Microbial Diversity in the Gastrointestinal Tract of a Bat, Hypsugo alaschanicus

Bats are a vital part of natural ecosystems as they play essential roles in pollination, seed dispersal and controlling forest and agricultural insect pests, among other roles (Kunz et al., 2011). Although bats play positive roles in ecosystems, they are considered to be reservoirs of viruses and pathogens. As the number of emerging infectious diseases increases in a manner that is detrimental to human health, a growing number of studies have found that bats act as natural reservoirs for many emerging viruses and pathogens (Calisher et al., 2006; Mühldorfer et al., 2011; Gulraiz et al., 2017). A mass of pathogenic viruses colonize bats, such as henipaviruses, SARS (severe acute respiratory syndrome), MERS (Middle East respiratory syndrome), Ebola and Marburg virus or lyssaviruses (Calisher et al., 2006; He et al., 2013). However, bat bacterial flora and its zoonotic threat remain ill defined (Veikkolainen et al., 2014). Studies have shown that the best studied microorganisms in mammals are in the gastrointestinal tract (Hanning and Diaz-Sanchez, 2015). To date, there are only a few studies on the microbiota of bats originating from the gastrointestinal tract (Graves et al., 1988; Heard et al., 1997; Prem-Anand and Sripathi, 2004; Mühldorfer et al., 2010; Daniel et al., 2013; Hatta et al., 2016). Most previous approaches have been limited to culturable microbial communities, while systematically analyzing the bat bacterial community using a next-generation sequencing approach has only been reported in recent years. Veikkolainen et al. (2014) found Bartonella spp. in peripheral blood, fecal droppings, and ectoparasites of Myotis daubentonii. de Mandal et al. (2015) first reported on a bacterial community from bat guano using Illumina next-generation sequencing. Banskar et al. (2016) performed a microbiome analysis of the abundance of bacterial pathogens in Rousettusle schenaultii guano. In addition, Hatta et al. (2016) detected Campylobacter jejuni in rectal swab samples from Rousettus amplexicaudatus in Philippines. However, systematic analysis of the bacterial community in Hypsugo alaschanicus using a next-generation sequencing approach has not been reported.

The majority of microbial species could not be cultured in the laboratory using traditional technology, while high-throughput sequencing technologies can overcome the limit and identify the microbial community efficiently even in complex systems, such as the gastrointestinal tract (Human Microbiome Project Consortium, 2012). MiSeq high-throughput sequencing technology combines the advantages of Illumina HiSeq 2500 and Roche 454.

In China, H. alaschanicus is distributed in the central and northeast regions. H. alaschanicus lives mainly on insects and has various habitats. For instance, this species is relatively common in urban or human-associated areas, and can be found roosting in abandoned houses and buildings as well as in natural caves. For the past decades, some bat caves or houses have been explored for tourism, which not only threaten the habitats of bats but also could increase the risk of the transmission of bat pathogenic bacteria to humans. This transmission may cause zoonosis (Valdez and Salata, 1999; Veikkolainen et al., 2014; Bu et al., 2015). With the possibility of human to wildlife transmission, the study of the gastrointestinal microbiota composition in H. alaschanicus and especially the diversity of pathogens are extremely crucial.

To comprehensively analyze the microbiota of the gastrointestinal tract of H. alaschanicus, MiSeq high-throughput sequencing technology was used to sequence the V1−V2 variable regions of the 16s rRNA gene of gastrointestinal tract bacteria.

In September 2015, bats were captured using mist nets placed along the Weihe River in Xinxiang City, Henan Province, China (N35°35′, E113°92′). In this study, five H. alaschanicus specimens (three males and two females, all were adults) were captured in the field. The bat species were first identified based on the morphology according to “A Guide to the Mammals of China” (Smith and Xie, 2009), and then were verified using sequence analysis of the cytochrome B (cytb) gene amplified from total extracted DNA of the five H. alaschanicus. All fieldwork was conducted in accordance with the Law of the People’s Republic of China on the Protection of Wildlife.

An overdose of chloroform was used to euthanize the bats. The bats were then soaked with 70% ethanol for surface sterilization and rinsed with sterile water. Then the stomach and intestinal contents (including the ileum, jejunum, duodenum, and hind gut) were removed using sterile surgical forceps and a scalpel under aseptic conditions. The organs were placed in sterile tubes and immediately stored in liquid nitrogen until DNA extraction.

The genomic DNA of the stomach and intestinal fluid of five H. alaschanicus was extracted using a Stool DNA Kit (Feiyang, Guangzhou, China). The 16S rRNA gene V1−V2 regions were amplified with the primers 8F (5′-GCTTTGATGGACAT GGAAGAAGACAT-3′) and 338R (5′-GAGCCATCCCTCT CAATAATTTCAGG-3′). Polymerase chain reactions (PCRs) were performed in volume of 50 μl. The cycling parameters for the V1−V2 hypervariable regions of the 16S rRNA gene were as follows: 98 °C for 30 s; 98 °C for 10 s, 50 °C for 30 s, and 72 °C for 30 s for 20 cycles; and a final extension at 72 °C for 7 min. Amplicons were extracted from 2% agarose gels and purified using the QIA quick Gel Extraction kit (Comwei, Beijing, China) according to the manufacturer’s instructions. Purified amplicons were paired-end sequenced on an Illumina MiSeq platform (Zhongyijinda, Jiangsu, China).

With the use of the rRNA Detection pipeline in MG - RAST V3.3.6 for data analysis, the original data were subjected to filter processing and sequence optimization, and the optimized sequence was classified according to operational taxonomical unit (OTU) via clustering analysis and classification analysis. Based on the results of OTU clustering analysis, the diversity indices and depth of the sequences were detected. Using this taxonomic information, statistical analyses of the community structure at the level of the phylum and genus were conducted. Calculating the OTU number of each sample at a 97% similarity level, the analyses included optimized sequence statistics and calculated diversity indices and allowed for the generation of a high-throughput sequencing heat map.

After data analysis of gastrointestinal tract bacteria in H. alaschanicus, a total of 21, 336 effective sequences were obtained from the stomach fluid, and the effective sequences were clustered into 2, 468 OTUs with 97% identity. The sequencing coverage rate was 99.001%, indicating that the majority of the bacterial species in the stomach were included. A total of 34, 188 effective sequences were obtained from the intestinal fluid, and the effective sequences were clustered into 3, 511 OTUs with 97% identity. The sequencing coverage rate was 98.890%, indicating that the majority of the bacterial species in the intestine were included. Thus, there were species diversity in the stomach and intestine of H. alaschanicus (Table І).

The richness indices (Chao, 7803.176; ACE, 12051.440) in the stomach were significantly lower than those of the intestinal tract (Chao, 10749.844; ACE, 23799.700). The Shannon diversity index (3.536) in the stomach was lower than that of the intestinal tract (5.472). The Simpson diversity index (0.199) in the stomach was higher than that of intestinal tract (0.027). These indices show that the bacterial abundance and diversity in the intestine are higher than those in the stomach (Table І).

The bacteria from the samples (intestine and stomach) of H. alaschanicus belonged to 24 different phyla and were mainly distributed in four phyla: Bacteroidetes, Firmicutes, Proteobacteria and Actinobacteria (Fig. 1). The four phyla were the dominant taxa in the gastrointestinal tract, but the proportion for each group varied. The two dominant phyla were Proteobacteria (accounting for 27.8% of the total bacteria in the stomach and 39.7% of the total bacteria in the intestine) and Firmicutes (accounting for 12.7% of the total bacteria in the stomach and 59.5% of the total bacteria in the intestine) (Table II). More than 80% of the bacteria in the gastrointestinal tract belong to Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria. The other bacteria phyla only accounted for a small number of the bacteria in the gastrointestinal tract.

Only the top 5 enriched class categories are shown in the table. *Others, represents the rare genera.

The gastrointestinal tract flora diversity at the level of the genus was analyzed by drawing a heat map, and the bacterial genera that were found at concentrations greater than 0.1% in at least one of the samples are shown (the percentage is based on log10 and shown on a heat map) (Fig. 2). The deeper the red color, the higher the percentage of the bacteria. There were 20 bacteria genera present at more than 0.1% in the intestine and stomach (Fig. 2), and the changes in color demonstrates that the genera percentages in the intestine and stomach flora were different. In the stomach, the predominant bacteria genus was Enterococcus, accounting for 46.1% of the flora, and the second largest group was Bacillus, accounting for 7.4% of the flora. In the intestine, the predominant bacteria genus was Sphingomonas, accounting for 10.5% of the flora, and the subdominant group was Mycobacterium, accounting for 7.3% of the flora (Table III).

Genera obtained over 0.1% of the total reads are shown. *Others, represents the rare genera. - is representative of a very low bacterial content.

Next generation sequencing platforms are rapidly changing the methods of characterizing microbial communities from various sources. Metagenomic studies using these sequencing technologies have contributed enormously to our understanding of the structure and composition of bacterial communities (Banskar et al., 2016). This study provides an in-depth identification of the bacterial communities that are present in the gastrointestinal tract of H. alaschanicus. The richness indices (Chao and ACE indices) and Shannon diversity index in the stomach were significantly lower than those of the intestinal tract. The Simpson diversity index in the stomach was higher than that of the intestinal tract. These indices suggested that the bacteria abundance and diversity in the intestine were higher than those in the stomach. The composition differences of the gastrointestinal tract flora in H. alaschanicus may be related to gastrointestinal tract environment discrepancies or be associated to the differences of gastrointestinal functions (Daniel et al., 2013).

Using MiSeq high-throughput sequencing, bacteria from samples (intestine and stomach) of H. alaschanicus belonged to 24 defined different phyla and were dominated by Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria (Fig. 1). In previous studies, the bacteria in Myotis daubentonii (location: Finland) were dominated by three phyla: Chlamydiae, Proteobacteria and Bacteroidetes (Veikkolainen et al., 2014). The bacteria in Rousettus leschenaultii (location: India) were composed of 27 bacterial phyla, with Firmicutes, Actinobacteria and Proteobacteria predominating (Banskar et al., 2016). This is similar to those of other wildlife like rodents, for example C57BL/6 mice, whose intestinal bacteria contain Actinobacteria, Proteobacteria, Spirochaetes, Deferribacteres, Tenericutes, Verrucomicrobia, and unclassified bacteria (Kim et al., 2015). In this study, totally 387 genera were identified, which was over three times than that of the Philippine bat (Rousettus amplexicaudatus). Rousettus amplexicaudatus (location: Philippines) consisted of 103 genera of rectal swab bacterial flora, and the predominant genera were Clostridium and Campylobacter (Hatta et al., 2016). H. alaschanicus and R. amplexicaudatus shared 52 genera, and the dominant genera were different between them. Thus, the bacteria in different bat species included different phyla and genera numbers, and the dominant phyla and genera were also different. These differences might be due to the habitat, host specificity and diet (Carrillo-Araujo et al., 2015). In terms of habitats, Myotis daubentonii inhabits in forests and farmhouses. R. leschenaultii resides in caves, forests and hardy banana. R. amplexicaudatus is common in caves, rock cracks and tombs. However, H. alaschanicus mainly lives in human-associated areas and natural caves (Simmons, 2005; Smith and Xie, 2008). As for feeding habits, R. leschenaultii and R. amplexicaudatus are frugivorous, while M. daubentonii and H. alaschanicus are insectivorous.

Some bacteria in the gastrointestinal tract of H. alaschanicus may be pathogenic bacteria or opportunistic pathogens. Studies have shown that some species of the genus Sphingomonas, which was found in the stomach of H. alaschanicus in this study, frequently cause inflammatory disease in animals (Hu et al., 2007). Mycobacterium tuberculosis is associated with tuberculosis. M. leprae and M. lepromatosis, is mainly responsible for leprosy (Jagielski et al., 2016). Some species of the predominant genus Sphingomonas and second dominant genus Mycobacterium in the stomach of H. alaschanicus could be pathogenic bacteria. Enterococcus strains isolated from patients were associated with inflammatory bowel disease (Golińska et al., 2013). Bacillus anthracis could cause a zoonotic disease of anthrax (Welkos et al., 2015). In the intestinal tract of H. alaschanicus, the dominant genus Enterococcus and the second predominant genus Bacillus, may also contain pathogens. Mycoplasma may cause pneumonia and urinary tract infections (Huang et al., 2010; You et al., 2013). Lactococcus garvieae can cause human sepsis, endocarditis and osteomyelitis (Hirakawa et al., 2011). Escherichia hermannii has mainly been found to be involved in the sepsis, diarrhea and other infections, especially in immune compromised individuals (Kaewpoowat et al., 2013). Thus it is reasonanle to argue that the genera (Mycoplasma, Lactococcus, Escherichia) in the gastrointestinal tract of H. alaschanicus probably also include pathogenic bacteria species. However, further studies are needed to test the prediction. Most of these bacterial isolates were opportunistic pathogens that usually do not harm the host unless the immune system is weakened (Peterson, 1996). However, some bacteria in this study have been reported to be pathogenic to humans (Mühldorfer, 2013).

The reported metagenomic analysis of the bat microbiota indicates that bats are reservoir hosts for several pathogenic bacterial genera (Veikkolainen et al., 2014; Hatta et al., 2016; Banskar et al., 2016). However, no comprehensive study has been published on how those potential pathogens are transmitted from bats to human hosts. H. alaschanicus mainly lives in urban, rural housing, forest and natural caves, which include overlapping habitats with human. We propose that bacteria from bat fecal droppings could be transmitted to human hosts. Bacteria can contaminate flowing streams, which could be a source of drinking water in China, posing a direct threat to the health of the residents that use the water. Furthermore, there are as many as 155 bat species in China (Liu et al., 2013). Most people have little understanding of this danger and often go to caves to collect the bats’ bodies and feces, which are used in medicine or as fertilizer (Liu et al., 2011). In addition, many caves harboring bats have been exploited for tourism (Bu et al., 2015). At the same time, with the increase of the population density and change of people’s lifestyles, the chance of people contacting bats is increasing, which may lead to higher risk of the transmission of pathogenic bacteria in bats to humans (Banskar et al., 2016). To avoid the occurrence of zoonosis, contacts between humans and bats should be highly controlled or reduced. This study could improve the knowledge of the gastrointestinal microflora of bats and lead to the acquisition of further information on the prevention of bat zoonotic disease. Moreover, it is necessary to identify bacterial pathogens that are continuously transmitted to humans from bats.

This project was supported by the National Natural Science Foundation of China (NSFC, No. 31172056). We thank all those who helped in the field, especially Zongxiao Zhang, Liumeng Zheng, Jie Wu, Yingying Liu, Junlou Li, and Lili Hu. We also thank Hongwei Zhou and Shuyi Zhang for gastrointestinal contents sampling. We are especially grateful to Professor Qingxiang Yang for guidance and assistance in the experiment.

The authors declare that there is no conflict of interests regarding the publication of this article.