-melibiose (XRM) containing MacConkey agars, no colorless colonies were observed on both the media. However, when E. albertii was spiked into these three diarrheal stools, the ratio of colorless colonies to red colonies was higher on CT-PS-XR-MacConkey agar compared to XRM-MacConkey agar in all three samples. Notably, out of 105 Eacdt-gene PCR negative diarrheal stools 56 yielded colorless colonies on MacConkey agar while out of these 56 diarrheal stools, nine yielded colorless colonies on XRM-MacConkey but no colorless colonies were observed on CT-PS-XR-MacConkey agar. Furthermore, evaluation of these two media with five E. albertii positive-stool specimens revealed that the number of red colonies were constantly less, whereas that of colorless colonies were constantly more on CT-PS-XR-MacConkey agar, thus aiding in efficient isolation. Altogether, these results suggest that the CT-PS-XR-MacConkey agar could be a useful selective differential medium for isolation of E. albertii from diarrheal stool specimens.
Keywords: Escherichia albertii, selective medium, cefixime-tellurite-phosphate-xylose-rhamnose MacConkey agar, isolation method, clinical specimens
INTRODUCTION
In the year 1991, Escherichia albertii was initially isolated in Bangladesh as an eae gene-positive Hafnia alvei from a 9-month-old infant with diarrhea [1]. Because atypical enteropathogenic E. coli (aEPEC), enterohemorrhagic E. coli (EHEC), cytolethal distending toxin II gene-positive E. coli (CTEC-II, now identified as E. albertii) and Shigella boydii serotype 13 are very similar to E. albertii in terms of virulence gene profiles in addition to biochemical and genetic properties, E. albertii has often been misidentified as aEPEC, EHEC, CTEC-II or S. boydii [5, 10, 11, 28, 31]. E. albertii has been isolated not only from children but also from adults with gastroenteritis [6, 11, 26, 31]. Oaks et al. [29] reported isolation of E. albertii from dead wild birds. Subsequently, numerous reports have described isolation of E. albertii not only from various wild birds but also from wild mammals [14, 15, 27], indicating that E. albertii is an emerging zoonotic pathogen. Moreover, E. albertii has recently been isolated from patients with extra-intestinal infections such as urinary tract infection, sepsis, etc. [9, 18, 23, 40]. E. albertii carrying stx2a and stx2f genes have been isolated from patients with diarrhea, respectively [8, 11, 26]. Furthermore, stx2f gene-positive E. albertii has been isolated from a patient with hemolytic-uremic syndrome [19]. Given its pathogenic potential, zoonotic nature, and ability to cause infections, it is necessary to rapidly detect and isolate E. albertii from clinical specimens which in turn will help us to better understand the characteristics of clinical E. albertii isolates and managing the infection.
In recent past, at least 11 outbreaks of food-poisoning in Japan have been reported [5, 25, 26]. Suspected causative agents were environmental water, vegetables, etc. but not chicken and beef as in the case of Campylobacter and EHEC infections. Indeed, E. albertii has frequently been detected in wild mammals such as raccoon and raccoon dogs as well as in wilds birds [7, 14,15,16, 21, 27, 30, 31]. Furthermore, E. albertii has also been detected in chickens, pork, oysters, vegetables and environmental water bodies [4, 20, 24, 34, 37], although the positive detection rate in these sources is relatively low.
Various PCR methods including conventional PCR [12, 17, 22], nested-PCR [32] and real-time PCR [3, 6, 34] have been developed and utilized for the detection of E. albertii not only from patient samples but also from food and environmental samples. These PCR methods have contributed to enhance the detection and subsequent successful isolation of E. albertii. However, despite having higher detection rate of E. albertii in tested samples, isolation rate is not always high [38]. Thus, enrichment media, differential media and selective differential media are urgently required to enhance the isolation rate of E. albertii from various specimens. Keeping this in mind, recently various enrichment cultures for the isolation of E. albertii from food samples and fecal specimens of wild animals have been developed [2, 36, 38]. In addition, xylose-rhamnose-melibiose (XRM)-MacConkey agar has been developed to differentiate between E. albertii and E. coli based on the colony color where E. albertii forms colorless colonies while E. coli forms red colonies [13].
XRM-MacConkey agar is a differential medium, therefore, for more efficient isolation of E. albertii a selective differential medium is needed. Very recently, our group has developed a selective differential medium, named cefixime–tellurite-phosphate-xylose-rhamnose-MacConkey or simply CT-PS-XR-MacConkey medium, for the efficient isolation of E. albertii from chicken meat samples [35]. In this medium, cefixime and tellurite inhibit the growth of most bacteria belonging to Enterobacterales while phosphate and soy peptone enhance the growth of E. albertii in the presence of tellurite. In this study, we evaluated this newly developed E. albertii selective differential medium to test whether this medium is applicable to human diarrheal specimens for efficient isolation of E. albertii.
MATERIALS AND METHODS
Collection and analysis of clinical samples
Diarrheal stool swabs (SWEEDSWAB γ1, Eiken chemical Co., Tokyo) were collected from adults and children at Mizushima Central Hospital, Okayama, Japan, between November 2020 and August 2021. Presence or absence of E. albertii was examined by culture method using XRM-MacConkey agar [13], Eacdt gene-based conventional PCR [12] and Eacdt gene-based real-time PCR as described previously [6]. Informed consent was always taken from patients for using their fecal specimens for research purpose. In case of children, consent was obtained from their respective parents or legal guardians. Clinical analysis conducted in this study was approved by the ethical committee of Osaka Prefecture University (OPUGSLES-19-E12 and OPUGSLES-20-E18).
Spiking of E. albertii to E. albertii-negative diarrheal stools for the evaluation of CT-PS-XR-MacConkey agar
E. albertii strain JCM 17328T was cultured in tryptic soy broth (TSB) at 37°C for 15 ± 1 hr. Subsequently, 20 µL of the overnight culture was transferred into 3 mL of fresh TSB and incubated at 37°C with shaking at 180 rpm for 3 hr. Bacterial cells were collected by centrifugation at 9,800 g for 5 min at 4°C, washed twice with phosphate-buffered saline (PBS; pH 7.4), and optical density at 600 nm (OD600) was adjusted to 0.1 (equivalent to ~7 log CFU/mL). E. albertii negative diarrheal stool swabs (n=3) were suspended in 1.0 mL of PBS and 450 µL suspension was spiked with 50 µL of either prepared E. albertii (~5.7 log CFU) or PBS (non-spike control). CFU enumeration was carried out using XRM-MacConkey, and CT-PS-XR-MacConkey agar. After incubation, the number of red and colorless colonies were enumerated on each media.
Colorless colonies were identified as E. albertii by colony hybridization assay using 32P-labelled EacdtB gene-probe [10]. In brief, all the colorless colonies obtained from XRM-MacConkey and CT-PS-XR-MacConkey agar were spotted on the nitrocellulose membrane (GE healthcare life sciences, Marlborough, MA, USA) which was placed onto LB agar. The spotted colonies were incubated at 37°C for 4–6 hr. Subsequently, colonies were lysed by alkaline lysis (1.0 N NaOH), neutralized by 1.0 M Tris-HCl buffer (pH 7.0) three times and 1.0 M Tris-HCl buffer (pH 7.0) containing 1.5 M NaCl, followed by UV cross-linking (Ultra-Violet Products Ltd., Upland, CA, USA). The EacdtB gene-probe from E. albertii strain AH-5 was prepared by PCR using primers as described previously [12]. The gene-probe was labeled with 111 TBq/ mmol [α-32P]-dCTP (Perkin Elmer, Wellesley, MA, USA) by random priming method using Multiprime DNA labeling kit (Cytiva, Marlborough, MA, USA). The processed membranes were hybridized with the 32P-labelled EacdtB gene-probe under high stringency conditions as reported previously [39], and radioactivity was visualized using the BAS FLA-3000 system (Cytiva).
Comparative evaluation of newly developed CT-PS-XR-MacConkey and XRM-MacConkey agars for colony types in E. albertii negative diarrheal fecal suspensions
E. albertii negative diarrheal fecal swabs were assessed for colony types on MacConkey, DHL, XRM-MacConkey and CT-PS-XR-MacConkey agars. Fecal swabs were suspended in 1.0 mL of PBS (pH 7.4), mixed well and serially diluted. Aliquots of 100 µL were spread onto these four agar media. The plates were incubated at 37°C for 20 ± 2 hr. Subsequently, number of red and colorless colonies were enumerated on each medium. Furthermore, the colorless colonies were identified using 16S rRNA gene sequencing using universal primers.
Analysis of Eacdt gene-positive stool specimens by culturing on CT-PS-XR-MacConkey and XRM-MacConkey agars
Five Eacdt gene-positive diarrheal fecal swabs were used for the evaluation of the newly developed CT-PS-XR-MacConkey medium. The fecal swab was suspended in 1.0 mL of PBS (pH 7.4), mixed well using vortex and serially diluted. Aliquots of 100 µL were spread onto XRM-MacConkey and CT-PS-XR-MacConkey agar plates, respectively. The plates were incubated at 37°C for 20 ± 2 hr. Subsequently, the number of red and colorless colonies were enumerated, respectively, on each medium. Furthermore, the colorless colonies were identified as E. albertii using Eacdt gene-based PCR [12]. The non-E. albertii colorless colonies were identified using 16S rRNA gene sequencing using universal primers as described below.
16S rRNA gene sequencing for bacterial identification
Non-E. albertii colorless colonies on XRM-MacConkey and CT-PS-XR-MacConkey were subjected to sequencing of 16S rRNA for identification of bacterial species using universal primers 9F (5′-GAGTTTGATCCTGCTC-3′) and 520R (5′-ACCGCGGCTGCTGGC-3′) [33] by Sanger sequencing.
RESULTS
Comparative evaluation of newly developed CT-PS-XR-MacConkey and XRM-MacConkey agars using E. albertii-spiked diarrheal fecal suspensions
For the spiking experiment, three E. albertii-negative diarrheal fecal specimens were selected. As shown in Table 1, without spiking, no colorless colonies were obtained on either CT-PS-XR-MacConkey or XRM-MacConkey agar plates in these three fecal specimens. However, plenty of red colonies were present on both the agar plates and the number of red colonies were constantly higher on XRM-MacConkey agar in all samples tested. When E. albertii was spiked into these stool specimens, colorless colonies were observed with higher numbers on CT-PS-XR-MacConkey agar plate compared to XRM-MacConkey agar. While red colonies were constantly more on XRM-MacConkey agar, CT-PS-XR-MacConkey agar consistently yielded more colorless colonies. All the colorless colonies obtained from both the agar plates were confirmed as E. albertii by colony hybridization assay using the Eacdt gene-probe as described above. Positive rate of E. albertii is summarized in Table 1 and was constantly higher on CT-PS-XR-MacConkey agar than that of XRM-MacConkey agar.
Table 1. Comparative analysis of CT-PS-XR-MacConkey and XRM-MacConkey agars using Escherichia albertii-spiked diarrheal fecal suspensions.
| Sample | Medium | Non-spike |
Spike |
||
|---|---|---|---|---|---|
| Red/colorless (CFU) |
Red/colorless (CFU) |
E. albertii*/colorless (%) | E. albertii*/Red+colorless (%) | ||
| OKY594 | CT-PS-XR-MacConkey | 89/0 | 63/35 | 35/35 (100) | 35/98 (35) |
| XRM-MacConkey | 126/0 | 117/16 | 16/16 (100) | 16/133 (12) | |
| OKY596 | CT-PS-XR-MacConkey | 162/0 | 181/29 | 29/29 (100) | 29/210 (13.8) |
| XRM-MacConkey | 190/0 | 186/26 | 26/26 (100) | 26/212 (12) | |
| OKY597 | CT-PS-XR-MacConkey | 151/0 | 146/24 | 24/24 (100) | 24/170 (14.1) |
| XRM-MacConkey | 217/0 | 267/8 | 8/8 (100) | 8/275 (2.9) | |
*E. albertii was confirmed by colony hybridization assay using the EacdtB gene-probe; CFU: colony forming unit; CT-PS-XR-MacConkey: cefixime-tellurite-phosphate-xylose-rhamnose MacConkey agar; XRM MacConkey: Xylose-Rhamnose-Mellibiose MacConkey agar.
Evaluation of CT-PS-XR-MacConkey and XRM-MacConkey agars by culturing with Eacdt-gene PCR negative diarrheal stool specimens
To compare the specificity of both CT-PS-XR-MacConkey or XRM-MacConkey agar plates, Eacdt-gene PCR-negative diarrheal stools (n=105) were first inoculated onto MacConkey agar plates to see colony characteristics such as colony color because some of E. albertii form colorless colony on MacConkey agar due to lack of lactose fermentation. As shown in Supplementary Table 1, five (4.8%) specimens did not produce any colonies on MacConkey. Forty-four (42%) specimens yielded only red colonies while 56 (53.3%) produced colorless colonies and out of the 56 specimens 47 specimens also produced red colonies. The 56 specimens which produced colorless colonies on MacConkey were further inoculated onto DHL (because these two media are often used for isolation of bacteria from diarrheal specimens), XRM-MacConkey and CT-PS-XR-MacConkey media as shown in Supplementary Table 2. On DHL, 53.6% (30/56) specimens produced only red colonies, whereas 46.4% (26/56) specimens produced red and colorless (due to lack of lactose and sucrose fermentations) colonies. When tested with XRM-MacConkey, 84% (47/56) specimens produced only red colonies, while 16% (9/56) of the specimens produced mixed (red and colorless) colonies. Interestingly, on CT-PS-XR-MacConkey, all 56 specimens did not produce any colorless colonies (Supplementary Table 2). Table 2 shows the number of red and colorless colonies from the nine samples (which produced mixed color colonies on XRM-MacConkey) on XRM-MacConkey and CT-PS-XR-MacConkey agars. Similar to the previous result as observed with spiking experiment, the number of red colonies were constantly higher on XRM-MacConkey than CT-PS-XR-MacConkey agar. The colorless colonies observed on XRM-MacConkey medium were streaked onto CT-PS-XR-MacConkey agars and it was found that they could not grow in this newly developed media (data not shown). These colorless colonies were identified as Pseudomonas aeruginosa, Morganella morganii or Providencia rettgeri by 16S rRNA gene sequencing (Table 2). Taken together, these results suggest the inhibitory nature of CT-PS-XR-MacConkey agar for non-E. albertii colorless colonies.
Table 2. Evaluation of CT-PS-XR-MacConkey and XRM-MacConkey agars.
| Sample | Medium | Red/colorless (CFU) | Colorless colony sequence* |
|---|---|---|---|
| OKY231 | CT-PS-XR-MacConkey | 156/0 | NA |
| XRM-MacConkey | 187/5 | Pseudomonas aeruginosa | |
| OKY261 | CT-PS-XR-MacConkey | 161/0 | NA |
| XRM-MacConkey | 214/5 | Pseudomonas aeruginosa | |
| OKY285 | CT-PS-XR-MacConkey | 174/0 | NA |
| XRM-MacConkey | 263/2 | Pseudomonas aeruginosa | |
| OKY290 | CT-PS-XR-MacConkey | 21/0 | NA |
| XRM-MacConkey | 66/6 | Morganella morganii | |
| OKY299 | CT-PS-XR-MacConkey | 113/0 | NA |
| XRM-MacConkey | 161/21 | Morganella morganii | |
| OKY303 | CT-PS-XR-MacConkey | 117/0 | NA |
| XRM-MacConkey | 143/5 | Morganella morganii | |
| OKY310 | CT-PS-XR-MacConkey | 125/0 | NA |
| XRM-MacConkey | 145/24 | Morganella morganii | |
| OKY345 | CT-PS-XR-MacConkey | 141/0 | NA |
| XRM-MacConkey | 250/2 | Providencia rettgeri | |
| OKY348 | CT-PS-XR-MacConkey | 283/0 | NA |
| XRM-MacConkey | 374/11 | Morganella morganii | |
Nine fecal samples which were negative by Eacdt-gene PCR but formed colorless colonies on XRM-MacConkey were evaluated by both CT-PS-XR-MacConkey and XRM-MacConkey agars, *Single colony was randomly selected and bacterial species was confirmed by 16S rDNA sequencing; CFU: colony forming unit; CT-PS-XR-MacConkey: cefixime-tellurite-phosphate-xylose-rhamnose MacConkey agar; XRM MacConkey: Xylose-Rhamnose-Mellibiose MacConkey agar. NA: not applicable.
Evaluation of XRM-MacConkey and CT-PS-XR-MacConkey agars using Eacdt gene-positive stool specimens
Since surveillance of E. albertii in stool specimens from diarrheal patients is routinely conducted in our laboratory, as soon as E. albertii positive stool specimen was obtained, it was inoculated onto both CT-PS-XR-MacConkey and XRM-MacConkey agars to evaluate the efficiency of isolating E. albertii. As shown in Table 3, when five Eacdt-gene positive fecal samples were analyzed, red colonies were consistently more abundant in XRM-MacConkey in all five samples. It should be noted that OKY361 yielded only colorless colonies but not red colonies on CT-PS-XR-MacConkey agar. All the eight randomly selected colorless colonies were identified as E. albertii, indicating that CT-PS-XR-MacConkey agar is highly specific. In another fecal specimen (OKY203), out of eight randomly selected colorless colonies from XRM-MacConkey, four were identified as E. albertii, however, remaining four were identified as M. morganii. All eight randomly selected colorless colonies from CT-PS-XR-MacConkey from this sample were identified as E. albertii. Overall, the number of CFUs obtained on the CT-PS-XR-MacConkey agar was consistent with the CFUs estimated by real-time PCR as previously reported [6], except one case, where the number of colonies was ~2 log lower (Table 3). Taken together, both CT-PS-XR-MacConkey and XRM-MacConkey agars seem to be reliable and useful isolation medium for E. albertii from clinical specimens, however, in some cases CT-PS-XR-MacConkey may have an advantage when interfering colorless colonies like M. morganii, P. aeruginosa or P. rettgeri are present in the fecal samples.
Table 3. Evaluation of CT-PS-XR-MacConkey and XRM-MacConkey agars using the Eacdt gene-positive stool specimens.
| Sample | Medium | Red colonies (CFU) |
Colorless colonies (CFU)/total colony (%) | Colorless colonies for Ea-PCR*1 | E. albertii*2 | Colorless colony sequence*3 | Ct (CFU/mL)*4 |
|---|---|---|---|---|---|---|---|
| OKY98 | CT-PS-XR-MacConkey | >300 | 124/>424 (29.2>) | 8 | 8 (100%) | NA | 23.1 (4.7 × 106) |
| XRM-MacConkey | >300 | 122/>422 (28.9>) | 8 | 8 (100%) | NA | ||
| OKY203 | CT-PS-XR-MacConkey | 88 | 105/193 (54.4) | 8 | 8 (100%) | NA | 23.8 (2.8 × 106) |
| XRM-MacConkey | >300 | 276/>576 (47.9>) | 8 | 4 (50%) | Morganella morganii | ||
| OKY361 | CT-PS-XR-MacConkey | 0 | 219/219 (100) | 8 | 8 (100%) | NA | 22.5 (7.1 × 106) |
| XRM-MacConkey | 17 | >300/>317 (94.6>) | 8 | 8 (100%) | NA | ||
| OKY374 | CT-PS-XR-MacConkey | 257 | 4/261 (1.7) | 4 | 4 (100%) | NA | 23.5 (3.5 × 106) |
| XRM-MacConkey | >300 | 70/>370 (18.9>) | 6 | 6 (100%) | NA | ||
| OKY375 | CT-PS-XR-MacConkey | 114 | 8/122 (6.6) | 8 | 8 (100%) | NA | 29.1 (6.9 × 104) |
| XRM-MacConkey | 212 | 2/214 (0.9) | 2 | 2 (100%) | NA | ||
*1 Colorless colonies were randomly selected and subjetced to Eacdt-gene PCR; *2 Bacterial species was confirmed by Eacdt-gene PCR; *3 Bacterial species of colorless colonies which were not Escherichia albertii was confirmed by 16S rDNA sequencing; *4 Ct and CFU values are according to Awasthi et al. [6]; CFU: colony forming unit; CT-PS-XR-MacConkey: cefixime-tellurite-phosphate-xylose-rhamnose MacConkey agar; XRM MacConkey: Xylose-Rhamnose-Mellibiose MacConkey agar. NA: not applicable.
DISCUSSION
E. albertii has often been misidentified as aEPEC, EHEC, CTEC-II or Shigella boydii serotype 13 due to lack of specific detection methods, enrichment culture methods, and selective medium that can selectively grow E. albertii and differentiate it from E. coli. PCR-based specific detection methods [3, 6, 12, 17, 22, 32] and enrichment culture protocols [2, 36, 38] for E. albertii have been developed and utilized for the detection and isolation of E. albertii in various samples [4, 5, 20, 37]. In addition, differential medium, XRM-MacConkey agar, which can differentiate E. albertii from E. coli and related bacteria by colony color has been developed [13]. Indeed, it has become much easier to differentiate E. albertii (colorless) from E. coli and other related bacteria which form red colonies on XRM-MacConkey agar than on MacConkey agar. However, isolation rate of E. albertii varied when XRM-MacConkey agar was used although its detection rate definitely enhanced using this selective medium. This could be due to presence of higher proportion of non-related red and colorless colonies in the samples [38]. Therefore, a medium that can selectively grow E. albertii and differentiate it from E. coli and related bacteria is highly desirable. As mentioned above, we have developed a selective differential medium, called CT-PS-XR-MacConkey agar, by which E. albertii (colorless) can be selectively grown and could be differentiated from E. coli (red) by the colony color. This medium could suppress the growth of some bacterial species showing red and colorless colonies on XRM-MacConkey due to cefixime and tellurite and thus, assisting in efficient isolation of E. albertii from food samples [35].
In this study, we evaluated, if this medium can also be used for clinical specimens such as diarrheal stool samples. As demonstrated by spiking experiment and shown in Table 1, CT-PS-XR-MacConkey agar inhibited the bacteria which form red colonies and the number of colorless colonies corresponding to E. albertii was constantly higher on CT-PS-XR-MacConkey than XRM-MacConkey agar. However, in this spiking experiment only one E. albertii was used, therefore, further studies using more strains are required.
It should be noted that 56, 26 and nine Eacdt-gene negative diarrheal stools yielded colorless colonies on MacConkey agar, DHL and XRM-MacConkey agar, respectively, but no colorless colonies were observed on CT-PS-XR-MacConkey agar, indicating that CT-PS-XR-MacConkey agar is highly selective for E. albertii (Supplementary Table 2).
Furthermore, as shown in Tables 2 and 3, for example, the growth of M. morganii, P. aeruginosa and P. rettgeri which were present in stool specimens were suppressed on CT-PS-XR-MacConkey agar. Altogether these data indicate that XRM-MacConkey agar is an excellent differential medium which can differentiate E. albertii from E. coli. However, CT-PS-XR-MacConkey agar is a more useful selective differential medium for the efficient isolation of E. albertii in comparison to XRM-MacConkey agar as demonstrated by spiking experiment (Table 1) and culturing Eacdt-gene negative diarrheal stool specimens (Supplementary Table 2 and Table 2). Awasthi et al. [6] reported that in one clinical case, although isolation of E. albertii using XRM-MacConkey agar was failed but E. albertii could be detected by real-time PCR but not by conventional PCR. In that clinical specimen, the number of E. albertii cells were low. If this newly developed CT-PS-XR-MacConkey agar was used, E. albertii might successfully be isolated from such samples. Apart from this, CFUs of E. albertii obtained on CT-PS-XR-MacConkey agar from E. albertii gene-positive clinical specimens was largely consistent with the CFU estimates obtained via real-time PCR (Table 3), as previously reported [6]. However, an exception was observed in one case, where the number of colonies was approximately 2 logs lower than expected based on the real-time PCR results (Table 3). This variation suggests the possibility of differences in the detection methods. This may be attributed to the characteristics of the E. albertii or presence of dead bacterial cells in the specimens which were detected by real-time PCR but not culture method. Further analysis is required to understand the reason for this inconsistency and to know what factors might affect the accuracy of CFU and PCR-based methods in some samples.
In conclusion, we found that CT-PS-XR-MacConkey agar is a more selective differential medium for the isolation of E. albertii than XRM-MacConkey agar from clinical specimens such as diarrheal feces. However, further studies with more clinical specimens are required to establish a standard isolation protocol for E. albertii from various specimens.
CONFLICT OF INTEREST
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Supplementary
Acknowledgments
We thank Dr. Rupak K. Bhadra, former Chief Scientist, CSIR-Indian Institute of Chemical Biology, Kolkata, India for critically reading the manuscript. This work was supported in part by JSPS KAKENHI grant numbers 17H04651, 20K06396 and 24K09249 to SY and AH. This study was performed in partial fulfillment of the requirements of a Ph.D. thesis for Keiji Takehira from the Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan.
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