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Citation: Foti, M.; Spena, M.T.;
Fisichella, V.; Mascetti, A.; Colnaghi,
M.; Grasso, M.; Piraino, C.; Sciurba,
F.; Grasso, R. Cultivable Bacteria
Associated with the Microbiota of
Troglophile Bats. Animals 2022,12,
2684. https://doi.org/10.3390/
ani12192684
Academic Editors: Ángela Magnet
and Fernando Izquierdo
Received: 26 August 2022
Accepted: 4 October 2022
Published: 6 October 2022
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animals
Article
Cultivable Bacteria Associated with the Microbiota of
Troglophile Bats
Maria Foti 1, * , Maria Teresa Spena 2, Vittorio Fisichella 1, Antonietta Mascetti 1, Marco Colnaghi 3, Maria Grasso 2,
Chiara Piraino 4, Franco Sciurba 4and Rosario Grasso 2
1Department of Veterinary Science, University of Messina, Via Palatucci 13, 98168 Messina, Italy
2Department of Biological, Geological and Environmental Sciences, University of Catania, Via Androne 81,
95124 Catania, Italy
3Centre for Mathematics and Physics in the Life Sciences and Experimental Biology (CoMPLEX),
Department of Genetics, Evolution and Environment, University College London, 610 Darwin Building,
Gower Street, London WC1E 6BT, UK
4Zooprophylactic Institute of Sicily, Via Gino Marinuzzi 3, 90129 Palermo, Italy
*Correspondence: malinvet@unime.it; Tel.: +39-090-6766720
Simple Summary:
Troglophile bats live in colonies, often in sites exploited for agro-pastoral purposes.
Determining the composition of the microbiome of bats is an important step in understanding their
ecology and biology and can also provide information on the spread of pathogenic bacteria in their
populations. This study aimed to determine how epidemiological factors shape the microbiome of
troglophile bats and evaluate the occurrence of potentially pathogenic bacterial species. A total of
413 Gram-negative and 183 Gram-positive strains were isolated from 189 individuals of four species
of troglophile bats living in Sicilian and Calabrian territory (Italy). Besides few potentially pathogenic
bacteria, several strains with a hypothesized symbiotic role were found.
Abstract:
Background: The study of bats is of significant interest from a systematic, zoogeographic,
ecological, and physiological point of view. The aim of this study is to investigate the culturable
aerobic enteric, conjunctival, and oral bacterial flora of bats to determine their physiological micro-
biome and to investigate the possible occurrence of pathogenic bacteria. Methods: Five hundred and
sixty-seven samples were collected from 189 individuals of four species of troglophile bats (Myotis
myotis,Myotis capaccinii,Miniopterus schreibersii, and Rhinolophus hipposideros) living in Sicilian and
Calabrian territory (Italy). All samples were tested for Gram-negative bacteria; conjunctival and
oral swabs were also submitted to bacteriological examination for Gram-positive bacteria. Results:
Four hundred thirteen Gram-negative strains were isolated. Of these, 377 belonged to 17 different
genera of the family Enterobacteriaceae and 30 to five other families. One hundred eighty-three
Gram-positive strains were isolated. Of these, 73 belonged to the Staphylococcaceae family, 72 to
the Bacillaceae family and 36 to four other families. Besides some potentially pathogenic strains,
several bacterial species have been found that are common to all the bat species studied. These could
perhaps play a physiological or nutritional role. Conclusion: A great variety of bacterial species
were identified in the cultivable microbiota of southern-Italian troglophile bats, including several
potentially pathogenic strains and numerous putatively symbiotic species.
Keywords:
bats; bacteriological test; Gram-negative bacteria; Gram-positive bacteria; public health;
Southern Italy
1. Introduction
In Europe, the Chiroptera represent the mammalian order with the largest number
of species, but almost all of them are threatened or at extinction risk. For this reason, the
list of strictly protected species (Annex II) by the “Berne Convention on the Conservation
of European Wildlife and Natural Habitats” (1979) includes all Microchiroptera, with
Animals 2022,12, 2684. https://doi.org/10.3390/ani12192684 https://www.mdpi.com/journal/animals
Animals 2022,12, 2684 2 of 14
the exception of Pipistrellus pipistrellus. The species referable to the Italian territory are
currently 35 [
1
]. They belong to the Italian fauna virtually all European species divided
into three families: Rhinolophidae, Vespertilionidae, and Molossidae. To these is added the
family Miniopteridae, recently considered as distinct from that of the Vespertilionids [
2
].
All Italian Chiroptera feed on Arthropods and are nocturnal even if several species come
out of shelters in the presence of light [
1
]. The diet of Myotis capaccinii also includes aquatic
larvae of Diptera and fish fry [1].
Interest in bats is growing exponentially as a result of their alleged involvement in
human and domestic animals’ viral diseases emergence, including severe acute respiratory
syndrome (SARS), Middle East respiratory syndrome (MERS), disease of Nipah and Hen-
dra [
3
,
4
], rabies [
5
], and, more recently, CoV disease 2019 (COVID-19) [
6
]. Furthermore,
an increasing number of other viruses, whose pathogenic potential is still unknown, is
constantly being reported in bats globally [
7
,
8
]. Compared to a great number of studies
conducted on viral agents, the possible role of bats as potential carriers of pathogenic
bacteria has not been explored in great depth, especially in European bats [
9
,
10
]. Most
studies on the pathogenetic diversity of bats have focused on single pathogens and very
little research has been carried out on the European continent. In European bats, infections
by Leptospira spp. [
11
], Borrelia spp. [
12
], Micoplasma spp. [
13
], Bartonella spp. [
10
,
11
,
14
,
15
],
and Rickettsia spp. [
16
] have been sporadically reported. Analysis of the intestinal microbial
flora [
17
,
18
], typically carried out by isolation from guano [
19
], has revealed the presence
of single pathogenic species such as Campylobacter spp. [
20
] and Salmonella spp. [
9
]. Data
on the occurrence of Gram-positive bacteria are even scarcer, and most studies have fo-
cused on enteric bacteria from genera Enterococcus,Lactococcus, and Lactobacillus [
17
,
21
].
Walther et al. (2008) [
22
] describe the isolation of a methicillin-resistant Staphylococcus
aureus strain from a bat wound. Vandžurováet al. (2013) [
19
] isolated high percentages of
Staphylococcus nepalensis in the guano of Myotis spp. in Slovakia. The aim of this study is to
study culturable aerobic bacteria of bats living in caves in Southern Italy to investigate the
composition of their microbiota and evaluate the possible occurrence of pathogenic bacteria.
As the microbiota can influence the state of health or disease through the modulation
of the immune system and the competition with pathogenic bacteria, it is important to
determine its normal constitution in bat populations. Moreover, this knowledge can help
to identify harmful pathogens that can potentially lead to declines in bat populations. We
have considered pathogenic the bacteria belonging to Risk Group 2 or higher, as per the
National Institutes of Health (NIH) guidelines [23].
2. Materials and Methods
2.1. Sampling
From September to November 2018, 567 samples (189 rectal swabs (R), 189 conjunctival
swabs (C) and 189 oral swabs (O)) were collected from 189 individuals of four different
species of troglophile bats (Table 1).
Table 1. Individuals sampled.
Superfamily Family Subfamily Species n. Individuals
Vespertilionoidea Vespertilionoidae Myotinae Myotis myotis 47
Myotis capaccinii 8
Miniopteridae Miniopterus schreibersii 91
Rhinolophoidea Rhinolophidae Rhinolophus hipposideros 43
Total 189
2.1.1. Study Population
The greater mouse-eared bat Myotis myotis (Borkhausen, 1797) is a European-Mediter-
ranean species with a range including Eastern, Southern, and Central Europe (up to
Animals 2022,12, 2684 3 of 14
Southern England), many Mediterranean islands and Asia Minor. All Italian regions are
considered to be included in the range of the species [
1
]. Colonies present mainly in areas
with a high percentage of woods [
24
]. M. myotis lives in forest environments, open pastures,
and meadows. Its diet is mostly based on insects caught on the ground. It forages over
deciduous woodland edge, open deciduous woodland, and pasture, preying on large,
ground-dwelling arthropods such as beetles, crickets, and spiders, gleaning them from the
ground. This bat typically roosts in underground sites during the whole year, although
Northern populations are also known to dwell in buildings (loft-spaces) during the summer.
Occasionally, M. myotis can also form small colonies in trees. It is an occasional migrant;
the longest recorded movement is 436 km [25].
The long-fingered bat Myotis capaccinii (Bonaparte, 1837) is found in the Mediterranean
basin, in north-western Africa and on almost all the Mediterranean islands. In Italy it is
found throughout the country, especially in karst environments. It has a fragmented
range, from the Iberian Peninsula to Asia Minor, Israel, Lebanon, Jordan, Turkey, Iran,
and Iraq [
24
]. It feeds on insects, particularly Trichoptera, Neuroptera, Diptera, and
occasionally on small fish caught just above water surfaces or just below them. M. capaccinii
generally roosts in underground habitats (principally caves) and depends strongly on
aquatic habitats, as it forages mainly over wetlands and waterways (including artificial
waterbodies, such as canals and reservoirs). It seems to prefer clutter-free water surfaces
when foraging, probably because this facilitates the echolocation of preys [
26
]. It feeds on
insects, particularly Trichoptera, Neuroptera, Diptera (Chironomidae), and occasionally
on small fish (Gambusia spp.) caught just above water surfaces or just below them [
24
].
Movements between summer and winter colonies are mostly within a distance of 50 km
(maximum 140 km) [27].
The Schreiber’s bent-winged bat Miniopterus schreibersii (Kuhl, 1817) in Europe is
present in all Mediterranean regions, including the major islands. It frequents all Mediter-
ranean habitats but with a preference for areas rich in broad-leaved trees. It typically
dwells in karst caves and other underground sites [
24
]. M. schreibersii forages mainly
in deciduous woodlands and mature orchards (including olive groves), gardens, along
hedgerows separating pastures and riverine forests, and in urban areas. In the Mediter-
ranean area it occasionally forages in grasslands but avoids arable land and maquis. It
feeds on insects caught in flight. With a highly specialized trophic regime, it preys mainly
on Lepidoptera. Non-flying preys are also reported in its diet. Dipterans were the second
most consumed prey. Several taxa of Coleoptera, Neuroptera, Orthoptera, and Trichoptera
were also recorded. Prey also included many pest arthropod species [
28
]. Schreiber’s bat
is a migrant species that changes its roosts several times during the year; long-distance
movements occur occasionally [27].
Lesser horseshoe bat Rhinolophus hipposideros (Bechstein, 1800) is now almost absent in
much of central Europe but is still widespread in the Mediterranean countries. Summer
roosts (breeding colonies) are found in natural and artificial underground sites in the
southern part of the range, and in attics and buildings in the northern part of it. Southern
populations have nurseries in caves. In winter, R. hipposideros hibernates in underground
sites (including cellars, small caves, and burrows). It feeds on small insects (Diptera,
Lepidoptera, and Neuroptera), captured either in flight or on the ground (e.g., spiders).
Members of this species generally hunt individually, very close to the ground, within and
along the edges of broadleaf deciduous woodland (which represents their primary foraging
habitat), but also in riparian vegetation, Mediterranean and sub-Mediterranean shrubland.
Foraging activities take place nearly exclusively within woodland areas, while open areas
are avoided [
29
,
30
]. Habitat loss and fragmentation may therefore reduce the number of
suitable habitats for the lesser horseshoe bat and pose a threat to this species [31].
Miniopterus schreibersii is the only one of the four species to be present in all 6 sampling
cavities. The studied caves are occupied starting from March–April and abandoned in
July–September or until November in the Grotta dei Pipistrelli (SR), depending on the
climate [32].
Animals 2022,12, 2684 4 of 14
In all sampling sites, these species share the same emergency points from the refuge
and similar hours of activity. Once out of the caves, they occupy different trophic niches.
The sampling was carried out in six caves of the Southern Italy territory (Table 2).
Table 2. The number of sampled individuals in the 6 study areas by species.
Site Number of Sampled Individuals
Myotis myotis Miniopterus
schreibersii
Rhinolophus
hipposideros
Myotis
capaccinii Total
Grotta dei Pipistrelli (SR) 13 12 17 8 50
Grotta Palombara (SR) 5 9 14
Grotta Chiusazza (SR) 12 13 7 32
Grotta dei Pipistrelli (CS) 16 15 31
Grotta del Burrò(CT) 1 15 17 33
Grave Grubbo (KR) 27 2 29
Total 47 91 43 8 189
•
Grotta dei Pipistrelli (Sortino, SR, 37
◦
08
0
30
00
N, 15
◦
01
0
48
00
E, cadastral number: Si
SR 3526). Located in a protected area (Natural Reserve “Pantalica, Valle dell’Anapo
e Torrente Cava Grande”), this cave is not far from the town of Sortino, in areas
where semi-wild breeding is practiced. The area is characterized by a natural plateau,
deeply engraved by the Anapo River and the Calcinara stream. Pantalica Nature
Reserve (over 37 kmq) was awarded in 2005 as UNESCO World Heritage Site for its
history, archaeology, speleology, and landscape (UNESCO, 1992–2019). It consists of
various natural and semi-natural environments (riparian forest, woodland, shrubland,
grassland, and steppe) along with cultivated land [
33
], which are essential habitats
for many invertebrate and vertebrate communities. Grotta dei Pipistrelli opens on a
rocky wall overhanging the Calcinara stream, approximately 10 m from the left bank
of the river, in a Miocene formation known as “Calcari di Siracusa”. The karst cavity
has a sub-horizontal development with a 7.3% west–east average slope and it has
been explored for approximately 260 m: between the entrance of the cave, at 234 m
above sea level (a.s.l.), and the ending point (253 m a.s.l). The cave hosts very large
colonies of Chiroptera and it represents the biggest nursery roost of this region [
32
,
34
].
From 2012 to date, Grotta dei Pipistrelli is the only systematically monitored bat cave
in Sicily [
32
,
34
]. The maximum number of bats recorded was around 10,000 in 2013.
Minioptera and large Myotis are the predominant genera, with a lower prevalence
of Rhinolophids [
32
]. Below the entrance flows two streams of water (Anapo and
Calcinara) from which the bats drink after emerging from the cave.
•
Grotta Palombara (Melilli, SR, 37
◦
06
0
22
00
N, 15
◦
11
0
39
00
E, cadastral number: Si SR 3536).
The Integral Nature Reserve “Grotta Palombara” falls within the area of the Climiti
Mountains in the eastern sector of the Ibleo plateau, in the territory of the Municipality
of Melilli (Syracuse). The area of the Reserve covers an area of 11.25 hectares and
was established in 1998 “in order to protect the most important karst cave in eastern
Sicily for its underground development and the complexity of the cavity systems,
with a varied cave fauna that includes an important Guanobia component”. A fossil
karst cavity that develops for approximately 800 m. Palombara cave is located near
the biggest Italian petrochemical plant, known as “Augusta-Priolo-Melilli”. The cave
hosts a colony of bats belonging to the species Myotis myotis,Miniopterus schreibersii,
Rhinolophus euryale, and Rhinolophus ferrumequinum. The maximum number of Chi-
roptera recorded was approximately 1000 specimens [
32
]. Human activity, especially
the presence of numerous illegal landfills, has significantly degraded the environment
around the cave [34].
Animals 2022,12, 2684 5 of 14
•
Grotta Chiusazza (Floridia, SR, 37
◦
01
0
29
00
N, 15
◦
09
0
35
00
E, cadastral number: Si SR
3533). The total length of this cave is approximately 250 m. The area, located in south-
eastern Sicily, falls within the territory of Syracuse in the “Grotta Perciata-Chiusazza”
district on the eastern edge of the Hyblean plateau. The altitudes vary between 200
and 50 m above sea level. The morphology of the area varies from hilly to the west, to
sub-flat to the east. The area in which the cave opens is characterized by the presence of
intensive monocultures, arable land, and small ponds fed by the runoff from farmland,
where the bats go to feed and drink. The cave is populated by a large colony of
Minioptera and dozens of Rhinolophids.
•
Grotta del Burrò(Randazzo, CT, 37
◦
49
0
35
00
N, 14
◦
56
0
04
00
E, cadastral number: Si
CT 1024). Volcanic cave created as a result of the eruptive activity of Etna. It is a
large lava flow tunnel, over 200 m long. Grotta del Burròdevelops in prehistoric
lava formations of uncertain age (15,000 years–3930
±
60 years), of which it is not
possible to identify the eruptive system. This was probably located a few kilometers
south of Monte Spagnolo, today covered by the lava of 1614- 24 (Monte Pomiciaro
locality) and the eruptive apparatus of 1536. The surrounding area is characterized
by an extensive shrub–herbaceous vegetation cover dominated by the Genista and
Ferula genera, with the presence of isolated Quercus ilex trees and no arable land [
35
].
In the area, semi-wild cattle breeding is commonly practiced. The cave is inhabited by
a large mixed colony of bats (approximately 600–700 Minioptera, along with several
dozen large Myotis and Rhinolophids) [35].
•
Grotta dei Pipistrelli (Cassano allo Jonio, CS, 39
◦
47
0
11
00
N, 16
◦
18
0
28
00
E, cadastral
number: Cb CS 110). Large cavity consisting of a succession of caverns whose bottom
is occupied by debris and guano. It hosts a breeding colony of bats with a prevalence
of Myotis myotis and Miniopterus schreibersii. The territory in which the cavity opens
is hilly in the western part and slopes down towards the Piana di Sibari in the East,
characterized by the presence of agricultural activity.
•
Grave Grubbo (Verzino, KR, 39
◦
15
0
41.4
00
N, 16
◦
51
0
45.1
00
E, cadastral number: Cb
KR 258), opens at 285 m a.s.l. and extends for 1926 m; the cave belongs to the extensive
Le Grave Complex, the second longest system developing in gypsum deposits in Italy.
The cave is active and water flows into one of its branches from an inlet point during
rainy periods. The cave hosts a large, mixed breeding colony of bats (Minioptera and
Rhinolophids). The surrounding area is characterized by extensive crops and olive
groves, as well as cattle and sheep breeding.
2.1.2. Handling and Sampling
The capture is an unavoidably invasive technique and was carried out only by expert
and authorized personnel, according to current legislation (Permit of Istituto Superiore per
la Protezione e la Ricerca Ambientale (ISPRA) Prot. N. 14589/T-A31). When possible, the
bats were caught by hand in order to minimize the risk of disturbance to the colony (Grotta
Chiusazza, Grotta Burrò, and Grave Grubbo) [
36
]. In some cavities a hand-held net with a
telescopic handle was used to capture static and non-flying bats (Grotta Chiusazza, Grotta
Burròand Grave Grubbo). Only in 2 cavities (Grotta dei Pipistrelli of Sortino and Grotta
dei Pipistrelli of Cassano allo Ionio) was it possible to use the harp-trap to catch specimens
that flew off in the exit from the cave. After capture, each bat was placed for a short time
inside a numbered canvas bag whose mouth was closed by a cord. Each bag contained only
one subject. All operations, including the safety devices used by the operators, complied
with the Guidelines for the monitoring of Chiroptera drawn up by the Istituto Nazionale
Fauna Selvatica [1].
Rectal, conjunctival, and oral swabs for bacteriological survey were obtained from
each bat using individually packed sterile microbiological swabs moistened with sterile
saline solution 0.9%, inserting the tip and gently rotating it against the mucosa. The swabs
were subsequently inserted into tubes containing Amies transport medium (Copan Italia,
Brescia, Italy) and kept in a cooler with frozen gel packs for purposes of transport for a
Animals 2022,12, 2684 6 of 14
maximum of 8 h before culture-plate inoculation, or further storage in a refrigerator at
4
◦
C for a maximum of another 24 h, if no earlier processing was possible due to logistical
reasons. All bats were handled for the shortest time possible and were released immediately
after sampling.
2.2. Bacterial Isolation and Identification
The samples were transported in conditions of refrigeration to the laboratory and
examined to detect the presence of potential pathogens. All samples (n. 567) were examined
for Gram-negative bacteria; conjunctival and oral swabs (n. 378) were also submitted to
bacteriological examination for Gram-positive bacteria. Rectal swabs (n. 189), after an
enrichment in buffered peptone water, were streaked into MacConkey agar plates (Biolife
Italiana, Milano, Italy). Conjunctival and oral swabs were cultured in nutritive broth and,
after, streaked into MacConkey agar plates and into Staphylococci 110 Medium plates
(Biolife Italiana, Milano, Italy). Colonies demonstrating distinctive macroscopic appearance
were considered separate organisms and isolated on new plates. Isolates were subcultured
in blood agar plates for identification by mass spectrometry MALDI-TOF (matrix assisted
laser desorption/ionisation–time of flight mass spectrometry). The isolated colonies were
seeded in a 48-well metal plate with disposable loops, using as a reference strain Escherichia
coli ATCC 8739. The spectra were analysed by VITEK MS system (bioMérieux SA, Marcy
l’Etoile, France) using the software Axima (Shimadzu Kyoto, Japan)-SARAMIS database
(Spectral Archive and Microbial Identification System) (AnagnosTec, Berlin, Germany).
Eighty-eight strains, unidentified by MALDI-TOF mass spectrometry, after being grown
on blood agar base (Biolife Italiana, Milano, Italy) and diluted in physiological solution,
were typed at the Laboratory of Specialized Bacteriology of the Zooprophylactic Institute
of Sicily, using the traditional macro test tube method [
37
–
39
]. The bacteria of the genus
Bacillus spp. (POS BAT 19/Rev 0) have been characterized by carbohydrates oxidation
and fermentation, motility, urease, gelatinase, nitrate reduction, and Voges Proskauer (VP)
tests; Staphylococcus and Streptococcus spp. (POS BAT 05/Rev 0 and POS BAT 30/Rev 0)
were characterized by catalase, hemolysis, coagulase, oxidase, VP tests, and carbohydrate
fermentation. The enterobacteria and Gram-negative glucose nonfermenting bacteria (POS
BAT 09/Rev 0) were identified by oxidation–fermentation, mobility, catalase, oxidase, ure-
ase and tryptophanase tests, and utilization/fermentation/oxidation of carbohydrates. The
serological typing of Salmonella spp. strains (POS BAT 04/Rev.4) was performed following
the Kauffmann–White–Le Minor method, in agreement with the National Salmonellosis
Center of Padua, Italy [40].
2.3. Statistical Analysis
We evaluated the difference in the number of strains belonging to pathogenic species
isolated from different sampling sites and different bat species using Fisher’s exact test,
using a significance level of
α
= 0.05. The data were analysed using RStudio Version 1.0.153
for macOS (https://github.com/rstudio/rstudio, accessed on 21 September 2022). The
built-in function fisher.test was used to calculate the p-values.
3. Results
In 64 samples out of 378 tested (16.9%), the coexistence of Gram-positive and-Gram
negative bacteria was found (12/189 conjunctival swabs (6.3%); 52/189 oral swabs (27.5%)).
3.1. Gram-Negative Strains
Four hundred thirteen Gram-negative strains were isolated from 567 tested samples.
Of these, 377 belonged to 17 different genera of the family Enterobacteriaceae and 30 to five
other families (Figure 1).
Animals 2022,12, 2684 7 of 14
Animals2022,12,xFORPEERREVIEW7of14
3.Results
In64samplesoutof378tested(16.9%),thecoexistenceofGram‐positiveand‐Gram
negativebacteriawasfound(12/189conjunctivalswabs(6.3%);52/189oralswabs(27.5%)).
3.1.Gram‐NegativeStrains
FourhundredthirteenGram‐negativestrainswereisolatedfrom567testedsamples.
Ofthese,377belongedto17differentgeneraofthefamilyEnterobacteriaceaeand30to
fiveotherfamilies(Figure1).
Figure1.OccurrenceofGram‐negativebacteria.(A)NumberofGram‐negativebacterialisolates
belongingtodifferent23generafoundinoral(lightbrown),conjunctival(yellow),andrectal(green)
samples.(B)Foreachgenus,thepercentageofisolatesfoundindifferentbatspeciesisindicated:
Myotismyotis(cyan),Miniopterusschreibersii(orange),Rhinolophushipposideros(blue),andMyotisca‐
paccinii(pink).
ThemostisolatedspecieswereEnterobactercloacae(71strains,17.2%),Hafniaalvei(47
strains,11.4%),Citrobacterspp.(46strains,11.1%),andSerratiamarcescensandCitrobacter
freundii(21strains,5.1%).PotentiallypathogenicspeciesincludingSalmonellaenterica,
Klebsiellapneumoniae,andPseudomonasaeruginosahavealsobeenidentified.Onlyseven
generaoutof23(Citrobacter,Enterobacter,Escherichia,Hafnia,Klebsiella,Morganella,and
Providencia)weredetectedatallthreesamplingsites(rectus,eye,andmouth).Inmost
samples(294;83.5%)asinglebacterialstrainwasisolated.Samplesinwhichtwoorthree
strainswereisolatedwerefew,55(15.6%)and3(0.9%),respectively(see
DataAvailability
Statement
).Figures1Band2showtheresultsofbacteriologicaltestsforthespeciesofbat
andthesamplingsite,respectively.Sixstrainshavenotbeenidentified.
Figure 1. Occurrence of Gram-negative bacteria.
(
A
) Number of Gram-negative bacterial isolates
belonging to different 23 genera found in oral (light brown), conjunctival (yellow), and rectal (green)
samples. (
B
) For each genus, the percentage of isolates found in different bat species is indicated:
Myotis myotis (cyan), Miniopterus schreibersii (orange), Rhinolophus hipposideros (blue), and Myotis
capaccinii (pink).
The most isolated species were Enterobacter cloacae (71 strains, 17.2%), Hafnia alvei
(47 strains, 11.4%), Citrobacter spp. (46 strains, 11.1%), and Serratia marcescens and Citrobacter
freundii (21 strains, 5.1%). Potentially pathogenic species including Salmonella enterica,
Klebsiella pneumoniae, and Pseudomonas aeruginosa have also been identified. Only seven
genera out of 23 (Citrobacter,Enterobacter,Escherichia,Hafnia,Klebsiella,Morganella, and
Providencia) were detected at all three sampling sites (rectus, eye, and mouth). In most
samples (294; 83.5%) a single bacterial strain was isolated. Samples in which two or three
strains were isolated were few, 55 (15.6%) and 3 (0.9%), respectively (see Data Availability
Statement). Figures 1B and 2show the results of bacteriological tests for the species of bat
and the sampling site, respectively. Six strains have not been identified.
Only three species (Enterobacter cloacae,Morganella morganii, and Serratia marcescens)
were present in all six caves. No significant difference was found in the number of strains
belonging to pathogenic species isolated from different sampling sites. Six bacterial species
(Citrobacter freundii,Hafnia alvei,Klebsiella oxytoca,Morganella morganii,Serratia liquefaciens,
and Serratia marcescens) were present in all the bat species. Among potentially pathogenic
species, the most frequently isolated were Escherichia coli,Serratia marcescens, and Pseu-
domonas aeruginosa. Table 3summarizes the comparison of the percentages of pathogenic
strains found in the different species of bats and p-value of significant differences calculated
using Fisher’s Exact Test.
Animals 2022,12, 2684 8 of 14
Animals2022,12,xFORPEERREVIEW8of14
Figure2.DistributionofGram‐negativebacteriaindifferentsamplinglocations.Thepercentage
ofisolatesbelongingtothe10generaofGram‐negativebacteriamostcommonlyoccurringinour
datasetisshownfor6differentsamplingsites:GrottadeiPipistrelli,SR(cyan),GrottaPalombara,
SR(orange),GrottaChiusazza,SR(blue),GrottadeiPipistrelli,CS(pink),GrottaBurrò,CT(green),
andGraveGrubbo,KR(yellow).
Onlythreespecies(Enterobactercloacae,Morganellamorganii,andSerratiamarcescens)
werepresentinallsixcaves.Nosignificantdifferencewasfoundinthenumberofstrains
belongingtopathogenicspeciesisolatedfromdifferentsamplingsites.Sixbacterialspe‐
cies(Citrobacterfreundii,Hafniaalvei,Klebsiellaoxytoca,Morganellamorganii,Serratialiquefa‐
ciens,andSerratiamarcescens)werepresentinallthebatspecies.Amongpotentiallypath‐
ogenicspecies,themostfrequentlyisolatedwereEscherichiacoli,Serratiamarcescens,and
Pseudomonasaeruginosa.Table3summarizesthecomparisonofthepercentagesofpatho‐
genicstrainsfoundinthedifferentspeciesofbatsandp‐valueofsignificantdifferences
calculatedusingFisher’sExactTest.
Table3.Comparisonbetweenthepercentagesofpathogenicstrainsisolatedindifferentbatspecies
andp‐valueofsignificantdifferences,calculatedwiththeFisher’sexacttest.
MS/MCMS/MMMS/RHMC/MMMC/RHMM/RH
Escherichia
coli4.4%/0%4.4%/4.3%4.4%/16%
p=0.03740%/4.3%0%/16%4.3%/16%
Serratiamar‐
cescens
7.7%/50%
p=0.00487.7%/6.4%7.7%/16.3%50%/6.4%
p=0.005950%/16.3%6.4%/16.3%
Pseudomonas
aeruginosa3.3%/0%3.3%/21.3%
p=0.00123.3%/0%0%/21.3%0%/0%21.3%/0%
p=0.0012
Legend:MS=Miniopterusschreibersii;MC=Myotiscappaccinii;MM=Myotismyotis;andRH=Rhi‐
nolophushipposideros.
Figure 2. Distribution of Gram-negative bacteria in different sampling locations.
The percentage
of isolates belonging to the 10 genera of Gram-negative bacteria most commonly occurring in our
dataset is shown for 6 different sampling sites: Grotta dei Pipistrelli, SR (cyan), Grotta Palombara, SR
(orange), Grotta Chiusazza, SR (blue), Grotta dei Pipistrelli, CS (pink), Grotta Burrò, CT (green), and
Grave Grubbo, KR (yellow).
Table 3.
Comparison between the percentages of pathogenic strains isolated in different bat species
and p-value of significant differences, calculated with the Fisher’s exact test.
MS/MC MS/MM MS/RH MC/MM MC/RH MM/RH
Escherichia coli 4.4%/0% 4.4%/4.3% 4.4%/16%
p= 0.0374 0%/4.3% 0%/16% 4.3%/16%
Serratia
marcescens
7.7%/50%
p= 0.0048 7.7%/6.4% 7.7%/16.3% 50%/6.4%
p= 0.0059 50%/16.3% 6.4%/16.3%
Pseudomonas
aeruginosa 3.3%/0% 3.3%/21.3%
p= 0.0012 3.3%/0% 0%/21.3% 0%/0% 21.3%/0%
p= 0.0012
Legend: MS = Miniopterus schreibersii; MC = Myotis cappaccinii; MM = Myotis myotis; and RH = Rhinolo-
phus hipposideros.
Escherichia coli is more common among R. hipposideros than in M. myotis,M. schreibersii,
and M. cappaccinii. Fisher ’s exact test showed significant differences between R. hipposideros
and M. schreibersii.Serratia marcescencens occurs more frequently in M. cappaccinii than
in M. myotis,M. schreibersii, and R. hipposideros. The same test also showed significant
differences between M. cappaccinii and M. myotis as well as M. cappaccinii and M. schreibersii.
M. myotis is characterised by a higher occurrence of Pseudomonas aeruginosa compared to
M. schreibersii,R. hipposideros, and M. cappaccinii.P. aeruginosa levels are also significantly
higher in M. myotis than M. schreibersii and R. hipposideros.
Animals 2022,12, 2684 9 of 14
3.2. Gram-Positive
One hundred eighty-three Gram-positive strains belonging to six different genera
were isolated from a total of 378 samples (Figure 3). Of these, 73 belonged to the Staphy-
lococcaceae family, 72 to the Bacillaceae family, and 36 to four other families. Two strains
have not been identified. The most frequently isolated species were Enterococcus faecalis
(28 strains, 15.3%), Bacillus licheniformis (15 strains, 8.2%), Bacillus megaterium (14 strains,
7.7%), and Staphylococcus sciuri (12 strains, 6.6%). Only one strain belonging to a potentially
pathogenic species (Staphylococcus aureus) was found in a specimen of Myotis myotis.
Animals2022,12,xFORPEERREVIEW9of14
EscherichiacoliismorecommonamongR.hipposiderosthaninM.myotis,M.
schreibersii,andM.cappaccinii.Fisher’sexacttestshowedsignificantdifferencesbetween
R.hipposiderosandM.schreibersii.SerratiamarcescencensoccursmorefrequentlyinM.cap‐
pacciniithaninM.myotis,M.schreibersii,andR.hipposideros.Thesametestalsoshowed
significantdifferencesbetweenM.cappacciniiandM.myotisaswellasM.cappacciniiand
M.schreibersii.M.myotisischaracterisedbyahigheroccurrenceofPseudomonasaeruginosa
comparedtoM.schreibersii,R.hipposideros,andM.cappaccinii.P.aeruginosalevelsarealso
significantlyhigherinM.myotisthanM.schreibersiiandR.hipposideros.
3.2.Gram‐Positive
Onehundredeighty‐threeGram‐positivestrainsbelongingtosixdifferentgenera
wereisolatedfromatotalof378samples(Figure3).Ofthese,73belongedtotheStaphy‐
lococcaceaefamily,72totheBacillaceaefamily,and36tofourotherfamilies.Twostrains
havenotbeenidentified.ThemostfrequentlyisolatedspecieswereEnterococcusfaecalis
(28strains,15.3%),Bacilluslicheniformis(15strains,8.2%),Bacillusmegaterium(14strains,
7.7%),andStaphylococcussciuri(12strains,6.6%).Onlyonestrainbelongingtoapoten‐
tiallypathogenicspecies(Staphylococcusaureus)wasfoundinaspecimenofMyotismyotis.
Figure3.OccurrenceofGram‐positivebacteria.(A)NumberofGram‐positivebacterialisolates
belongingtodifferentsixgenerafoundinoral(lightbrown)andconjunctival(green)samples.(B)
Foreachgenus,thepercentageofisolatesfoundindifferentbatspeciesisindicated:Myotismyotis
(cyan),Miniopterusschreibersii(orange),Rhinolophushipposideros(blue),andMyotiscapaccinii(pink).
Figures3Band4showtheresultsofbacteriologicaltestsforthespeciesofbatand
thesamplingsite,respectively.BacteriabelongingtothegeneraBacillusandStaphylococcus
werepresentinallthecaves,buttheonlycommonspeciesfoundwasEnterococcusfaecalis.
ThebacterialspeciesfoundinallfourspeciesofbatswereEnterococcusfaecalis,Bacillus
cereus,andStaphylococcusepidermidis.In11samples(6.4%),twodifferentstrainswereiso‐
lated(see
DataAvailabilityStatement
).
Figure 3. Occurrence of Gram-positive bacteria.
(
A
) Number of Gram-positive bacterial isolates
belonging to different six genera found in oral (light brown) and conjunctival (green) samples.
(
B
) For each genus, the percentage of isolates found in different bat species is indicated: Myotis
myotis (cyan), Miniopterus schreibersii (orange), Rhinolophus hipposideros (blue), and Myotis
capaccinii (pink).
Figures 3B and 4show the results of bacteriological tests for the species of bat and
the sampling site, respectively. Bacteria belonging to the genera Bacillus and Staphylococcus
were present in all the caves, but the only common species found was Enterococcus faecalis.
The bacterial species found in all four species of bats were Enterococcus faecalis,Bacillus
cereus, and Staphylococcus epidermidis. In 11 samples (6.4%), two different strains were
isolated (see Data Availability Statement).
Animals2022,12,xFORPEERREVIEW10of14
Figure4.DistributionofGram‐positivebacteriaindifferentsamplinglocations.Thepercentage
ofisolatesbelongingtosixgeneraofGram‐negativebacteriaisshownforsixdifferentsampling
sites:GrottadeiPipistrelli,SR(cyan),GrottaPalombara,SR(orange),GrottaChiusazza,SR(blue),
GrottadeiPipistrelli,CS(pink),GrottaBurrò,CT(green),andGraveGrubbo,KR(yellow).
4.Discussion
Inrecentyears,anewfieldofresearchhasemergedthatoutlinestheimportanceof
themicrobiotainhealthanddisease.Themicrobiomeplaysakeyroleinhostevolution
andcansignificantlycontributetovariousfunctionsoftheorganism,suchassugarme‐
tabolism[41],digestionandtheabsorptionofnutrients[42,43],andtheproductionofmet‐
abolicenzymes[44].
WeinvestigatedthecompositionofthecultivablemicrobiomeofSouthernItalian
Troglophilebats,usingaculture‐basedapproach.Thepresentstudyalsoinvestigatesthe
presenceofpotentiallypathogenicGram‐negativeandGram‐positivebacteria.Thereisa
growingawarenessofhowthespreadofpathogensinwildanimalscanimpacthuman
andanimalhealth.Theemergenceofnewinfectiousdiseasesisnotonlyaconservation
issue,duetothedangersitposestoprotectedspecies,butalsoapotentialthreattopublic
health.Theisolationofinfectiousagentsinbatsindicatestheimportanceofmonitoring
thepresenceofpotentiallypathogenicbacteriainthisorder,isolatedfromdifferentbat
populations.
Ouranalysisledtotheidentificationofapproximatelysixhundredbacterialstrains
belongingtoninetydifferentbacterialspecies(50Gram‐negativeand40Gram‐positive)
and29genera(Figures1and3).Ourresultsdemonstratethepresenceofawidervariety
ofbacterialspeciesthanindicatedbypreviousresearchonEuropeanbats.DiBellaetal.
(2003)[17]isolated26bacterialspeciesbelongingto13generafromfaecalsamples(Aci‐
netobacter,Alcaligenes,Citrobacter,Enterobacter,Escherichia,Hafnia,Klebsiella,Kluyvera,Mor‐
ganella,Proteus,Pseudomonas,Streptococcus(nowEnterococcus),andYersinia),allofwhich
werealsofoundinoursurvey,withtheexceptionofYersiniaspp.Someofthespecies
isolatedinthepresentstudycanbeconsideredcommensalsorenvironmentalcontami‐
nants(e.g.,Bacillusspp.),butothers(e.g.,Salmonellaenterica,Klebsiellapneumoniae,Pseudo‐
monasaeruginosa,andStaphylococcusaureus)arepotentiallypathogenic,bothforbatsand
forotheranimalsandalsoforhumans[9,45].Enterococcusfaecaliswasthemostisolated
Figure 4. Cont.
Animals 2022,12, 2684 10 of 14
Animals2022,12,xFORPEERREVIEW10of14
Figure4.DistributionofGram‐positivebacteriaindifferentsamplinglocations.Thepercentage
ofisolatesbelongingtosixgeneraofGram‐negativebacteriaisshownforsixdifferentsampling
sites:GrottadeiPipistrelli,SR(cyan),GrottaPalombara,SR(orange),GrottaChiusazza,SR(blue),
GrottadeiPipistrelli,CS(pink),GrottaBurrò,CT(green),andGraveGrubbo,KR(yellow).
4.Discussion
Inrecentyears,anewfieldofresearchhasemergedthatoutlinestheimportanceof
themicrobiotainhealthanddisease.Themicrobiomeplaysakeyroleinhostevolution
andcansignificantlycontributetovariousfunctionsoftheorganism,suchassugarme‐
tabolism[41],digestionandtheabsorptionofnutrients[42,43],andtheproductionofmet‐
abolicenzymes[44].
WeinvestigatedthecompositionofthecultivablemicrobiomeofSouthernItalian
Troglophilebats,usingaculture‐basedapproach.Thepresentstudyalsoinvestigatesthe
presenceofpotentiallypathogenicGram‐negativeandGram‐positivebacteria.Thereisa
growingawarenessofhowthespreadofpathogensinwildanimalscanimpacthuman
andanimalhealth.Theemergenceofnewinfectiousdiseasesisnotonlyaconservation
issue,duetothedangersitposestoprotectedspecies,butalsoapotentialthreattopublic
health.Theisolationofinfectiousagentsinbatsindicatestheimportanceofmonitoring
thepresenceofpotentiallypathogenicbacteriainthisorder,isolatedfromdifferentbat
populations.
Ouranalysisledtotheidentificationofapproximatelysixhundredbacterialstrains
belongingtoninetydifferentbacterialspecies(50Gram‐negativeand40Gram‐positive)
and29genera(Figures1and3).Ourresultsdemonstratethepresenceofawidervariety
ofbacterialspeciesthanindicatedbypreviousresearchonEuropeanbats.DiBellaetal.
(2003)[17]isolated26bacterialspeciesbelongingto13generafromfaecalsamples(Aci‐
netobacter,Alcaligenes,Citrobacter,Enterobacter,Escherichia,Hafnia,Klebsiella,Kluyvera,Mor‐
ganella,Proteus,Pseudomonas,Streptococcus(nowEnterococcus),andYersinia),allofwhich
werealsofoundinoursurvey,withtheexceptionofYersiniaspp.Someofthespecies
isolatedinthepresentstudycanbeconsideredcommensalsorenvironmentalcontami‐
nants(e.g.,Bacillusspp.),butothers(e.g.,Salmonellaenterica,Klebsiellapneumoniae,Pseudo‐
monasaeruginosa,andStaphylococcusaureus)arepotentiallypathogenic,bothforbatsand
forotheranimalsandalsoforhumans[9,45].Enterococcusfaecaliswasthemostisolated
Figure 4. Distribution of Gram-positive bacteria in different sampling locations.
The percentage
of isolates belonging to six genera of Gram-negative bacteria is shown for six different sampling sites:
Grotta dei Pipistrelli, SR (cyan), Grotta Palombara, SR (orange), Grotta Chiusazza, SR (blue), Grotta
dei Pipistrelli, CS (pink), Grotta Burrò, CT (green), and Grave Grubbo, KR (yellow).
4. Discussion
In recent years, a new field of research has emerged that outlines the importance
of the microbiota in health and disease. The microbiome plays a key role in host evolu-
tion and can significantly contribute to various functions of the organism, such as sugar
metabolism [
41
], digestion and the absorption of nutrients [
42
,
43
], and the production of
metabolic enzymes [44].
We investigated the composition of the cultivable microbiome of Southern Italian
Troglophile bats, using a culture-based approach. The present study also investigates the
presence of potentially pathogenic Gram-negative and Gram-positive bacteria. There is
a growing awareness of how the spread of pathogens in wild animals can impact human
and animal health. The emergence of new infectious diseases is not only a conservation
issue, due to the dangers it poses to protected species, but also a potential threat to public
health. The isolation of infectious agents in bats indicates the importance of monitor-
ing the presence of potentially pathogenic bacteria in this order, isolated from different
bat populations.
Our analysis led to the identification of approximately six hundred bacterial strains
belonging to ninety different bacterial species (50 Gram-negative and 40 Gram-positive)
and 29 genera (Figures 1and 3). Our results demonstrate the presence of a wider variety
of bacterial species than indicated by previous research on European bats. Di Bella et al.
(2003) [
17
] isolated 26 bacterial species belonging to 13 genera from faecal samples (Acineto-
bacter,Alcaligenes,Citrobacter,Enterobacter,Escherichia,Hafnia,Klebsiella,Kluyvera,Morganella,
Proteus,Pseudomonas,Streptococcus (now Enterococcus), and Yersinia), all of which were also
found in our survey, with the exception of Yersinia spp. Some of the species isolated in the
present study can be considered commensals or environmental contaminants (e.g., Bacillus
spp.), but others (e.g., Salmonella enterica,Klebsiella pneumoniae,Pseudomonas aeruginosa, and
Staphylococcus aureus) are potentially pathogenic, both for bats and for other animals and
also for humans [
9
,
45
]. Enterococcus faecalis was the most isolated species among Gram-
positive bacteria, according to other authors [
46
]. It has been held responsible for several
diseases in bats (septicemia; pneumonia; myocarditis; and wound infection) [46].
Such bacteria could probably be endemic to bats and play a mutually beneficial
role, providing the host with stable growing conditions and additional nutrients [
47
].
For example, we have found the presence of Serratia marcescens in the oral cavity of all
four species of bats examined. Galizia et al. (2014) [
47
] found Serratia marcescens in the oral
cavity of hematophagous bats but not in frugivorous and nectivorous, hypothesizing a
role of Serratia in the production of an extracellular protein that binds to the hemo-hasA
group, which allows the release of the heme group from hemoglobin and thus acquire
the iron necessary for its metabolism. This bacterial species had been isolated by other
authors in the intestine of frugivorous [
48
,
49
]. Shigella spp. are rarely detected in animals
Animals 2022,12, 2684 11 of 14
other than primates. The presence of these bacterial species may be due to water or food
source contamination, or also through transmission from other bats within colonies [
33
]. In
addition, the high number of species isolated from faecal samples is potentially related to
the presence of endemic bacteria in ingested insects [
49
]. Habitat preference, geographic
origin, and eating habits are factors that can influence the colonization of bats with different
bacteria. Our results indicate a highly varied distribution of bacterial species in the different
sampling sites (only three species in common for Gram-negative and one species for Gram-
positive) but no significant difference was found in the distribution of pathogenic species.
The most commonly isolated bacterial genus in our study was Enterobacter, which has
the ability to break down a large number of sugars [
41
]. Five bacterial genera (Citrobacter,
Hafnia,Klebsiella,Morganella, and Serratia) were isolated from all bat species. Hafnia produce,
among others, enzymes that allow the degradation of chitin, favouring digestion and the
assimilation of nutrients in insectivorous bats [
42
]. The Serratia genus also appears to play
an active role in food degradation in several bats [43].
It would be useful to compare our results with data on the microbiota of further
species and populations living in other regions that have habitats and eating habits similar
to the subjects of our study. Further studies are needed to identify any bacterial species that
constitute a common core of troglophile bat microbiomes and to evaluate which species
play an important physiological or nutritional role.
Several bacteria detected in our investigation have also been found by other authors
in Europe, reinforcing the hypothesis of a common bacterial core [
17
,
44
]. Furthermore,
Hughes et al. (2018) demonstrated that there are no distinct shifts in microbiome composi-
tion between adult and juvenile insectivorous bats; the authors hypothesize that, through
the production of specific enzymes, this core of bacteria contributes to the fast metabolism
necessary to rapidly provide energy for flight [44].
The variety of bacterial species that make up the intestinal microbiome of insectivorous
bats depends on the environment, diet, and numerous other factors [
21
]. Experimental
studies have shown that the microbiomes of insectivorous bats placed in captivity con-
verge within a six-week period [
50
]. We believe it is important to continue studying the
microbiome of insectivorous bats from other regions with similar habitats to verify the
hypothesis that there is a symbiotic microbial core that has evolved hand in hand with
the hosts.
5. Conclusions
This study provides novel data on the cultivable microbiome of Southern Italian bats,
revealing a greater diversity of bacterial strains than measured by previous studies. Several
physiological bacteria were found to be common to all the studied bat species. These strains
may have evolved hand in hand with their hosts and thus constitute a common core of
symbiotic bacteria, but further studies are needed to evaluate their biological and ecological
functions. The results of the present study also indicate the presence of pathogenic and
potentially pathogenic strains in wild bat populations in Italy, but not in alarming numbers.
There is no evidence that the bats examined constitute a spread hazard of zoonotic bacterial
agents we researched. Nevertheless, troglophile bats populations can be considered a good
indicator of environmental contamination by potentially pathogenic bacteria and should
be regularly monitored for conservation and public health purposes.
Author Contributions:
M.F.: Project Administration; Conceptualization, Methodology, Writing—
Original draft preparation. M.T.S.: Methodology, Writing—Review and Editing. V.F.: Investigation
(laboratory procedures), Resources. A.M.: Investigation (sampling). M.C.: Data Curation, Formal
Analysis, Writing—Review and Editing. M.G.: Investigation (sampling). C.P.: Investigation (labora-
tory procedures). F.S.: Investigation (laboratory procedures). R.G.: Conceptualization, Investigation
(sampling), Project Administration. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Animals 2022,12, 2684 12 of 14
Institutional Review Board Statement:
The animal study protocol was approved by the Ethics Com-
mittee of Istituto Superiore per la Protezione e la Ricerca Ambientale (ISPRA)
(Prot. N. 14589/T-A31)
for studies involving animals.
Informed Consent Statement: Not applicable.
Data Availability Statement:
The data that support this study are available in Mendeley Data
repository at doi: 10.17632/tct4zh9fy6.1].
Acknowledgments:
R.G. and M.T.S. wish to express their sincere thanks to: A.M. De Marinis
(Istruttoria), P. Genovesi (Gestione Patrimonio Faunistico Nazionale e mitigazione danni e impatti)
and E. Morroni (ISPRA) for supporting the study of Chiroptera (Prot. 14589/T-A31). R.G. and M.T.S.
would like to tank: Mario Vincenzo Benedetto, Fabio Selvaggi and Gruppo Speleo-Archeologico
“Aquila Libera” (Cassano allo Ionio, CS); Antonio Larocca and Gruppo Speleologico “Sparviere”
(Alessandria del Carretto, CS); Francesco Ferraro and Gruppo Speleologico “Le Grave” (Verzino, KR);
Angelo Iemmolo and Speleo Club Ibleo (Ragusa, RG).
Conflicts of Interest: The authors declare no conflict of interest.
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