1. IntroductionEscherichia albertii is a newly described Escherichia species and emerging foodborne pathogen causing watery diarrhea, abdominal distention, vomiting, fever, and even bacteremia in humans [1]. E. albertii was first identified in diarrheal children from Bangladesh and has been associated with several human gastroenteritis outbreaks in Japan [2,3]. Due to the lack of distinguishing biochemical characteristics, E. albertii strains were often misidentified as E. coli, Hafnia alvei, Salmonella enterica, or Shigella boydii serotype 13 [4]. Thus, the prevalence of E. albertii in different hosts may well have been underestimated.E. albertii possesses an outer membrane protein intimin encoded by eae gene, which is responsible for the formation of attaching effacing (A/E) lesions on host intestinal epithelium [1]. In addition, almost all E. albertii isolates harbor a cdtABC locus which encodes the cytolethal distending toxin (CDT) [5]. The cdtB gene has been divided into five subtypes (cdtB-I to cdtB-V) in E. coli. A new subtype, cdtB-VI, was recently reported in E. albertii [6]. Shiga toxins (stx2a and stx2f) were identified in some E. albertii isolates [7,8]. Other virulence factors reported in E. albertii, such as enteroaggregative E. coli heat-stable enterotoxin encoded by astA, may contribute to pathogenicity, but they have not been systematically investigated [6].E. albertii has been isolated from various animal sources, such as poultry, pigs, cats, dogs, bats, and raccoons as well as animal-derived raw meats [9,10], yet its natural reservoirs and transmission route to humans remain uncertain. Previous epidemiological investigations demonstrated that birds may be considered as potential reservoirs of E. albertii [11,12,13]. Migratory birds can travel over long distances and carry pathogens from one region to another, spreading pathogens by feeding and excretion [14,15]. Poyang Lake, a globally important wintering and transfer point for migratory birds in the East Asian–Australasian Flyway (EAAF), harbors more than 400,000 birds belonging to around 87 species in the winter season [16]. Cranes, geese, and swans are the most abundant migratory birds found in Poyang Lake [17]. Every year, they start their northward migration at Poyang Lake and progress through inland China, Korea, Japan, or Russia from March to May [18].

In this study, for the first time, we reported the identification of E. albertii in migratory birds from Poyang Lake, China. Genomic characterization indicated the genetic diversity of migratory bird-derived E. albertii isolates, and some isolates may have the potential to cause human disease.

4. DiscussionE. albertii is known to be an emerging zoonotic foodborne pathogen and has been isolated from several species of wild birds (Redpoll finches, European wigeon, Pine siskins, Magpies, Pigeons, and others) worldwide, demonstrating its diverse reservoirs and global distribution [12,44]. In the present study, for the first time, we reported E. albertii in different migratory birds in Poyang Lake, China. Besides Eurasian wigeon, E. albertii were first identified from Taiga bean goose, Greater white-fronted goose, Northern pintail, Lesser white-fronted goose, and Tundra swan. The overall culture-positive rate was 22.2% (18/81) in this study, which was higher than that reported in birds from other countries (0.7–3.2%) [12,44].Serotyping plays an important role in diagnosis and epidemiological studies for pathogens of public health importance. For example, most reported outbreaks of E. coli have been attributed to several serogroups (e.g., O26, O111, and O157) [45]. The diversity of O-antigen biosynthesis gene clusters (O-AGCs) provides the primary basis for serotyping. Instead of the conventional agglutination test, forty O-genotypes (named EAOg1–EAOg40) and four H-genotypes (EAHg1-EAHg4) unique to E. albertii have been proposed [27,28]. The O-antigen genotypes of E. albertii were associated with virulence genes. For example, the EAOg18 strains were predominant in human-derived strains and often harbored stx2f gene [46]. In this study, 38 isolates belonged to five known EAOgs (EAOg1, EAOg2, EAOg6, EAOg8, and EAOg21) and three EAHgs (EAHg1, EAHg3, and EAHg4). Three novel O-AGCs, named as EAOg41–43, were identified among nine isolates in this study, indicating the high diversity of E. albertii in migratory birds.The eae and cdtB genes were commonly considered as the key virulence determinants of E. albertii [5]. Currently, at least 30 eae subtypes have been described in E. coli. Some subtypes such as beta1, gamma1, and sigma are common in E. albertii, but several novel subtypes have also been identified in E. albertii, implying the pathogenic difference between E. coli and E. albertii [6,13]. The CDT is encoded by the cdtABC genes which were widely distributed in E. albertii [1]. The cdtB gene has been divided into six subtypes (cdtB-I to cdtB-VI), with cdtB-II and cdtB-VI being the most common subtypes [6,13]. Recently, several cdtB-II-positive E. coli isolates were reclassified as E. albertii, suggesting that previously identified cdtB-II-positive E. coli isolates might be E. albertii. [47]. E. albertii cdtB-II gene (Eacdt) was used to develop a PCR assay for the detection of E. albertii [20]. In this study, all isolates possessed eae and cdtB, but none carried stx2. The predominant eae and cdtB subtypes were sigma and cdtB-II, which were found to be common in clinical strains of E. albertii (Table S3). Moreover, heat-stable enterotoxin gene astA, an important virulence gene in diarrheagenic E. coli [48], was also presented in several isolates of migratory birds. These indicated that the migratory bird-derived isolates may have pathogenic potential for humans.The E. albertii isolates from migratory birds were classified into different phylogenetic clusters, indicating the genomic diversity of E. albertii in migratory birds in Poyang Lake. Several genotypes of E. albertii coexisted in a single individual, similar to other findings in raccoons and other birds [44,49]. Isolates belonging to the same clone were identified in two sampling sites (A and B) about 15 km apart, indicating clonal transmission in Poyang Lake.Several gastroenteritis outbreaks caused by E. albertii have been reported [3]. Environmental water and vegetables were identified as some of the transmission vehicles in previous outbreaks [3,50]. Migratory birds are known to be involved in the maintenance and dissemination of zoonotic pathogens such as viruses, tick-borne pathogens, Vibrio, Listeria monocytogenes, Salmonella enterica, Escherichia coli, Campylobacter jejuni, and Mycobacterium avium [15,51,52,53]. These pathogens can also be transmitted to humans, animals, and poultry by contaminated water. Humans may come in contact with contaminated water for household or agricultural purposes. Therefore, migratory birds with a higher occurrence rate of E. albertii might contaminate environmental water, leading to human infections or outbreaks. In the present study, the isolates from migratory birds were genetically related to those isolated from human, poultry, bird, and dog from different regions or countries. Human, animal, and poultry might be infected by contact with contaminated water, soil, and food. The strains with an average of 101 cgSNPs indicated a highly close relationship with each other. Though the estimated number of cgSNPs per year for E. albertii or E. coli was unclear, a cutoff of ≤21 cgSNPs per genome per year for Klebsiella pneumoniae has been proposed [54]. These isolates with 101 cgSNP differences might share a recent common ancestor and clonal transmission. These data further proved that migratory birds might play a significant role in pathogen dissemination.Our proposed method of bacterial isolation and identification faces several key limitations that must be acknowledged. First, the E. albertii specific primers proposed by Lindsey et al. [19] targeting EAKF1_ch4033 can only correctly identify 96.5% (305/316) and missed 11 strains [9]. Second, the selection of white or colorless colonies in MacConkey agar might result in the exclusion of other lactose-fermenting E. albertii isolates [9]. These limitations might result in underestimation of the actual pathogen occurrence rate. We additionally acknowledge the limited sample size in this study; further large-scale epidemiological and more in-depth studies on migratory birds, other animal species, the environment and humans around Poyang Lake region are highly warranted to understand the significance of birds, and the transmission potential to the environment and humans due to E. albertii being carried.

In conclusion, this study proved that migratory birds may serve as an important reservoir of heterogeneous E. albertii with potential transmission sources to cause human infections. Considering the limited number of samples in this study, in the future, integrated, global, and ‘One Health’ approaches are critically needed to study E. albertii, an emerging zoonotic bacterial pathogen important in public health.