Physiological and potentially pathogenic microbial flora in bats

Abstract

The study of bats has a significant interest from a systematic, zoogeographic, ecological and physiological point of view, but their possible role as potential carriers of pathogenic bacteria is little explored and very little research has been carried out on the European continent. The aim of this study is to investigate the culturable aerobic enteric, conjunctival and oral bacterial flora of bats living in southern Italy to determine the physiological bacterial microflora and to investigate the possible occurrence of pathogenic bacteria. Five hundred and sixty-seven samples were collected from 189 individuals of 4 species of bats ( Myotis myotis, Myotis capaccinii, Miniopterus schreibersii and Rhinolophus hipposideros ). The sampling was carried out in six areas of the territory of Sicily and Calabria (southern Italy). All samples were examined for Gram negative bacteria; conjunctival and oral swabs were also submitted to bacteriological examination for Gram positive bacteria. Four hundred thirteen Gram negative strains were isolated. Of these, 377 belonged to 17 different genera of the Enterobacteriaceae Group and 30 to 5 other Families. One hundred eighty three Gram positive strains were isolated. Of these, 73 belonged to Staphylococcaceae Family, 72 to Bacillaceae Family and 36 to 4 other Families. To the best of our knowledge, this is the first time that some of these genera have been isolated from bats. The results confirmed that bats play an important role in the ecology and circulation of potentially pathogenic bacteria not only for wild species but also for domestic animals and for humans.

Full text

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Physiological and potentially pathogenic microbial ora in bats
Maria Foti (  malinvet@unime.it )
University of Messina: Universita degli Studi di Messina https://orcid.org/0000-0001-5591-8976
Mariateresa Teresa Spena
University of Catania: Universita degli Studi di Catania
Vittorio Fisichella
University of Messina: Universita degli Studi di Messina
Antonietta Mascetti
University of Messina: Universita degli Studi di Messina
Marco Colnaghi
UCL: University College London
Maria Grasso
University of Catania: Universita degli Studi di Catania
Chiara Piraino
Istituto Zooprolattico Sperimentale della Sicilia Adelmo Mirri
Franco Sciurba
Istituto Zooprolattico Sperimentale della Sicilia Adelmo Mirri
Rosario Grasso
University of Catania: Universita degli Studi di Catania
Research Article
Keywords: Bats, Bacteriological test, Gram negative bacteria, Gram positive bacteria, Public health, Southern Italy
Posted Date: September 28th, 2021
DOI: https://doi.org/10.21203/rs.3.rs-932107/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License.  Read Full License

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Abstract
The study of bats has a signicant interest from a systematic, zoogeographic, ecological and physiological point of view, but their possible
role as potential carriers of pathogenic bacteria is little explored and very little research has been carried out on the European continent. The
aim of this study is to investigate the culturable aerobic enteric, conjunctival and oral bacterial ora of bats living in southern Italy to
determine the physiological bacterial microora and to investigate the possible occurrence of pathogenic bacteria. Five hundred and sixty-
seven samples were collected from 189 individuals of 4 species of bats (
Myotis myotis, Myotis capaccinii, Miniopterus schreibersii
and
Rhinolophus hipposideros
). The sampling was carried out in six areas of the territory of Sicily and Calabria (southern Italy). All samples were
examined for Gram negative bacteria; conjunctival and oral swabs were also submitted to bacteriological examination for Gram positive
bacteria. Four hundred thirteen Gram negative strains were isolated. Of these, 377 belonged to 17 different genera of the Enterobacteriaceae
Group and 30 to 5 other Families. One hundred eighty three Gram positive strains were isolated. Of these, 73 belonged to Staphylococcaceae
Family, 72 to Bacillaceae Family and 36 to 4 other Families. To the best of our knowledge, this is the rst time that some of these genera
have been isolated from bats. The results conrmed that bats play an important role in the ecology and circulation of potentially pathogenic
bacteria not only for wild species but also for domestic animals and for humans.
Introduction
In Europe, the Chiroptera represent the Mammals 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 the exception of
Pipistrellus pipistrellus
. The species referable to the
Italian territory are currently 35 (Agnelli et al. 2004). 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 (Tiunov 1989). All Italian Chiroptera feed on Arthropods and are nocturnal even if several species come out of shelters in the
presence of light. The diet of
Myotis capaccinii
also includes aquatic larvae of Diptera and sh fry.
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 Hendra (Banerjee et
al. 2019; Ochani et al. 2019), Rabies (Rupprecht et al. 2011) and, more recently, CoV disease 2019 (COVID-19) (Zhou et al. 2020).
Furthermore, an increasing number of other viruses, whose pathogenic potential is still unknown, is constantly being reported in bats globally
(Chen et al. 2014; Fagre and Kading 2019). In spite of this growing evidence, the possible role of bats as potential carriers of pathogenic
bacteria has not been explored in great depth (Mühldorfer 2013; Veikkolainen et al. 2014). 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. (Tagi-Zade et al. 1970; Fennestad and Borg-Petersen 1972; Bai et al. 2017),
Borrelia
spp. (Evans et al. 2009),
Micoplasma
spp. (Millán et al. 2015),
Bartonella
spp. (Concannon et al. 2005; Veikkolainen et al. 2014; Bai et al. 2017; Stuckey et al. 2017; Corduneanu et
al. 2018) and
Rickettsia
spp. (Hornok et al. 2018) have been sporadically reported. Analysis of the intestinal microbial ora (Di Bella et al.
2003; Wolkers-Rooijackers et al. 2019), typically carried out by isolation from guano (Vandžurová et al. 2013), has revealed the presence of
single pathogenic species such as
Campylobacter
spp. (Hazeleger et al. 2018) and
Salmonella
spp. (Mühldorfer 2013). Data on the
occurrence of Gram positive bacteria is even scarcer, and most studies have focused on enteric bacteria from genera
Enterococcus
,
Lactococcus
and
Lactobacillus
(Di Bella et al. 2003; Vengust et al. 2018). Walther et al. (2008) describe the isolation of a methicillin-resistant
Staphylococcus aureus
strain from a bat wound. Vandžurová et al. (2013) isolated high percentages of
Staphylococcus nepalensis
in the
guano of
Myotis
spp. in Slovakia. The aim of this study is to investigate the culturable aerobic enteric, conjunctival and oral bacterial ora of
bats living in southern Italy to determine the physiological bacterial microora and to investigate the possible occurrence of pathogenic
bacteria. We have considered pathogenic the bacteria belonging to Risk Group 2 or higher, as per NIH guidelines (NIH, 2019).
Materials And Methods
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 4 different species of troglophile bats (Permit of Istituto Superiore per la Protezione e la Ricerca Ambientale (ISPRA)
Prot. N. 14589/T-A31) (Table n. 1).
Myotis myotis
lives in forest environments, open pastures and meadows. Its diet is mostly based on insects caught on the ground.

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Myotis capaccinii
feeds on insects, particularly Trichoptera, Neuroptera, Diptera and occasionally on sh fry caught just above water
surfaces or just below them.
Miniopterus schreibersii
feeds on insects caught in ight. With a highly specialized trophic regime, it preys mainly on Lepidoptera. Non-ying
preys are also reported in its diet.
Rhinolophus hipposideros
feeds on small insects (Diptera, Lepidoptera and Neuropoptera), which they capture in ight or on the ground (e.g.
spiders). They generally hunt individually, very close to the ground.
The sampling was carried out in six different caves of the southern Italy territory (Table n. 2).
Grotta dei Pipistrelli (SR). Although located in a protected area (Natural Reserve "Pantalica"), this cave is not far from the town of
Sortino, where semi-wild breeding is practiced. The cave is known for its large colonies of bats and it is the only systematically
monitored Sicilian cave. 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. Below the entrance to the cave ow two streams of water (Anapo and
Calcinara) from which the bats drink after emerging from the cave. The waste water from the municipality of Palazzolo Acreide (SR)
ows into the Anapo.
Grotta Palombara (Melilli, SR). Fossil karst cavity that develops for about 800 meters. Palombara cave is located near the biggest
petrochemical plant of Italy, 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 Chiroptera recorded was
about 1000 specimens. Human activity, especially the presence of numerous illegal landlls, has signicantly degraded the environment
around the cave.
Grotta Chiusazza (Floridia, SR). The total development is about 250 m. The area in which the cave opens is characterized by the
presence of intensive monocultures and, 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). Volcanic cave created as a result of the eruptive activity of Etna. It is a large lava ow tunnel, over 200
m long. The surrounding area has a dense shrub-herbaceous vegetation cover and no arable land. In the territory, semi-wild cattle
breeding is commonly practiced. The cave is inhabited by a large mixed colony of bats (about 600–700 Minioptera along with several
dozen large
Myotis
and Rhinolophids).
Grotta dei Pipistrelli (Cassano allo Jonio, CS). 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). Opens at 285 m a.s.l. and extends for 1926 m. The cave is active and water ows 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.
The samples were collected between September and November 2018.The bats were caught, in some cases, simply with the hands in order to
minimize the risk of disturbance to the colony. In some cavities a hand-held net with a telescopic handle was used to capture static and non-
ying bats. Only in 2 cavities (Grotta dei Pipistrelli of Sortino and Grotta dei Pipistrelli of Cassano allo Ionio) it was possible to use the harp-
trap to catch specimens that ew off the roosts. Rectal, conjunctival and oral swabs for bacteriological survey were obtained from each bat
using individually packed sterile microbiological swabs pmoistened with sterile saline solution 09%, 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 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 released
immediately after sampling.
Bacterial Isolation and Identication
The samples were transported in conditions of refrigeration to the laboratory and examined for potentially pathogens detection. All samples
(n. 567) were examined for Gram - 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 identication by mass

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spectrometry MALDI-TOF (matrix assisted laser desorption/ionisation - time of igt 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 are analysed by VITEK
MS system (bioMérieux SA, Marcy l'Etoile, France), using the software Axima (Shimadzu Kyoto, Japan)-SARAMIS database (Spectral
ARchive And Microbial Identication System) (AnagnosTec, Berlin, Germany). Eighty-eight strains, unidentied 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. 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 identied by OF, mobility, catalase, oxidase, urease and
triptophanase 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 (Grimont and Weill, 2007).
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, xing the signicance limit at P = 0.05.
Results
Four hundred forty-six samples (78.7%) were positive for bacteria and 121 (21.3%) were negative (Tab. n. 3).
In 64 samples out of 378 tested (16.9%) coexistence of Gram + and Gram - bacteria was found [12/189 conjunctival swabs (6.3%); 52/189
oral swabs (27.5%)].
Gram negative
Four hundred thirteen Gram negative strains from 567 tested samples were isolated. Of these, 377 belonged to 17 different genera of the
Enterobacteriaceae Group and 30 to 5 other Families (Table n. 4). Six strains have not been identied. The most isolated species was
Enterobacter cloacae
(71 strains, 17.2%),
Hafnia alvei
(47 strains, 11.4%)
Citrobacter
spp (46 strains, 11.1%),
Serratia marcescens and
Citrobacter freundii
(21 strains, 5.1%). Potentially pathogenic species including
Salmonella enterica
,
Klebsiella pneumoniae
and
Pseudomonas aeruginosa
have also been identied. Only 7 genera out of 23 (
Citrobacter
,
Enterobacter
,
Escherichia
,
Hafnia
,
Klebsiella
Morganella
and
Providencia
) were detected at all 3 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 (Foti et al. 2021).
Table n. 5 shows the results of bacteriological tests for sampling site and Table n. 6 for species of bat. Only 3 species (
Enterobacter cloacae,
Morganella morganii, Serratia marcescens
) were present in all the caves. No signicant 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
Pseudomonas aeruginosa
.
Escherichia coli
is more common among
Rhinolophus hipposideros
(16% of the samples) than in
Myotis myotis
,
Miniopterus schreibersii
and
Myotis
cappaccinii
(4.3%, 4.4% and 0% respectively). Fisher’s Exact Test showed signicant differences between
Rhinolophus hipposideros
and
Miniopterus schreibersii
(P = 0.0374).
Serratia marcescencens
occurs most frequently in
Myotis cappaccinii
(50%) than in
Myotis myotis
(6.4%),
Miniopterus schreibersii
(7.7%) and
Rhinolophus hipposideros
(16.3%). Fisher’s Exact Test showed signicant differences between
Myotis cappaccinii
and
Myotis myotis
(P = 0.0059) and
Myotis cappaccinii
and
Miniopterus schreibersii
(P = 0.0048).
Myotis myotis
is
characterised by a higher occurrence of
Pseudomonas aeruginosa
(21.3%) compared to
Miniopterus schreibersii
(3.3%),
Rhinolophus
hipposideros
(0%) and
Myotis cappaccinii
(0%). Fisher’s Exact Test showed signicant differences between
Myotis myotis
and
Miniopterus
schreibersii
(P = 0.0012) and
Rhinolophus hipposideros
(P = 0.0012).
Gram positive
One hundred eighty three Gram positive strains were isolated from a total of 378 samples (Table n. 7). Of these, 73 belonged to
Staphylococcaceae Family, 72 to Bacillaceae Family and 36 to 4 other Families. Two strains have not been identied. The most frequently
isolated species was
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%). Potentially pathogenic species including
Staphylococcus aureus
have also been
identied. Table n. 8 shows the results of bacteriological tests for sampling site and Table n. 9 for species of bat. Bacteria belonging to the

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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 4 species of bats were
Enterococcus faecalis, Bacillus cereus
and
Staphylococcus epidermidis
. In 11 samples
(6.4%) two different strains were isolated (Foti et al. 2021).
Discussion
The spread of pathogens in wild animals is gaining increasing interest around the world for their role in the animal and human 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 monitoring the presence of potentially
pathogenic bacteria in these species. But because of the scarcity of data and lack of experimental studies, there is a concrete risk that wild
bat populations become, unbeknown to the scientic community, environmental reservoirs of infectious diseases. The present study
investigates the presence of potentially pathogenic Gram negative and Gram positive bacteria, isolated from different bat populations using
culture-based approach. Our analysis led to the identication of about six hundred bacterial strains belonging to ninety different bacterial
species (50 Gram negative and 40 Gram positive) and 29 genera. To the best of our knowledge, this is the rst time that some of these
genera (
Pantoea
in Gram negative and
Aerococcus
,
Alloicoccus
,
Paenibacillus
in Gram positive) have been isolated from bats. Our results
demonstrate the presence of a wider variety of bacterial species than indicated by previous research on European bats. In Italy, Di Bella et al.
(2003) isolated 26 bacterial species belonging to 13 genera from faecal samples (
Acinetobacter
,
Alcaligenes
,
Citrobacter
,
Enterobacter
,
Escherichia
,
Hafnia
,
Klebsiella
,
Kluyvera
,
Morganella
,
Proteus
,
Pseudomonas
,
Streptococcus
(now
Enterococcus
) and
Yersinia
), all of which,
except for
Yersinia
spp, were also found in our survey. Some of the species isolated in the present study can be considered commensals or
environmental contaminants (e.g.
Bacillus
spp.), but several others (e.g.
Escherichia coli
,
Salmonella enterica
,
Klebsiella pneumoniae,
Pseudomonas aeruginosa, Staphylococcus aureus
) are potentially pathogenic, both for bats and for other animals and humans (Mühldorfer
2013). Such bacteria could be endemic to bats and play a mutually benecial role, providing the host with stable growing conditions and
additional nutrients (Galicia et al. 2014). For example, we have found the presence of
Serratia marcescens
in the oral cavity of all 4 species
of bats examined. Galizia et al. (2014) 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-
has
A 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 (Daniel et al. 2013; Banskar et al. 2016a) and hematophagous (Chaverri et al. 2006).
Shigella
spp. are rarely detected in animals other than primates (Edwards 1999).
Shigella exneri
has been previously isolated in rectal
swabs in bats from Poland (Ròzalska et al. 1998) and in the intestine of bats in Madagascar (Brygoo et al. 1971). The presence of these
bacterial species may be due to water or food source contamination, or also through transmission from other bats within colonies (Daniel et
al. 2013). In addition, the high number of species isolated from faecal samples is potentially related to the presence of endemic bacteria in
ingested insects (Banskar et al. 2016). Habitat preference, geographic origin and eating habits are factors that can inuence the colonization
of bats with different bacteria. Our results indicate a highly varied distribution of bacterial species in the different sampling sites (only 3
species in common for Gram negative and 1 species for Gram positive) but no signicant difference was found in the distribution of
pathogenic species.
Conclusions
Recent events related to the Covid-19 pandemic have underlined the importance of bats role in the pathogenic microorganisms spread. The
results of the present study conrm the presence of pathogenic and potentially pathogenic bacteria in wild bat populations in Italy. Our study
indicates that such populations can act as an environmental reservoir of infectious agents and therefore constitute a potential health threat
not only to wild species, but also to domestic animals and humans.
Declarations
Funding
This research did not receive any specic grant from funding agencies in the public, commercial, or not-for-prot sectors.
Conicts of Interest
The authors have no nancial or proprietary interests in any material discussed in this article..
Ethical approval
All applicable institutional and/or national guidelines for the care and use of animals were followed.

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Consent to participate
Not applicable
Consent for publication
Not applicable
Authors' contributions
Maria Foti: Project Administration; Conceptualization, Methodology, Writing- Original draft preparation. Maria Teresa Spena: Methodology,
Writing – Review & Editing. Vittorio Fisichella: Investigation (laboratory procedures), Resources. Antonietta Mascetti: Investigation
(sampling). Marco Colnaghi: Data Curation, Formal Analysis. Maria Grasso: Investigation (sampling). Chiara Piraino: Investigation
(laboratory procedures). Franco Sciurba: Investigation (laboratory procedures). Rosario Grasso: Conceptualization, Investigation (sampling),
Project Administration.
Data Availability Statement
The data that support this study are available in Mendeley Data repository at doi: 10.17632/tct4zh9fy6.1
Acknowledgements
RG and MTS 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). RG and MTS 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).
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Tables
Table n. 1: Individuals sampled
Superfamily Family Subfamily Specie n. individuals
Vespertilionoidea Vespertilionoidae Myotinae
Myotis myotis
47
Myotis capaccinii
8
Miniopteridae
 Miniopterus schreibersii
91
Rhinolophoidea Rhinolophidae
 Rhinolophus hipposideros
43
Total 
 
189

Table n. 2: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
Table n. 3:Number of samples
Sample Positive Negative Total
Rectal swabs 148 41 189
Conjunctivalswabs 120 69 189
Oral swabs 178 11 189
Total 446 121 567
Table n. 4: Results of bacteriological tests for Gram negative detection in different samples

Page 9/23
Bacterial Family Bacterial species Number of isolates
R C O Total
Aeromonadaceae
Aeromonas hydrophila
  3 3
Aeromonas
spp   1 1
Alcaligenaceae

Achromobacter denitricans
 1  1
Achromobacter insolitus
 1 1 2
Alcaligenes faecalis
 1  1
Alcaligenes xylosoxidans
  1 1
Enterobacteriaceae Group

Citrobacter amalonaticus
3  2 5
Citrobacter farmeri
1  1 2
Citrobacter freundii
16  5 21
Citrobacter sedlakii
2   2
Citrobacter
spp 31 3 12 46
Citrobacter youngae
1   1
Cronobacter muytjensis
 1  1
Cronobacter zurichensis
  1 1
Enterobacter aerogenes
2 1 9 12
Enterobacter agglomerans
2   2
Enterobacter asburiae
  1 1
Enterobacter cancerogenus
11 2 6 19
Enterobacter cloacae
38 5 28 71
Enterobacter gergoviae
  1 1
Enterobacter kobei
4 1 5 10
Enterobacter ludwigii
7 1 4 12
Enterobacter
spp 6 1 4 11
Escherichia coli
6 1 6 13
Hafnia alvei
19 4 24 47
Klebsiella oxytoca
3  10 13
Klebsiella pneumoniae
2 1 2 5
Kluyvera intermedia
  2 2
Leclercia adecarboxilata
  1 1
Moellerella wisconsensis
  2 2
Morganella morganii
2 1 13 16
Pantoea agglomerans
 1  1
Proteus mirabilis
1  4 5
Proteus vulgaris
1 1  2
Providencia alcalifaciens
  2 2
Providencia rettgeri
8 2 7 17
Providencia rustigianii
1 1 1 3

Page 10/23
Providencia
spp   1 1
Rahnella aquatilis
  1 1
Salmonella
Manhattana1   1
Serratia liquefaciens
1  3 4
Serratia marcescens
2  19 21
Serratia plymuthica
1   1
Shighella exneri
 1  1
Moraxellaceae
Acinetobacter
spp.  1  1
Pasteurellaceae
Pasteurella aerogenes
1   1
Pseudomonadaceae

Pseudomonas aeruginosa
2  11 13
Pseudomonas aureofaciens
  1 1
Pseudomonas
spp  1  1
Pseudomonas stutzeri
 2 2 4

Not id
1 1 4 6
Total

178 36 199 413
aSalmonella isolate named by the respective serotype of
Salmonella enterica
ssp.
enterica
.
Table n. 5: Results of bacteriological tests for sampling site.

Page 11/23
Site Bacterial species Number of isolates
R C O Total
G. pipistrelli (SR)
Achromobacter insolitus
  1 1
Aeromonas hydrophila
  1 1
Aeromonas
spp   1 1
Alcaligenes xylosoxidans
  1 1
Citrobacter amalonaticus
3  2 5
Citrobacter freundii
4   4
Citrobacter sedlakii
2   2
Citrobacterspp
5 2  7
Enterobacter aerogenes
 1 5 6
Enterobacter cancerogenus
 1  1
Enterobacter cloacae
9 1 4 14
Enterobacter kobei
1 1 3 5
Enterobacter ludwigii
1 1 1 3
Hafnia alvei
3  2 5
Klebsiella oxytoca
  2 2
Kluyvera intermedia
  1 1
Morganella morganii
  1 1
Pasteurella aerogenes
1   1
Proteus vulgaris
 1  1
Providencia rettgeri
  1 1
Pseudomonas aeruginosa
  7 7
Serratia liquefaciens
  1 1
Serratia marcescens
  7 7
N.I. 1   1
Total 30 8 41 79
Palombara
Alcaligenes faecalis
 1  1
Cronobacter muytjensis
 1  1
Cronobacter zurichensis
  1 1
Enterobacter aerogenes
1  1 2
Enterobacter cloacae
8 2 6 16
Leclercia adecarboxilata
  1 1
Morganella morganii
  1 1
Proteus mirabilis
  2 2
Pseudomonas aeruginosa
1  1 2
Serratia marcescens
  1 1
Total 10 4 14 28
Chiusazza
Citrobacter freundii
3   3

Page 12/23
Citrobacter
spp 12  3 15
Enterobacter aerogenes
  2 2
Enterobacter cancerogenus
1  1 2
Enterobacter cloacae
10 1 9 20
Enterobacter gergoviae
  1 1
Enterobacter ludwigii
1  1 2
Enterobacter spp
2 1 1 4
Escherichia coli
2 1 2 5
Hafnia alvei
  1 1
Klebsiella oxytoca
1  1 2
Klebsiella pneumoniae
2 1  3
Morganella morganii
1 1 3 5
Pantoea agglomerans
 1  1
Proteus mirabilis
1  2 3
Providencia alcalifaciens
  1 1
Providencia rettgeri
5 1 2 8
Pseudomonas aeruginosa
  3 3
Salmonella
spp 1   1
Serratia liquefaciens
  1 1
Serratia marcescens
  2 2
Shighella exneri
 1  1
Total 42 8 36 86
G. pipistrelli (CS)
Acinetobacter
spp  1  1
Citrobacter freundii
3  1 4
Citrobacter
spp 5  5 10
Enterobacter aerogenes
1   1
Enterobacter cancerogenus
3 1 2 6
Enterobacter cloacae
3 1 1 5
Enterobacter ludwigii
3   3
Enterobacter spp
2   2
Escherichia coli
1   1
Hafnia alvei
4  2 6
Klebsiella oxytoca
  2 2
Klebsiella pneumoniae
  1 1
Morganella morganii
1  3 4
Providencia rettgeri
2  2 4
Providencia
spp   1 1
Pseudomonas aeruginosa
1   1
Rahnella aquatilis
  1 1

Page 13/23
Serratia liquefaciens
  1 1
Serratia marcescens
  4 4
Total 29 3 26 58
Burrò
Citrobacter freundii
1  3 4
Citrobacter
spp 5 1 4 10
Enterobacter aerogenes
  1 1
Enterobacter cancerogenus
2  1 3
Enterobacter cloacae
2  8 10
Enterobacter kobei
2  2 4
Enterobacter ludwigii
1   1
Enterobacter
spp 1   1
Escherichia coli
1  3 4
Hafnia alvei
4 2 12 18
Klebsiella oxytoca
2  3 5
Moellerella wisconsensis
1   1
Morganella morganii
  3 3
Proteus vulgaris
1   1
Providencia alcalifaciens
  1 1
Providencia rettgeri
1  2 3
Providencia rustigianii
1 1 1 3
Pseudomonas
spp  1  1
Pseudomonas stutzeri
  2 2
Serratia marcescens
  2 2
Serratia plymuthica
1   1
Total 26 5 48 79
Grave Grubbo
Achromobacter denitricans
 1  1
Achromobacter insolitus
 1  1
Aeromonas hydrophila
  2 2
Citrobacter farmeri
1  1 2
Citrobacter freundii
5  1 6
Citrobacter
spp 4   4
Citrobacter youngae
1   1
Enterobacter agglomerans
2   2
Enterobacter asburiae
  1 1
Enterobacter cancerogenus
5  2 7
Enterobacter cloacae
6   6
Enterobacter kobei
1   1
Enterobacter ludwigii
1  2 3
Enterobacter
spp 1  3 4

Page 14/23
Escherichia coli
2  1 3
Hafnia alvei
8 2 7 17
Klebsiella oxytoca
  2 2
Klebsiella pneumoniae
  1 1
Kluyvera intermedia
  1 1
Moellerella wisconsensis
1   1
Morganella morganii
  2 2
Providencia rettgeri
 1  1
Pseudomonas aureofaciens
  1 1
Pseudomonas stutzeri
 2  2
Serratia liquefaciens
1   1
Serratia marcescens
2  3 5
N.I.  1 4 5
Total 41 8 34 83
Total

178 36 199 413

Table n. 6: Results of bacteriological tests for species of bat

Page 15/23
  Number of isolates 
Bacterial Family Bacterial species
Myotis
myotis Miniopterus
schreibersii Rhinolophushipposideros Myotis
capaccinii
Total
Aeromonadaceae

Aeromonas hydrophila
 1 2  3
Aeromonas
spp    1 1
Alcaligenaceae

Achromobacter
denitricans
  1  1
Achromobacter
insolitus
 1 1  2
Alcaligenes faecalis
 1   1
Alcaligenes
xylosoxidans
  1  1
Enterobacteriaceae
Group
Citrobacter
amalonaticus
4 1   5
Citrobacter farmeri
 2   2
Citrobacter freundii
3 8 8 2 21
Citrobacter sedlakii
2    2
Citrobacter
spp 17 19 10  46
Citrobacter youngae
 1   1
Cronobacter
muytjensis
1    1
Cronobacter
zurichensis
1    1
Enterobacter
aerogenes
9 2 1  12
Enterobacter
agglomerans
 2   2
Enterobacter asburiae
  1  1
Enterobacter
cancerogenus
5 9 5  19
Enterobacter cloacae
20 42 9  71
Enterobacter
gergoviae
  1  1
Enterobacter kobei
2 6 2  10
Enterobacter ludwigii
4 6 2  12
Enterobacter
spp 2 8 1  11
Escherichia coli
2 4 7  13
Hafnia alvei
6 21 18 2 47
Klebsiella oxytoca
2 2 7 2 13
Klebsiella pneumoniae
4 1   5
Kluyvera intermedia
1 1   2
Leclercia
adecarboxilata
1    1
Moellerella
wisconsensis
  2  2
Morganella morganii
1 8 6 1 16

Page 16/23
Pantoea agglomerans
 1   1
Proteus mirabilis
1 2 2  5
Proteus vulgaris
 1 1  2
Providencia
alcalifaciens
 1 1  2
Providencia rettgeri
8  8 1 17
Providencia rustigianii
 3   3
Providencia
spp 1    1
Rahnella aquatilis
 1   1
Salmonella enterica
1    1
Serratia liquefaciens
1 1 1 1 4
Serratia marcescens
3 7 7 4 21
Serratia plymuthica
  1  1
Shighella exneri
  1  1
Moraxellaceae
Acinetobacter
spp.  1   1
Pasteurellaceae
Pasteurella aerogenes
  1  1
Pseudomonadaceae

Pseudomonas
aeruginosa
10 3   13
Pseudomonas
aureofaciens
 1   1
Pseudomonas
spp  1   1
Pseudomonas stutzeri
 2 2  4
 Not id.  4 1 1 6
 Total 112/47 175/91 111/43 15/8 413
Table n. 7: Results of bacteriological tests for Gram positive detection in different samples

Page 17/23
Bacterial Family Bacterial species Number of isolates
C O Total
Aerococcaceae
Aerococcus viridans
1 2 3
Bacillaceae
Bacillus amiloliquefaciens
3 1 4

Bacillus atrophaeus
1  1

Bacillus brevis
1 1 2

Bacillus cereus
2 3 5

Bacillus circulans
 2 2

Bacillus clausii
2  2

Bacillus coagulans
1  1

Bacillus fastidiosus
1  1

Bacillus rmus
1  1

Bacillus laterosporus
1  1

Bacillus licheniformis
4 11 15

Bacillus macerans
1  1

Bacillus megaterium
11 3 14

Bacillus pantothenticus
1  1

Bacillus pasteurii
1  1

Bacillus pumilis
3 2 5

Bacillus simplex
2  2

Bacillus sphaericus
2  2

Bacillus spp
2  2

Bacillus subtilis
6 2 8
Enterococcaceae
Enterococcus casseliavus
2 1 3

Enterococcus faecalis
9 19 28
Lactobacillaceae
Alloicoccus otitis
 1 1
Paenibacillaceae
Paenibacillus durus
 1 1
Staphylococcaceae
Staphylococcus aureus
 1 1

Staphylococcus capitis
2 1 3

Staphylococcus cohnii
subsp.1 2 2 4

Staphylococcus cohnii
subsp. 2 5  5

Staphylococcus epidermidis
9  9

Staphylococcus gallinarum
1 1 2

Staphylococcus haemolyticus
2 2 4

Staphylococcus hominis
1  1

Staphylococcus koosi
 3 3

Staphylococcus lentus
2 5 7

Staphylococcus saprophyticus
2 1 3

Staphylococcus sciuri
1 11 12

Page 18/23

Staphylococcus simulans
2  2

Staphylococcus warneri
8 2 10

Staphylococcus xylosus
5 2 7
 Not id. 1 1 2
Total  101 81 183

Table n. 8: Results of bacteriological tests for sampling site.

Page 19/23
Site Bacterial species Number of isolates
C O Total
G. pipistrelli (SR)
Aerococcus viridans
 2 2
Bacillus amyloliquefaciens
2  2
Bacillus atrophaeus
1  1
Bacillus brevis
 1 1
Bacillus cereus group
1  1
Bacillus licheniformis
3 4 7
Bacillus megaterium
 1 1
Bacillus pumilis
 1 1
Bacillus subtilis
3  3
Enterococcus faecalis
1 3 4
Staphylococcus aureus
 1 1
Staphylococcus capitis
 1 1
Staphylococcus cohnii
subsp.1 2  2
Staphylococcus cohnii
subsp.2 2  2
Staphylococcus epidermidis
3  3
Staphylococcus gallina rum
1 1 2
Staphylococcus haemolyticus
1 1 2
Staphylococcus hominis
1  1
Staphylococcus saprophyticus
1  1
Staphylococcus sciuri
1 3 4
Staphylococcus simulans
1  1
Staphylococcus warneri
1 1 2
Staphylococcus xylosus
1 1 2
Total 26 21 47
Palombara
Bacillus amiloliquefaciens
 1 1
Bacillus coagulans
1  1
Bacillus licheniformis
 2 2
Bacillus macerans
1  1
Bacillus pasterii
1  1
Bacillus pumilus
1 1 2
Enterococcus faecalis
 2 2
Paenibacillus durus
 1 1
Staphylococcus cohnii
subsp.
1
 1 1
Staphylococcus epidermidis
1  1
Staphylococcus saprophyticus
1  1
NI
1  1
Total 7 8 15

Page 20/23
Chiusazza
Alloiococcus otitis
 1 1
Bacillus clausii
2  2
Bacillus megaterium
3  3
Bacillus simplex
1  1
Bacillus subtilis
1  1
Enterococcus casseliavus
1  1
Enterococcus faecalis
3 1 4
Staphylococcus cohnii
subsp. 1  1 1
Staphylococcus cohnii
subsp. 2 1  1
Staphylococcus epidermidis
2  2
Staphylococcus sciuri
 1 1
Staphylococcus warneri
1  1
Total 15 4 19
G. pipistrelli (CS)
Bacillus amiloliquefaciens
1  1
Bacillus brevis
 1 1
Bacillus cereus
 1 1
Bacillus fastidiosus
1  1
Bacillus rmus
1  1
Bacillus laterosporus
1  1
Bacillus licheniformis
 1 1
Bacillus megaterium
 2 2
Bacillus pumilus
1  1
Bacillus sphaericus
2  2
Bacillus subtilis
1 1 2
Enterococcus casseliavus
1  1
Enterococcus faecalis
1 4 5
Staphylococcus cohnii
subsp 2 1  1
Staphylococcus haemolyticus
1  1
Staphylococcus lentus
 4 4
Staphylococcus sciuri
 3 3
Staphylococcus warneri
1  1
Total 13 17 30
Burrò
Bacillus cereus
 1 1
Bacillus cereus
subsp
mycoides
 1 1
Bacillus licheniformis
 5 5
Bacillus megaterium
1  1
Bacillus pantothenticus
1  1
Bacillus pasteuri
1  1
Bacillus pumilus
1  1

Page 21/23
Bacillus simplex
1  1
Enterococcus casseliavus
 1 1
Enterococcus faecalis
4 6 10
Staphylococcus capitis
1  1
Staphylococcus epidermidis
1  1
Staphylococcus lentus
 1 1
Staphylococcus warneri
2  2
Staphylococcus xylosus
1 1 2
NI  1 1
Total 14 17 31
Grave Grubbo
Aerococcus viridans
1  1
Bacillus cereus subsp mycoides
1  1
Bacillus circulans
 2 2
Bacillus megaterium
7  7
Bacillus
spp 2  2
Bacillus subtilis
1 1 2
Enterococcus faecalis
 3 3
Staphylococcus capitis
1  1
Staphylococcus cohnii
subsp 2 1  1
Staphylococcus epidermidis
2  2
Staphylococcus haemolyticus
 1 1
Staphylococcus koosi
 3 3
Staphylococcus lentus
2  2
Staphylococcus saprophyticus
 1 1
Staphylococcus sciuri
 4 4
Staphylococcus simulans
1  1
Staphylococcus warneri
3 1 4
Staphylococcus xylosus
3  3
Total 25 16 41
Total

100 83 183

Table n. 9: Results of bacteriological tests for species of bat

Page 22/23
  Number of isolates 
Bacterial Family Bacterial species Myotis Miniopterus Rhinolophus Capaccinii Total
Aerococcaceae
Aerococcus viridans
 2  1 3

Bacillaceae
Bacillus amyloliquefaciens
2 2   4
Bacillus atrophaeus
1    1
Bacillus brevis
 1 1  2
Bacillus cereus
1 1 2 1 5
Bacillus circulans
 2   2
Bacillus clausii
 1 1  2
Bacillus coagulans
1    1
Bacillus fastidiosus
 1   1
Bacillus rmus
 1   1
Bacillus laterosporus
 1   1
Bacillus licheniformis
3 7 5  15
Bacillus macerans
1    1
Bacillus megaterium
1 12 1  14
Bacillus pantothenticus
 1   1
Bacillus pasteurii
 1 1  2
Bacillus pumilis
2 2 1  5
Bacillus simplex
  2  2
Bacillus sphaericus
 2   2
Bacillus
spp  2   2
Bacillus subtilis
3 4 1  8
Enterococcaceae
Enterococcus casseliavus
2   1 3
Enterococcus faecalis
7 14 5 2 28
Lactobacillaceae
Alloicoccus otitis
  1  1
Paenibacillaceae
Paenibacillus durus
 1   1
Staphylococcaceae

Staphylococcus aureus
1    1
Staphylococcus capitis
 1 2  3
Staphylococcus cohnii
subsp.1 2 1 1  4
Staphylococcus cohnii
subsp. 2 4 1   5
Staphylococcus epidermidis
1 4 3 1 9
Staphylococcus gallinarum
1 1   2
Staphylococcus haemolyticus
 3 1  4
Staphylococcus hominis
   1 1
Staphylococcus koosi
 3   3
Staphylococcus lentus
3 2 2  7
Staphylococcus saprophyticus
1 2   3
Staphylococcus sciuri
7 3 2  12

Page 23/23
Staphylococcus simulans
 2   2
Staphylococcus warneri
 8 1 1 10
Staphylococcus xylosus
 5 1 1 7
 Not id.  1  1 2
 Total 44/47 95/91 34/43 10/8 183/189
