DeLeon, 2019-PowerSoil DNA Isolation Kit .Pone

RESEARCH ARTICLE
Bacterial diversity of bat guano from

Cabalyorisa Cave, Mabini, Pangasinan,
Philippines: A first report on the metagenome
of Philippine bat guano
Marian P. De Leon1☯*, Andrew D. Montecillo2☯, Dale S. Pinili3☯, Maria Auxilia T. Siringan4‡,
Doo-Sang Park5‡
1 Microbial Culture Collection, Museum of Natural History, University of the Philippines Los Baños, College,
Laguna, Philippines, 2 Microbiology Division, Institute of Biological Sciences, College of Arts and Sciences,
a1111111111 University of the Philippines Los Baños, College, Laguna, Philippines, 3 Plant Breeding, Genetics and
a1111111111 Biotechnology Division, International Rice Research Institute, Los Baños, Laguna, Philippines,
a1111111111 4 Microbiological Research and Services Laboratory, Natural Sciences Research Institute, University of the
a1111111111 Philippines Diliman, Quezon City, Philippines, 5 Korean Collection for Type Cultures, Biological Resource
a1111111111 Center, Korea Research Institute of Bioscience and Biotechnology, Jeongeup, South Korea
☯ These authors contributed equally to this work.

‡ These authors also contributed equally to this work.
* [email protected]
OPEN ACCESS
Citation: P. De Leon M, Montecillo AD, Pinili DS,

Siringan MAT, Park D-S (2018) Bacterial diversity
Abstract
of bat guano from Cabalyorisa Cave, Mabini,
Bats are highly diverse and ecologically valuable mammals. They serve as host to bacteria,
Pangasinan, Philippines: A first report on the
metagenome of Philippine bat guano. PLoS ONE viruses and fungi that are either beneficial or harmful to its colony as well as to other groups
13(7): e0200095. https://doi.org/10.1371/journal. of cave organisms. The bacterial diversity of two bat guano samples, C1 and C2, from
pone.0200095 Cabalyorisa Cave, Mabini, Pangasinan, Philippines were investigated using 16S rRNA gene
Editor: Wanda Markotter, University of Pretoria, amplicon sequencing. V3-V4 hypervariable regions were amplified and then sequenced
SOUTH AFRICA using Illumina MiSeq 250 PE system. Reads were processed using Mothur and QIIME pipe-
Received: January 4, 2018 lines and assigned 12,345 OTUs for C1 and 5,408 OTUs for C2. The most dominant OTUs
Accepted: June 19, 2018 in C1 belong to the Proteobacteria (61.7%), Actinobacteria (19.4%), Bacteroidetes (4.2%),
Firmicutes (2.7%), Chloroflexi (2.5%), candidate phylum TM7 (2.3%) and Planctomycetes
Published: July 19, 2018
(1.9%) while Proteobacteria (61.7%) and Actinobacteria (34.9%) dominated C2. Large pro-
Copyright: © 2018 P. De Leon et al. This is an open
portion of sequence reads mainly associated with unclassified bacteria indicated possible
access article distributed under the terms of the
Creative Commons Attribution License, which occurrence of novel bacteria in both samples. XRF spectrophotometric analyses of C1 and
permits unrestricted use, distribution, and C2 guano revealed significant differences in the composition of both major and trace ele-
reproduction in any medium, provided the original ments. C1 guano recorded high levels of Si, Fe, Mg, Al, Mn, Ti and Cu while C2 samples
author and source are credited.
registered high concentrations of Ca, P, S, Zn and Cr. Community structure of the samples
Data Availability Statement: Metagenome were compared with other published community profiling studies from Finland (SRR868695),
sequence data of C1 and C2 guano are available at
Meghalaya, Northeast India (SRR1793374) and Maharashtra State, India (CGS). Core
NCBI accession no. SRP101645.
microbiome among samples were determined for comparison. Variations were observed
Funding: The post-doctoral research fellowship
among previously studied guano samples and the Cabalyorisa Cave samples were attributed
was funded by the Department of Agriculture-
Bureau of Agricultural Research (DA-BAR) and to either bat sources or age of the guano. This is the first study on bacterial diversity of guano
University of the Philippines Diliman, Natural in the Philippines through high-throughput sequencing.
Sciences Research Institute (UP NSRI) and the
Next Generation Sequencing by the project of Dr.
PLOS ONE | https://doi.org/10.1371/journal.pone.0200095 July 19, 2018 1 / 17

Bacterial metagenome of bat guano from Cabalyorisa Cave, Pangasinan, Philippines
Doo-Sang Park through the National Research Introduction

Foundation of Korea (NRF-2016M3A9A5919255).
Bats (Order: Chiroptera) are highly diverse and ecologically valuable comprising 25% of living
Competing interests: The authors have declared
mammalian species. The most abundant group of mammals based on the number of individu-
that no competing interests exist.
als, they evolved into an incredibly rich diversity that roost in foliages, caves, rock crevices
even in man-made structures[1, 2]. They feed on insects, nectar, fruits, seeds, fish, frogs and
small mammals [3, 4].
Bats serve the ecosystem as pollinators, agents of seed dispersal and sources of guano-based
fertilizers. As biological agents to control pest population, they limit spread of human diseases
and prevent significant economic losses on crops and livestock [4–7]. Through the production
of guano, their accumulated excrement, bats are also responsible for nutrient cycling. As the
main source of energy for a wide range of unique species found on specific caves, guano depos-
its form the basis of cave ecosystems [7]. The formation of bat guano is an interesting interplay
among bats as primary producers, the nutrients present in the food they eat and the biological
and environmental changes inside the cave ecosystem. Guano is noted for its agricultural ben-
efits as an ideal fertilizer attributed to its richness in carbon, nitrogen, potassium and phospho-
rus. In the US, Asia, Cuba and South America, guano is being marketed as the best organic
fertilizer available. In the Philippines, caves in the Bicol Region, Pangasinan and Samal (Davao
del Norte) are few places that produce abundant guano in high risk of disturbance due to ille-
gal extraction. According to local government officials, guano-mining is inevitable since it is
the source of income among the locals and, likewise, a free organic fertilizer for agricultural
crops within the vicinity of the caves.
As prey and predator, bats have been implicated in epidemiologic cycles of several emerging
and re-emerging zoonoses [8] and carriers of pathogenic agents that include more than 200
different types of viruses such as rabies [9], Ebola and Marburg viruses [10–12], and Severe
Acute Respiratory Syndrome (SARS) coronavirus [5, 13, 14]. Fungi like Histoplasma capsula-
tum [15–18], Geomyces destructans [19] and Pseudogymnoascus destructans [20] were reported
to be isolated, considered as cause of infection and death in hibernating bats. Although bacte-
rial colonization has been observed only on samples of rocks, cave wall and/or ceiling paint-
ings, dripping waters, springs and underwater passages, oligotrophic cave-dwelling microbial
species have been described as phylogenetically diverse with lineages across the breadth of the
bacteria. Presence of pathogenic enteric bacteria mainly from the family Enterobacteriaceae
and some bacterial pathogens common in human and animal diseases (e.g. Pasteurella, Salmo-
nella, Escherichia and Yersinia spp.) [21, 22] was reported to be affected by the foraging habits,
diet and activities of the bats inside and outside of caves. Other bacterial pathogens (e.g. Barto-
nella, Borrelia, Leptospira spp., Serratia, Pseudomonas, Enterobacter, Acinetobacter, Bacillus,
Arthrobacter and Micrococcus spp.) provided evidence for novel species that seem to be specific
for bat hosts with probable medical importance to humans and other animals [23–26]. These
harmful microorganisms may also contribute to continuous decline of bat population in caves.
In addition, human-induced environmental stresses such as habitat destruction and fragmen-
tation, overhunting for bush meat, and increased use of pesticides also have negative impact to
bat population in caves [4]. Thus a persistent call for global bat conservation is necessary.
So far, the discovery of novel and potentially pathogenic bacteria and its interaction with
the environment is limited by culture-based approaches. Culture-independent methods are
essential to understand the genetic diversity, population structure, and ecological roles of the
majority of microorganisms [27]. High-throughput screening allows the paradigm shift on
microbiology and bioinformatics towards modern metagenomics that augment the limitations
of culture-based methods [28, 29]. Availability and accessibility of cost-effective next-genera-
tion high-throughput sequencing methods improved understanding of the assembly, evolution

and functions of ecological communities. Metagenomic studies using 16S rRNA gene sequenc-
ing were used to characterize gut and guano microbiome of several bat species [22, 27, 30–32]
and feces of different animals to determine their diet as well as the diversity of metabolic func-
tional genes (i.e. enzymes) [33–35].
To date, there have been no reports on the bacterial diversity of guano from Philippine
caves. There is limited local information in terms of benefits and harmful effects as well as eco-
logical importance. Bacterial diversity of guano may give insights on its potential as source of
nutrients for plant growth and their impacts on human health. This is the first report on the
bacterial diversity of bat guano using metagenomics in the Philippines particularly at Caba-
lyorisa Cave, Mabini, Pangasinan.
Materials and methods

Sample collection
Guano samples were collected from two large chambers, Chamber 1 (C1) and Chamber 2 (C2)
in Cabalyorisa Cave Complex, Mabini, Pangasinan, Philippines (N16˚00’22.6"E and N119˚
56’35.2"E, respectively at 100 msl). Prior to sampling, a Wildlife Gratuitous Permit (WGP)
(2015–002) has been granted by the Department of Environment and Natural Resources
(DENR), La Union, Philippines. The WGP allowed the conduct of field collection for the
assessment of bat guano community in selected caves in Mabini, Pangasinan, Philippines with
clearances and endorsements of the local government unit.
The first guano sample, C1, was collected from a chamber located 50 meters from the cave
entrance. Patches and pool of powdery guano were collected in between rocks, rock crevices
and on pot holes of the breakdown pile of about 3–4.5 m high and 3–5 m from the ceiling
using sterile hand trowel. The chamber was about 9 m at its widest and near a pool of water.
The second sample, C2, was collected 77 m further up near the flyway of bats. It is 6 m at its
widest with ceiling of 3 to 7 m high [36] and host sizable roost of bats of about 10–100 bat indi-
viduals. This chamber is an important flyway for bats roosting further in the cave and host
about 1–10 bat individuals. The floor of the site was uneven, made of large boulders and rock
formations that holds pool of guano. Guano were collected from seven sites in each chamber,
pooled and placed inside re-sealable zipper plastic bags using sterile shovel. Composite sam-
ples were then brought to the laboratory inside an ice chest and subjected to immediate DNA
extraction and X-ray Fluorescence (XRF) spectrophotometric analysis.
Bat species identification

Bats were identified through a combination of cave bat survey methods such as live-trapping
with mist nets, acoustic analysis using a bat detector (Dodotronic Ultranic 250K, Brazil) and
visual surveys. Six meter long mist nets were placed along the entrance and flyways inside the
cave. Individual bats were collected and identified by the Zoological and Wildlife Museum of
the University of the Philippines Los Baños Museum of Natural History (UPLB-MNH) based
from the guide “Key to the Bats of the Philippine Islands” [37]. Bats were immediately released
upon identification. No bats were killed in this study.
Elemental analysis of bat guano

The elemental compositions of bat guano C1 and C2 were analyzed semi-quantitatively using
handheld X-Ray Fluorescence Spectrometer (Delta Professional, Olympus, Japan) at the
Earth Material Science Laboratory of the National Institute of Geological Science, University
of the Philippines Diliman. Guano samples were dried at 105˚C and elemental analysis was

done in triplicates. Light elements, Silver (Ag), Cadmium (Cd), Tin (Sn) and Antimony (Sb)
were removed to normalize the results and reported as semi-quantitative or based on a relative
composition.
DNA extraction, amplicon library construction and sequencing

Total DNA was extracted using the PowerSoil DNA Isolation Kit (MoBio, Solana Beach, CA,
USA) and used as template for polymerase chain reaction (PCR) amplification with primer set
341F (5’-CCTACGGGNGGCWGCAG-3’ and 805R: 5’-GACTACHVGGGTATCTAATCC-3’)
targeting the V3-V4 region of the 16S rRNA gene. Amplifications were performed in a final
volume of 50 μl containing 10X Taq buffer, dNTP mixture, 10 mM of each primer and 2 U of
Taq polymerase (ExTaq, Takara, Japan). Cycling conditions in C1000 Touch thermal cycler
(Bio-Rad, Hercules, CA, USA) were: initial denaturation at 95˚C for 3 min; followed by 25
cycles of 95˚C for 30 s, 55˚C for 30 s, 72˚C for 30 s; with final extension at 72˚C for 5 min [38].
The PCR products were confirmed using gel electrophoresis and purified using the Agencourt
AMPure XP Reagents beads (Bechman Coulter, Brea, CA, USA). Equal amounts of purified
product were pooled, and final product quality were assessed on a Bioanalyser 2100 (Agilent,
Palo Alto, CA, USA) using a DNA 7500 chip. Sequencing was performed by Chunlab Inc.
(Seoul, Republic of Korea) with Illumina MiSeq 250 paired-end system (Illumina, San Diego,
CA, USA), in accordance with the manufacturer’s instructions.
Bioinformatic and statistical analyses of C1 and C2 guano

Data preprocessing and taxonomic classification of C1 and C2 sequence reads were carried
out in Mothur pipeline [39]. Forward and reverse reads, after primer and barcodes removal,
were aligned to form one continuous DNA sequence. Sequences that contained ambiguous
bases less than 430 bp in length, or with homopolymers of greater than 8 bp were discarded.
Chimeras were identified and removed using UCHIME [40]. SILVA reference database release
128 [41] was used for sequence alignments and classification. The microbial diversity was ana-
lyzed using QIIME software [42]. In addition, the upstream analyses of microbial sequences
through Mothur pipeline were also complemented with QIIME tool.
Alpha diversity analysis included Shannon index, Simpson, Chao1, and observed number
of bacterial species. The functional profiles of microbial communities were predicted by the
software “Phylogenetic Investigation of Communities by Reconstruction of Unobserved States
(PICRUSt)” and Kyoto Encyclopedia of Genes and Genomes (KEGG) Database [43]. The phy-
loseq, biom, and pheatmap R packages were used for data analysis and plotting. Metagenome
sequence data of C1 and C2 guano are available at NCBI with accession no. SRP101645.
Comparative analysis of bat guano microbiome

C1 and C2 bat guano data were compared with published microbiome profiles retrieved
from NCBI SRA (Sequence Read Archives) in fastq or fasta format [22, 35, 44], according to
protocol. Briefly, data were quality filtered using Mothur. Good quality sequences, defined as
sequences having a length of at least 100 bases, contain homopolymers of not more than 5 bp,
and having zero ambiguity were used for comparison. All the filtered sequences were merged
into a single fasta file and processed using QIIME pipeline. SILVA123_QIIME_release was
used as reference to pick OTUs employing a closed reference approach based from different
studies that utilized different regions of the 16S rRNA gene. Non-bacterial OTUs were
removed from the dataset. Core OTU was defined as an OTU present in 100% of the samples.
Correspondence analysis of the samples was done in R using the FactoMineR package [45].

Results and discussion

Cabalyorisa Cave, Mabini, Pangasinan
Mabini is a third-class municipality located in the Western district of the Province of Pangasi-
nan, Philippines. Mabini, named after the sublime paralytic Philippine hero, was once part of
the Province of Zambales and formerly known as Balincaguin or “Bali lan Caguin”, a Zambal
phrase which translates to “Abode of Bats” [46]. The municipality lies at about 15˚55’00” and
16˚52’00” longitudes and about 119˚56’00” and 120˚04’00” latitudinal lines and bounded on
the north by the City of Alaminos and town of Sual, on the northwest by the municipalities of
Agno and Burgos and on the southwest by the municipality of Dasol [36, 46]. The town was
formerly named Balincaguin because of its numerous karstic and limestone caves that include
Cacupangan Cave, Binmatya Cave, Ara-saas and Santo Rosario Caves, and Tinmori Tower
Karst [47] which serve as roosting sites for various species of bats.
Bat guano
A combination of several approaches on bat survey, namely, live-trapping with mist nets,
acoustic monitoring using bat detector and visual surveys, were used. Bats inside Cabalyorisa
Cave were mainly insectivorous and identified as Miniopterus australis, little long-fingered bat
or little bent-winged bat; M. schreibersii, a common bent wing bat and Rhinolophus amplixe-
dectus, the horse shoe bat. Encinares (2016) documented that aside from macroarthropods,
microarthropods observed included isotomid, sminthurid and an unidentified springtail, Geo-
laelaps, Oplitis, Deraiophorus, two uropodids and one oribatd mite, and an unidentified brown
ant. Presence of these microarthropods confirmed the insect-feeding nature of the bats roost-
ing in Cabalyorisa Cave [36].
XRF spectrophotometric analyses of C1 and C2 samples revealed significant differences in
the composition of both the major and trace elements (Table 1). C1 guano had high levels of
Si, Fe, Mg, Al, Mn, Ti and Cu while C2 samples yielded high concentrations of Ca, P, S, Zn
and Cr. The differences may be attributed to the decaying process once the guano were
Table 1. Elemental composition of bat guano from Cabalyorisa Cave using XRF spectrophotomer.
Elementsa, %
Sample SiO2 CaO Fe3O4 P S MgO Al2O3 Mn Zn Ti Cu Cr2O3
C1 26.52 23.63 15.45 6.12 1.66 7.45 13.61 4.72 0.21 0.35 0.23 0.05
C2 15.83 38.68 11.51 13.01 4.94 5.68 8.19 1.15 0.40 0.17 0.20 0.25
a
Legend:
SiO—Silicon monoxide
Al2O3—Aluminum oxide
CaO—Calcium oxide
Mn—Manganese
Fe3O4—Iron oxide
Zn—Zinc
P—Phosphorus
Ti—Titanium
S—Sulfur
Cu—Copper
MgO—Magnesium Oxide
Cr2O3—Chromium oxide
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deposited on the ground or on rock crevices. Decomposition of bat guano is affected by vari-
ous physical and environmental factors [22] and the dynamic nature of the microorganism
present. The conversion of organic and inorganic matters by the microorganism present, in
turn, results in the change of pH due to the release of ammonia. Thus, the resulting high levels
of nutritional contents from major and trace elements are vital components of organic and
inorganic compounds used in biosynthesis and energy production supporting the growth and
diversification of guano-borne bacteria [48]. Sequentially, high nutrient levels of guano pro-
mote the growth of diverse groups of microorganisms.
Richness and diversity analysis of bacterial community composition from

two guano samples
A total of 220,152 and 169,161 reads were obtained from C1 and C2, respectively. After contig
assembly, trimming, and chimera removal, a total of 104,764 and 87,379 valid reads were
obtained for C1 and C2, respectively. Valid reads for C1 were assigned to 12,435 OTUs, while
valid reads for C2 were assigned to 5,408 OTUs.
Rarefaction was done to a maximum depth of 85,000 counts per sample and 10 replicates
per iteration. The results of the Shannon, Simpson, and Chao1 indices, and the number of
observed species are show apparent variations between the two samples (Fig 1). Analysis of
within-sample alpha diversity suggests that the bacterial community of C1 and C2 samples
exhibited high biodiversity in all tested metrics, with C1 being more diverse than C2. Compari-
son of observed species and Chao1 diversity indices suggest adequate sampling of the commu-
nities in both samples.
In order to determine the difference in bacterial diversity between two cave locations, the
true diversity was computed using Shannon-Weiner Index and Shannon entropy. Since diver-
sity indices alone cannot distinguish two or several unequal samples, it is desirable to deter-
mine the number of equally-common genus termed as “effective genus number.” In this study,
effective genus number was calculated as a function of the natural exponent raised to a Shan-
non entropy value, estimated according to Chao and Shen’s formula from the Shannon index
[49, 50]. C1 was computed to have an effective genus number of 11.33, while C2 effective
genus number was 2.04. Thus, the bacterial diversity at genus level in C1 is five times higher
compared to C2, despite yielding lesser number of OTUs.
Fig 1. Shannon, Simpson, and Chao1 indices, and the number of observed species in bat guano samples, C1 (red)
and C2 (blue).
https://doi.org/10.1371/journal.pone.0200095.g001

Fig 2. Comparison of the phylum level distribution of bat guano samples, C1 and C2.
Bacterial composition in the two guano samples

Bacterial OTUs of the representative sequences were taxonomically assigned to a total of 30
phyla excluding the unclassified reads. Most OTUs within these phyla belonged to unclassified
groups suggesting the presence of novel groups of microorganisms. Apparent dominance of
Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes, Chloroflexi, candidate phylum TM7
(or Saccharibacteria), and Planctomycetes in the C1 and C2 guano samples was observed (Fig
2). C1 and C2 shared 2,467 OTUs (24.8%) classified under 15 phyla, despite being obtained
from the same cave system inhabited by the same species of bats. The differences between C1
and C2 microbiome profiles were attributed to these factors: a) the physico-chemical proper-
ties of guano; b) decaying process or age of the sampled guanos; c) host species; d) surrounding
environment.
Core microbiome of C1 and C2 revealed 117 shared genera. Fourteen genera have relative
abundance greater than 1% which is dominated by unassigned Xanthomonadaceae and Myco-
bacterium (Fig 3). Other significantly abundant genera on both C1 and C2 samples include
Bacillus (1.9% and 0.18%), Luteibacter (3.8% and 5.2%) and Rhodococcus (1.6% and 0.14%).
Much of the core microbiome members are yet to be classified.
C1 and C2 samples harbored potentially pathogenic genera such as Burkholderia, Coryne-
bacterium, Francisella, Legionella, Mycobacterium, Pseudomonas, and Rickettsia. This sug-
gested that guano may act as source of microorganisms that could potentially be pathogenic
to humans and animals [51]. The frequent intra- and inter-roost movements and long dis-
tance migrations of bats enhance the potential of bacterial transmission among individuals
[17, 22, 25, 52]. Presence of beneficial bacteria in bats like probiotics can provide vital func-
tions to their host such as processing of skin proteins, freeing fatty acids to reduce invasion
of transient microorganisms and inhibiting pathogenic microorganisms [53–57]. Pseudomo-
nas spp. isolated from bats, amphibians and plants produce antifungal properties particu-
larly against Pseudogymnoascus destructans, a causative fungus of white-nose syndrome in
bats [57]. Dietary habits were, likewise, reported to affect the bat gut microbiome and allow

Fig 3. Relative abundance of dominant core bacterial genera present in bat guano samples, C1 and C2.
the acquisition and transmission of infectious agents to other bats and organisms inside a
cave. [53–55]. Indeed, several insects consumed by insectivorous bats have been found to
harbor harmful bacteria and indigenous bacteria common in human and animal diseases,
including Salmonella, Yersinia or Campylobacter species and several enteric pathogens
[22, 25, 54, 58–64]. Similarly, contaminated fruits [51] or water [25] might also be possible
sources of bacteria.
Many members of identified core microbiome of C1 and C2 were uncultured or unclassi-
fied. These data validate possible presence of novel microorganisms found in the guano sam-
ples from Cabalyorisa Cave, Mabini, Pangasinan.
Functional profiles of the two guano samples

A total of 1136 OTUs were used to predict the functional profile of the microbial communities.
Using PICRUSt, genes found to be involved in metabolism of carbohydrates, amino acids,
nucleotides, lipids, xenobiotics and other compounds were both equally abundant in C1 and
C2. Pathways involved in biosynthesis of other secondary metabolites such as antimicrobials
(i.e. streptomycin biosynthesis, novobiocin biosynthesis, stilbenoid, diarylheptanoid and gin-
gerol biosynthesis, penicillin and cephalosporin biosynthesis, etc.) are inferred based on the
samples. Other pathways involved in metabolism of xenobiotics and of other compounds (i.e.
polycyclic aromatic hydrocarbon degradation, chloroalkane and chloroalkene, naphthalene,
benzoate, aminobenzoate degradation, bisphenol, caprolactam, etc.) were also predicted
(Fig 4).
Based on predicted functions of C1 and C2 microbiome, the enrichment of genes related to
pathways involved in metabolism of carbohydrates, amino acids, lipid, and energy were com-
parably abundant in C1 and C2. This suggested role of microorganisms active in decay and
biogeochemical processes. Moreover, pathways involved in the biosynthesis of secondary
metabolites such as antimicrobials and other degrading recalcitrant compounds indicated
presence possible novel of microorganisms that produce these metabolites.

Fig 4. Relative abundance of predicted function of C1 and C2 guano microbiome.


Fig 5. Phylum level distribution of different microbiome studies compared.

Comparative analysis of bat guano microbiome

Bat guano microbiome have been reported from composite guano collected on the cave floor
(no information about the host bats) and on fresh and decaying guano (source bat is Rousettus
eschenaultia) from Robber’s Cave in India [22]. Analysis of C1 and C2 microbiome data along
with previous data from NCBI GenBank database revealed differences in terms of composition
and relative proportion of bacterial communities. The relative abundances of major phyla in
previous studies were different. Quality filtered and analyzed sequence reads (SRR868695)
from the study of Veikkolainen et al. (2014) [44] showed only four dominant phyla, namely,
Chlamydiae (50%), Proteobacteria (27.8%), Firmicutes (16.7%), and Bacteroidetes (5.6%). In
the SRR1793374 study of de Mandal et al. (2015) [34] and the CGS study of Banskar et al.
(2016) [22], at least 28 and 22 bacterial phyla were detected (Fig 5), respectively. Actinobac-
teria, Chloroflexi, Planctomycetes, and Proteobacteria dominated in the study of de Mandal
et al. (2015) [35] while Proteobacteria (54.2%), Bacteroidetes (24.4%) and Actinobacteria
(8.6%) were the most dominant phyla in the CGS sample of Banskar et al., (2016) [22].
To further compare the samples, beta diversity analysis using CGS sample of Banskar et al.,
(2016) [22], the composite guano samples of de Mandal et al., (2015) [35] and the C1 and C2
samples were done in QIIME. This comparison was conducted for the representatives of
decaying guano samples. The core microbiome of CGS, composite guanos, and C1 and C2
showed 35 core OTUs (Fig 6). At the Phylum level, the major phyla common to all samples
are Acidobacteria, Actinobacteria, Chloroflexi, Nitrospirae, Proteobacteria, and Saccharibac-
teria. Bray-Curtis distance and UPGMA cluster analyses revealed that C1 and C2 guano sam-
ples were more similar than the composite guano and CGS sample. This suggested that C1
and C2 microbiome exhibited some unique microbiome profiles, further illustrated in the

Fig 6. Venn diagram showing the shared OTUs among the different microbiome studies compared.
correspondence map of four guano samples shown in Fig 7. Moreover, CGS and SRR1793374
microbiome profiles were remarkably different from those of C1 and C2.
Across samples, relatively high abundance of Actinobacteria suggested that members of this
phylum were conserved in bat guano. Growth of acidophilic Actinobacteria was favored due
to nutrient-rich acidic bat guano [65].
At the genus level, core microbiome of CGS, SRR1793374, C1 and C2 samples shared 27
bacterial genera (Fig 8). Majority of the 27 identified core genera are from Actinobacteria, 12,
and from Proteobacteria, 11. Other genera include Telmatobacter (from Acidobacteria), uncul-
tured Chloroflexi, Leptospirillum (Nitrospirae), and an uncultured Saccharibacteria. Relative
abundance of each genus varied across the samples. Streptomyces was one of the most abun-
dant genera in the core microbiome, followed by Mycobacterium (Fig 8). A number of genera
under Actinobacteria isolated from limestone caves, such as Streptomyces and Actinomyces,
were known to produce antimicrobial compounds [66, 67]. Furthermore, some genera in the
core microbiome, such as Mycobacterium, Burkholderia and Leptospirillum, were known to

Fig 7. Correspondence analysis map of the decaying guano samples at genus level.
have pathogenic species. The observed four microbiomes (including this study) shared com-
mon OTU under the Enterobacteriaceae family (Genus: Cronobacter). Proteobacteria were
overrepresented in C1, C2 and CGS samples while Bacteroidetes were relatively most abun-
dant in CGS [22]. Chloroflexi and Planctomycetes were most abundant in SRR1793374 [35]
relative to other samples.
Comparing guano microbiome data of C1 and C2 samples with data on composite and
decaying guano revealed that it was more similar to each other and were highly different in
microbial composition. Differences among the samples can be explained by origin, host bat
species and its diet, duration of the decaying process or age of the guano and other physico-
chemical properties. In terms of diet, the identified bats (Miniopterus schebersii, M. australis
and Rhinolophus amplixedectus) were observed to be mainly insectivorous, while R. eschenaul-
tia from Robber’s Cave is frugivorous. Phillips et al. (2012) reported that herbivorous and
reproductively active bats carried more diverse microbiota than carnivorous and reproduc-
tively inactive individuals [68]. According to Carillo-Araujo et al. (2015), diet primarily
defined the gut microbiome [62]. Banskar et al. (2016) observed that dietary overlap among
frugivorous and insectivorous bats explained the similarities in gut microbial communities
[25, 48]. Microbiome profiles observed in fresh and decaying guano might have been affected

Fig 8. Genus level distribution of core microbiome of different guano samples.

by the age of the guano or by the decaying process dependent on physical and environmental
factors [25]. As the guano age, microorganisms initially present in the guano carry out various
biogeochemical processes such as organic and inorganic nutrient cycles leading to changes
in pH and nutritional contents determining increase in bacterial growth and diversity.
This could explain the difference in microbiome of fresh and decaying guano observed by
Banskar et al. 2016 [22]. Microorganisms present on the soil or cave floor where the guano,
was obtained also contributed to the differences.
Conclusion
High-throughput sequence-based analysis with Illumina MiSeq was used to describe the first
in-depth study on bacterial community profiles of bat guanos collected in a Philippine cave.
The microbiome profiles of the Cabalyorisa bat guano exhibited unique characteristics. Meta-
genomic analysis of OTUs from bat guano revealed it as a source of potentially pathogenic
bacteria and/or novel functional genes. Difference on bacterial diversity between C1 and C2
samples may be attributed to differences of host species, diet and other physico-chemical char-
acteristics of the guano and its environmental surroundings. This first report on bat guano col-
lected in Cabalyorisa Cave, Mabini, Pangasinan stirred scientific interest for possibilities of
isolating and characterizing novel bacteria with multiple functions for production of antibiot-
ics and enzymes. On the downside, the presence of potential pathogenic bacteria may impose
health hazards to local folks harvesting guano for agricultural purposes.
Acknowledgments
The authors acknowledge the Department of Agriculture-Bureau of Agricultural Research
(DA-BAR) and University of the Philippines Diliman Natural Sciences Research Institute (UP
NSRI) for providing the Post-Doctoral Fellowship to Dr. Marian P. De Leon. The fellowship
host scientist, Dr. Maria Auxilia T. Siringan, Curator of the University of the Philippines
Culture Collection (UPCC) and Head of Microbiological Research and Services Laboratory
(MRSL) and the MRSL Researchers and Administrative staff for the assistance in the conduct

of the laboratory experiments. The Next Generation Sequencing was funded by the project
of Dr. Doo-Sang Park through the National Research Foundation of Korea (NRF-
2016M3A9A5919255). Mr. James DV. Alvarez, Mr. Edison A. Cosico and Mr. Manuel M.
Baldovino of the UPLB Museum of Natural History and Mr. John Mark A. Encinares of Insti-
tute of Biological Science, UPLB provided the technical and field assistance during the sample
collection and identification of bats in Cabalyorisa Cave. The Balincaguin Conservancy, Inc.
(BC), especially to Mr. Rawen Balmaña, Mr. Guillermo Rendon, Mr. Oliver Barao and his son,
Mr. Russel Barao, who accompanied Dr. De Leon in the fieldwork, conducted photo docu-
mentation and ensured the safety of the team. The local government of Mabini, headed by the
former Honorable Mayor Carlitos Reyes, for the provision of technical and manpower assis-
tance during the fieldwork and the Department of Environment and Natural Resources for the
issuance of gratuitous permit.
Author Contributions
Conceptualization: Marian P. De Leon.
Data curation: Marian P. De Leon, Andrew D. Montecillo.
Formal analysis: Marian P. De Leon, Andrew D. Montecillo, Dale S. Pinili.
Funding acquisition: Doo-Sang Park.
Investigation: Marian P. De Leon.
Methodology: Marian P. De Leon, Doo-Sang Park.
Project administration: Marian P. De Leon.
Resources: Doo-Sang Park.
Supervision: Maria Auxilia T. Siringan.
Validation: Marian P. De Leon, Andrew D. Montecillo.
Visualization: Andrew D. Montecillo, Dale S. Pinili.
Writing – original draft: Marian P. De Leon.
Writing – review & editing: Marian P. De Leon, Andrew D. Montecillo, Dale S. Pinili, Maria
Auxilia T. Siringan, Doo-Sang Park.
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RESEARCH ARTICLE

Bacterial diversity of bat guano from

☯ These authors contributed equally to this work.

Citation: P. De Leon M, Montecillo AD, Pinili DS,

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Doo-Sang Park through the National Research Introduction

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Materials and methods

Bat species identification

Elemental analysis of bat guano

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DNA extraction, amplicon library construction and sequencing

Bioinformatic and statistical analyses of C1 and C2 guano

Comparative analysis of bat guano microbiome

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Results and discussion

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Richness and diversity analysis of bacterial community composition from

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Bacterial composition in the two guano samples

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Functional profiles of the two guano samples

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Fig 4. Relative abundance of predicted function of C1 and C2 guano microbiome.

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Fig 5. Phylum level distribution of different microbiome studies compared.

Comparative analysis of bat guano microbiome

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Fig 8. Genus level distribution of core microbiome of different guano samples.

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