Characteristics of microbial communities and intestinal pathogenic bacteria for migrated Larus ridibundus in southwest China

Abstract Larus ridibundus, a migratory wild bird, has become one of the most popular gull species in southwest China in recent years. There has been no information on the gut microbiota and intestinal pathogenic bacteria configuration in wild L. ridibundus, even though the public are in close contact with this bird. In this study, 16S rRNA amplicon‐sequencing methods were used to describe the microbial community structure and intestinal pathogenic bacteria were isolated to identify their characteristics. The taxonomic results revealed that Firmicutes (86%), Proteobacteria (10%), and Tenericutes (3%) were the three most abundant phyla in the gut of L. ridibundus. Enterococcaceae, Enterobacteriaceae, and Mycoplasmataceae were the most predominant families, respectively. The number of operational taxonomic units (OTUs), the richness estimates and diversity indices of microbiota, was statistically different (p < 0.05). However, beta diversity showed that no statistical significance (p > 0.05) between all the fecal samples. The most frequently isolated intestinal pathogenic bacteria from L. ridibundus were enteropathogenic Escherichia coli (32%) and Salmonella (21%). Pulsed‐field gel electrophoresis (PFGE) results of Salmonella species revealed a high degree of similarity between isolates, which was not observed for other species. None of the potentially pathogenic isolates were identical to human‐isolated counterparts suggesting that there was little cross‐infection between humans and gulls, despite close proximity. In brief, this study provided a baseline for future L. ridibundus microbiology analysis, and made an understanding of the intestinal bacterial community structure and diversity.


| INTRODUC TI ON
Larus ridibundus is a migratory bird distributed in Eurasia and on the East Coast of North America. Its body and coat color is similar to pigeons, showing red beak and feet, and the feathers of most of the body are white (Rajaei, Esmaili Sari, Bahramifar, Ghasempouri, & Savabieasfahani, 2010;Ushine, Sato, Kato, & Hayama, 2017 March, tens of thousands L. ridibundus fly to Kunming (southwest China) to overwinter. Each year, many visitors come to watch and feed them on city lakes or rivers; and thus the public are in close contact with this wild bird. Therefore, we speculated and studied the possibility there may be intestinal pathogenic bacteria from L. ridibundus that may endanger public health (Broman et al., 2002;Sixl et al., 1997).
Metagenomic investigations can be used to estimate the distributions of microorganisms, the taxonomic diversity, and functional gene contents (Cornejo-Granados et al., 2017). Studies in the past on microbiota were performed by using culture-based methods, and research result in the field was limited. However, recent availabilities in next-generation sequencing (NGS) methods have allowed a more thorough analysis of the complex and diverse of gut microbial communities (Kwong, McCallum, Sintchenko, & Howden, 2015). Therefore, a large number of studies on the gut metagenomics, using NGS system have been reported since then, which provided a broad and deep understanding of the microbial community. Previous studies showed that gut microbial mutualisms, commensalisms, and pathogen interactions have been considered to be an important factor for human health and animals (Backhed, Ley, Sonnenburg, Peterson, & Gordon, 2005). Several studies on gut metagenomic analysis have been conducted on some domestic bird species (Waite & Taylor, 2014); and some referred to wild birds (Dewar, Arnould, Krause, Dann, & Smith, 2014;Dewar et al., 2013). So far, there has been no information on the gut microbiota and intestinal pathogenic bacteria configuration in migrated wild L. ridibundus.
In this study, microbial communities of recently migrated L. ridibundus were analyzed by 16S rRNA amplicon sequencing investigation, and intestinal pathogenic bacteria were isolated to identify their characteristics.

| Sample collection of and DNA extraction
Five hundred fecal samples from L. ridibundus were collected in Kunming (103°40′ E, 26°22′ N), southwest China. The collection was divided into two periods, November and December 2017. The samples were collected around 5-10 m far from each other along the Daguan River in the center of Kunming city. Each sample was handled in two ways; one was by isolation using methods for common intestinal pathogenic bacteria; the other was randomly selecting 86 samples to extract total DNA for 16S rRNA amplicon sequencing analysis. The whole genomic DNA was extracted by using the fecal samples' DNA extraction kit (Tiangen, Beijing) following the manufacturer's instructions. In this kit, the genomic DNA of fecal samples was extracted by centrifugal adsorption column with specific binding to total DNA. A unique inhibitor adsorption tablet (InhibitEX), combined with a specially developed buffer system was also used for extraction. The entire microorganism's DNA could be extracted efficiently from fecal samples by using this kit. All extracted DNA samples were stored at −20°C for microbial community analysis.

| PCR, amplification, Miseq library construction, and sequencing
The variable region V3-V4 of the 16S rRNA gene was used for the library construction of the bacterial community by Illumina Miseq sequencing. Standard amplicon primers were used (Klindworth et al., 2013), and Illumina adapter overhang nucleotide sequences were added to the gene-specific sequences according to the protocol of library preparation guideline. PCR amplifications were performed using KAPA HotStart PCR kits (Kapa, Biosystems). Each PCR reaction included 0.2 M Trehalose, 5 μl buffer, 0.75 μl dNTP mixtures, 0.3 μM of the primers, 0.5 U KAPA polymerase, 25 ng extracted DNA, and PCR water totally added up to 25 μl. The amplification conditions were as follows: 95°C for 3 min, followed by 25 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, and final extension at 72°C for 5 min (Bobrova, Kristoffersen, Oulas, & Ivanytsia, 2016). The PCR products were purified with AMPure XP magnetic beads (Beckman, Coulter) and quantified using the Qubit fluorometer (Invitrogen, Life Technologies). A secondary amplifications to attach the Illumina Nextera barcodes were then performed, using the i5 forward primer and i7 reverse primer following the manufacturer's instructions (Kim, An, Kim, Lee, & Cho, 2017). The nontarget baseline for future L. ridibundus microbiology analysis, and made an understanding of the intestinal bacterial community structure and diversity.

K E Y W O R D S
gut microbiota, intestinal pathogenic bacteria, Larus ridibundus, southwest China fragments were removed, using AMPure XP magnetic beads as mentioned above (Beckman, Coulter). The amplicons were normalized, pooled, and sequencing was conducted in our laboratory (Kunming, China) using an Illumina MiSeq sequencing system (Illumina, San Diego, USA).

Workbench. CLC Microbial Genomics Module and CLC Genomics
Workbench performed operational taxonomic units (OTU) clustering and estimated alpha and beta diversities in microbial samples.
All the sequences were clustered into OTUs, based on 97% sequence similarity against the Greengenes reference sequence collection (version 13.5) (Cornejo-Granados et al., 2017). Taxonomy summaries with relative abundance data were generated. The representative sequences were aligned to the Greengenes reference alignment, and the alignment was filtered, then a phylogenic tree was constructed with the UPGMA for tree building. Prior to statistical analysis, the sequencing depths between samples were normalized. Alpha and beta diversity metrics from the final OTUs table were obtained. The alpha diversity analysis, including rarefaction curve and diversity indices, was carried out. The beta diversity was analyzed based on Fast UniFrac (Hamady, Lozupone, & Knight, 2010). Differences in the alpha and beta diversity were investigated, including the number of OTUs, richness, and diversity. Furthermore, differences in taxonomic composition were analyzed from the phylum to the family level. Statistical analysis was performed using a Kruskal-Wallis H test in SPSS statistics package, version 16.0 (SPSS IBM, New York, NY, USA), and PERMANOVA analyzed two groups of sampling time (November and December) for multiple testing correction by CLC Microbial Genomics Module (version 1.6.1).
p value of <0.05 was recognized as statistical significance. Sequence data were deposited on the NCBI database by the SRA accession: SRP131711.

| Pulsed-field gel electrophoresis
PFGE was used to analyze the important isolated strains (diarrheogenic E. coli, Salmonella and Yersinia enterocolitica) from L. ridibundus. The procedures were performed according to the protocols of each bacteria published by PulseNet (Curran et al., 2017;Ferrari, Panzenhagen, & Conte-Junior, 2017;Liang et al., 2014). The plugs digested with different enzymes, and electrophoresed with a recommendatory pulse times and hours at 20 V. For data analysis, *.tiff images of the gels were imported into the database of PFGE patterns.
Clustering of the band patterns was performed, using BioNumerics software (version 6.6) with the unweighted-pair group method of average linkages (UPGMA) and the Dice coefficient at a 1.5% tolerance. The diarrhea patients' PFGE patterns for different surveillance pathogenic bacteria from our previous database were used for comparison purpose.

| Taxonomic results of the L. ridibundus gut microbiota
In total, 18,945,847 reads were obtained from all the fecal samples. After quality trimming, merging, primer trimming, and length trimming, 15,143,601 valid reads were obtained from 86 samples.

| Phylogenic analysis of OTUs
The representative family or genus of OTUs was showed in

| Diversity estimation
For the alpha diversity analysis, the numbers of OTUs, Chao1, Shannon entropy and Simpson's index of each sample were calculated, then statistical analysis performed using the SPSS software package with Kruskal-Wallis H test, revealing that diversity estimates microbiota were significantly different between all the samples (p < 0.05) ( Table 1). The minimum value of numbers of OTUs was 9.43, and max value was 177.43, showing a large difference.
PCoA plot according to the Fast UniFrac distance metric was used to investigate the composition of gut microbiota between all the samples. There appeared to be a division across the primary axis for samples distributed in plot, as Figure 3 shown.

Most of samples in clustering A were collected in December,
while November for clustering B. Therefore, the time of sampling was considered as potential cause for clustering phenomenon.

One hundred twenty-four enteropathogenic E. coli (EPEC), 84
Salmonella, 62 Aeromonas, 41 Enterobacter, 15 Y. enterocolitica, 13 Pantoea, and 38 other bacteria were isolated from the 500 fecal samples. However, EPEC, Salmonella, and Aeromonas accounted for 69% of all isolated strains (Figure 4a). Interestingly, all isolated EPEC strains were defined as atypical; these strains did not possessed the EAF plasmid (bfpB and escV gene negative), where the eae gene was positive using multiplex PCR. All the Salmonella isolated from L. ridibundus was nontyphoid Salmonella strains, among them, 81 (96.43%) were Salmonella typhimurium, one was Salmonella Waycross, and two were Salmonella Indiana. All of the isolated Y. enterocolitica were biotype 1A, and the virulence genes for ail, ystA, ystB, virF, and yadA were absent. Eleven strains were serotype O: 1, 2; three strains were serotype O: 8; and one strain could not be typed.  (Zhang, Yang, et al., 2016; for comparison purpose, and no similar patterns were found between this wild bird and patients, as Figure 4b shown. For Salmonella, 81 S. typhimurium were divided into three patterns groups, and the similarity of three patterns groups was greater than 95%. Here, 66 strains showed the same PFGE pattern in the study. Compared with the PFGE patterns with diarrhea patients in our database, we found that S. typhimurium isolated from L. ridibundus were different from patients, separated into two clustering groups (Figure 4c)  of the species lived around seashores or rivers, and therefore their feces could be considered as the major source of contamination in coastal and lake waters. Since their migratory character and feeding behavior (Berg & Anderson, 1972), public maybe concerned on the possibility of waterfowl fecal contamination in environmental waters for the potential spread of microbial pathogens to humans, domesticated animals and human food sources. However, our actual data in this study did not strongly support this hypothesis. Previous study (Lu et al., 2011) showed that gull fecal pollution was widespread in urban coastal and riverine areas in North America and that it significantly contributed to fecal indicator bacterial loads.

| D ISCUSS I ON
However, the risks associated with exposure to recreational waters impacted by fresh gull, chicken, or pig faeces appear substantially lower than waters impacted by human sources (Soller, Schoen, Bartrand, Ravenscroft, & Ashbolt, 2010). As one of species of gulls, every winter, thousands of L. ridibundus flied to warm Kunming from remote Siberia for overwintering where the primary habitats were the lakes or rivers in the city. These areas were centralized areas for tourists and citizens where people could contact L. ridibundus. In our study, none of the potentially pathogenic isolates were identical to human isolates counterparts suggested that there was little crossinfection between human and gulls regardless of the potential close encounters. It was further suggested that the public health risks of gull fecal pollution (at least this species) were less than human and other animal sources.
Several studies showed the gut microbiota of waterfowl, using culture-based methods and mainly concerned on some pathogens (Craven et al., 2000;Gaukler et al., 2009). Because culture-based studies only provide a limited finding of natural microbial communities, culture-independent molecular methods can be used to describe the composition of waterfowl gut microbiome. A study referred to 60 mammalian species showed that microbial communities at higher taxonomic levels were very similar between birds and mammals; and most researches showed two phyla, Firmicutes and Bacteroidetes, as dominant out of 75 known microbial phyla (Ley, Lozupone, Hamady, Knight, & Gordon, 2008). This analysis result was very interesting, since it was believed that the common ancestor of amniotes (reptiles, birds, and mammals) possessed a microbial community mostly comprising Firmicutes and Bacteroidetes (Costello, Gordon, Secor, & Knight, 2010). Another study (Lu, Santo Domingo, Lamendella, Edge, & Hill, 2008) (Broman et al., 2002;Sixl et al., 1997), Salmonella spp. (Baudart, Lemarchand, Brisabois, & Lebaron, 2000), pathogenic E. coli (Kullas, Coles, Rhyan, & Clark, 2002;Makino et al., 2000), Microsporidia (Slodkowicz-Kowalska et al., 2006), and Cryptosporidium spp. (Zhou, Kassa, Tischler, & Xiao, 2004 (Makino et al., 2000) found Shiga toxin (Stx)-producing E. coli (STEC) strains isolated from a seagull in Japan. Kullas et al. (2002) demonstrated that Canada geese had a high prevalence of diarrheagenic E. coli. In the study of Palmgren et al. (2006), 83% of isolated Salmonella was S. Typhimurium from L. ridibundus, and indicated this wild bird might play a role in the spread of S. Typhimurium in Sweden. These data have paid useful attention on the risks for wild birds to public health. Although no homology of pathogenic bacteria was found between diarrhea patients and L. ridibundus in our study, this information was critical in order to recognize potential hazards associated with waterfowl fecal pollution. In general, this was the first study to demonstrate the microbial community structure and intestinal pathogenic bacteria from migrated L. ridibundus in southwest China. Firmicutes, Proteobacteria, and Tenericutes were the three most abundant phyla in the gut of L. ridibundus. The most isolated intestinal pathogenic bacteria from L. ridibundus were enteropathogenic E. coli and Salmonella. Although no homology of pathogenic bacteria was found between diarrhea patients and L. ridibundus, these results provided a baseline for future L. ridibundus microbiology study, and make an understanding of the intestinal bacterial community, structure, and diversity.

ACK N OWLED G M ENTS
This work was supported by Yunnan Science and Technology Talent and Platform Program (2017HC019) and National Sci-Tech key project (2017ZX10103010). We sent our manuscript to American Journal Experts (www.aje.com) for English language revisions.

CO N FLI C T O F I NTE R E S T
The authors declare no competing financial interests.

DATA ACCE SS I B I LIT Y S TATE M E NT
All data generated or analyzed during this study are included in this published article.