Journal of Threatened
Taxa | www.threatenedtaxa.org | 26 May 2025 | 17(5): 26951–26962
ISSN 0974-7907 (Online) | ISSN 0974-7893 (Print)
https://doi.org/10.11609/jott.9449.17.5.26951-26962
#9449 | Received 09 October 2024 | Final received 25 February 2025 |
Finally accepted 21 April 2025
First characterization of the
bacteriological profile of the Mediterranean Pond Turtle Mauremys
leprosa (Schweigger,
1812) in Reghaïa Lake, Algeria
Feriel Benhafid
1, Badis Bakhouche
2, Abdenour Moussouni
3, Rayane Ahcene Reda
Belaidi 4,
Imed Djemadi
5, Amina Saadi 6 & Naouelle Azzag 7
1 Laboratory of Eco-Biology
Animals, École Normale Supérieure
de Kouba Bachir El Ibrahimi, BP 92, 16050 Algiers, Algeria.
2 Laboratory of Biological
Oceanography and the Marine Environment, University of Science and Technology
of Houari Boumediene, BP 32, 16111 Algiers, Algeria.
3 Laboratory of Algerian Forests
and Climate Change, Higher National School of Forests, Route de Hammam Essalhine 40000, Khenchela,
Algeria.
4 Laboratory of Biological
Oceanography and the Marine Environment, University of Science and Technology
of Houari Boumediene, BP 32, 16111 Algiers, Algeria.
5 Environment Research Center,
Biodiversity and Environment Division, Annaba, Algeria.
6 Dynamic and Biodiversity
Laboratory, University of Science and Technology of Houari
Boumediene, BP 32, 16024 Algiers, Algeria.
7 Research Laboratory for
Management of Local Animal Resources, Higher National Veterinary School of
Algiers, Rue Issad Abbes, El Alia, Algiers 16025,
Algeria.
1 ferielbenhafid@gmail.com, 2 badis_bakhouche@yahoo.fr,
3 moussouniabdenour@gmail.com (corresponding author), 4 rayaneahcenebelaidi@gmail.com,
5 i.djemadi@cre.dz, 6 amina.saadibak@gmail.com,
7 n.azzag@ensv.dz
Editor: Aniruddha Belsare, Auburn University College of Veterinary Medicine,
Auburn, Alabama, United States. Date of publication: 26
May 2025 (online & print)
Citation: Benhafid, F., B. Bakhouche, A. Moussouni, R.A.R. Belaidi, I. Djemadi, A. Saadi & N. Azzag (2025). First characterization of the
bacteriological profile of the Mediterranean Pond Turtle Mauremys
leprosa (Schweigger, 1812)
in Reghaïa Lake, Algeria. Journal of Threatened Taxa 17(5): 26951–26962. https://doi.org/10.11609/jott.9449.17.5.26951-26962
Copyright: © Benhafid et al. 2025. Creative Commons Attribution 4.0
International License. JoTT allows unrestricted use,
reproduction, and distribution of this article in any medium by providing
adequate credit to the author(s) and the source of publication.
Funding: None.
Competing interests: The authors declare no competing interests.
Author details: Feriel Benhafid is a PhD student at the High education school “Kouba”, where her research focuses on conservation of continental chelonians. Badis Bakhouche is an associate professor in Science and Technology Houari Boumediene university (USTHB). He leads “Environment and bio-monitoring” team within Ecology and Biodiversity Laboratory at High education school “Kouba”. Abdenour Moussouni an associate professor in Higher National School of Forests in Khenchela, Algeria. He leads the Biodiversity and Forest Ecosystems group at the Laboratory of Algerian Forests and Climate Change. His research focuses on biodiversity conservation and sustainable forest management. Rayane Ahcene Reda Belaidi hold a master degree in ecology and environment USTHB. He is currently focusing on research in the field of animal bacteriology. Imed Djemadi is an associate professor leading wildlife monitoring and protection team. His work combines research and conservation, contributing to ecological assessments and sustainable development, particularly in managing protected areas. Amina Saadi is an assistant professor at USTHB, working in the field of zootaxonomy and ecology. Her studies contribute to ecological monitoring and conservation efforts in protected area. Naouelle Azzag is an Associate Professor at the Higher National Veterinary School in Algiers, Algeria, with extensive experience in veterinary microbiology and genetics. She leads the Genetic Evolution of Pathogenic Microbial Agents research team. Her research focuses on pathogenic microorganisms affecting livestock.
Author contributions: Feriel Benhafid, Badis Bakhouche, and Abdenour Moussouni led the study design, software, data handling, and visualization. Methodology, analysis, and fieldwork involved all co-authors. Writing was led by Feriel Benhafid and Abdenour Moussouni, with contributions from all. Supervision: Abdenour Moussouni and Naouelle Azzag. All authors contributed equally to the article and approved the submitted version.
Acknowledgements: We sincerely thank Mr. Ramzi Abbada, director
of the Reghaïa Nature Reserve, for all the support and facilities provided during the fieldwork.
Abstract: The Mediterranean Pond Turtle Mauremys leprosa, a
‘Near Threatened’ species, is poorly studied in Algeria and no data exists on
its bacteriological carriage. However, knowledge about the microbiota of
wildlife species is essential to develop holistic conservation approaches that
integrate microbial health, habitat preservation, and species-specific needs.
Recent concerns regarding the potential transmission of zoonotic pathogens by
turtles have been highlighted in several studies. In this context, the current
study analyzed the aerobic cloacal/fecal bacteria associated with the
Mediterranean Pond Turtles, which were collected from Reghaïa
Lake. Samples collected from 24 turtles allowed the identification of 11
bacterial genera. Salmonella was the most frequent isolated genus with a
percentage of 22 of the total isolates, followed by Escherichia and Enterobacter.
The diversity of genera isolated from juveniles is relatively low compared with
adults. Turtle-bacterial genera relationships were tested by logistic regressions
and redundancy analysis (RDA). Results of RDA indicate a statistically
significant association (p-value <0.01) between morphological features and
bacterial genera frequency. Our results confirm the reputation of freshwater
turtles as a reservoir of several zoonotic bacterial pathogens. This microbiota
analysis offers a non-invasive, multi-faceted approach to conserving endangered
species by linking health, habitat, reproduction, and ecological dynamics. This
highlights the importance of establishing an epidemiological surveillance
system and an awareness program must be carried out to reduce the health risks
associated with owning pet turtles.
Keywords: Chelonian, cloacal microbiota,
freshwater ecosystems, health risk, wildlife conservation, zoonotic pathogens.
Introduction
Freshwater turtles are key
elements of aquatic ecosystems and play various ecological roles as predators
and herbivores. Their presence in, and interactions with, the environment are
crucial for maintaining the ecological balance and biodiversity of freshwater
ecosystems (Wilbur 1997; Garig et al. 2020).
Among the species of chelonians
in limnic ecosystems, the Mediterranean Pond Turtle Mauremys leprosa,
widely distributed in northern Africa, the Iberian Peninsula, and southern France,
stands out from other turtles occupying the same range (European Pond Turtle Emys orbicularis) due to its high tolerance
to salinity, environmental pollution, and its adaptation to anthropogenic
changes (Maran 1996; Bertolero & Busack 2017; El hassani et al.
2019). It is listed as Near Threatened in the IUCN Red List of Threatened
Species in 2023 (Luiselli 2024), considered
Vulnerable in the European Red List of Reptiles (Cox & Temple 2009), and
included in Appendix II of the Bern Convention.
However, compared to other
species, the Mediterranean Pond Turtle remains one of the least studied species
in ecological research (Nowakiewicz et al. 2015).
Indeed, only three studies focus on the ecology of this species in Algeria (Bakhouche et al. 2019; Ramedani
et al. 2024).
To the best of our knowledge,
none have examined the bacterial carriage of the Mediterranean Pond Turtle.
Recent concerns regarding the potential transmission of zoonotic pathogens by
turtles and reptiles were highlighted in several studies demonstrating the
presence of zoonotic fecal bacterial carriage in these animals (Kautman et al. 2016; Hernandez et al. 2021). These facts
raise concerns about the potential risks of transmitting dangerous pathogens to
humans in Algeria, due to the commercialization of wild species known to be
reservoirs of pathogens in several provinces (Tiar et
al. 2019).
On the other hand, the close
association of hosts and their microbiomes and the functional role of the
microbiota provide essential information for ecological aspects of conservation
biology (Zhu et al. 2021). Knowledge of microbiota can help identify specific
ecological needs of endangered species, such as dietary preferences or habitat
types that support a particular microbial community (Redford et al. 2012).
Our study seeks to characterize
the aerobic cloacal and fecal microbiota of the Mediterranean Pond Turtle,
focusing on identifying potential zoonotic pathogens within this microbial
community. By examining the turtle–bacteria interactions, we aim to better
understand the role of bacteria as pathogenic agents in wild turtles.
Methods
Study site
The study was conducted in Réghaïa’s Lake which was designated as a RAMSAR site during
2003. The site is located 30 km from the city of Algiers (36.785 0N,
3.342 0E). Bordered to the north by the Mediterranean Sea, this lake
takes the shape of a basin with a depth of 7–9 m for which Oued de Réghaïa constitutes the main tributary (Image 1). The
shores of the lake are slightly sloping. The immediate surroundings consist of
fallow fields, wild olive scrub, and mastic trees, as well as few groves of
Eucalyptus. It remains in the region the only witness and remnant of the
various biogeographical characteristics of the former coastal wetland areas of
the Mitidja Plain.
Study population and data
collection
All captured turtles were marked
to avoid duplicate sampling of the same individuals’ turtles during the same
study period. The surveys were carried out between March and May 2023, at a
rate of two monitoring sessions per week. This period is considered optimal for
studying the Mediterranean Pond Turtle of Reghaïa
Lake, according to our study already conducted on the site in 2019 (Bakhouche et al. 2019). As the site is classified as a Ramsar site and where many birds nest, we chose to sample
only at one station to avoid any disturbance. Capture of individuals was
carried out using a net and a fishing line. Once the individuals are captured,
they are marked. The marking involves making an incision with a file on the
marginal scales of the carapace. Recaptures allow tracking individuals over
time through their marking. Biometric measurements allow, on one hand, to
compare individuals with each other, and on the other hand, to test
relationship between bacterial carriage and species characteristics.
The method used for age
estimation is direct count of growth rings. Sexual maturity was assessed based
on the external secondary sexual characteristics of the shell and the sexual
behavior of individuals.
Comparison of different
morphological features was carried out using the Mann-Whitney U test for
independent samples. Statistical analysis was conducted using IBM SPSS software
version 27.
Analyses of bacterial communities
Samples were taken in situ for
each individual captured turtle. Two cloacal swabs were taken per specimen. One
was used for selective enrichment and the second for non-selective enrichment.
Samples are transported in an isothermal bag at 4 °C, then frozen at (-20) °C
until bacterial analysis.
Isolation and bacteriological
characterization were carried out at the medical microbiology laboratory of the
Higher National Veterinary School of Algiers, following the steps below:
- Pre-enrichment: Inoculation on
salmonella-selective Rappaport Vassiliadis Soy (RSV)
broth and non-selective Brain Heart Infusion (BHI) broth to amplify bacterial
numbers and increase chances of isolation. The swabs are immersed in the liquid
medium in a sterile manner, close to the Bunsen burner. The tubes are then
incubated at 37 °C for 18–24 hours.
- Isolation of bacteria on
selective media: The bacterial isolation was performed on selective media
(Microbiology agar, Merck KGaA, Germany) following
pre-enrichment and subsequent Gram staining. Inoculation of four prepared
culture media and incubation at 37 °C for 24–48 hours (Salmonella-Shigella (SS)
agar: for selective isolation of Salmonella. Eosin Methylene Blue (EMB) agar:
for the selective isolation of pathogenic Escherichia coli and other
Enterobacteriaceae. MacConkey agar: for the selective isolation of
lactose-positive Enterobacteriaceae and other gram-positive bacteria. Chapman
agar: for the selective isolation of Staphylococci and other gram-positive
bacteria.)
- Biochemical characterization of
isolated bacteria: Biochemical testing was conducted using API E20 test strips
(Biomerieux SA, Marcy l’Etoile,
France). The table 1 summarizes the various tests that were used.
Turtle-Bacterial genera
relationships
To highlight the relationships
between the turtles and bacterial genera, we first performed binary logistic
regressions using the ‘stepwise top-down’ method, aiming to maximize the
explanation of the dependent variable (bacterial genus) with the lowest number
of independent variables (Turtle characteristics). The logistic model belongs
to the family of generalized linear models and links, by a linear combination,
the environmental variables to the variable to be predicted by means of a
logistic link function. It is a statistical tool recommended for binary data
(in our case presence/absence of bacterial genera) (Guisan
& Zimmermann 2000). We used IBM SPSS version 27 software.
Secondly, we performed a
redundancy analysis (RDA) to examine relationships between the bacterial genera
frequency and characteristics of Mediterranean Pond Turtle. RDA can highlight
associations between specific morphological characteristics (such as weight,
shell length) and microbiota composition. It is particularly useful when the
data are multivariate, which is our case (several microbial genera and several
morphological traits) and allows us to better understand the interactions
between the species, its microbiota and the environment.
To test the relationship
significance between mentioned variables, we performed a Monte Carlo
permutation test. For the purposes of RDA, we constructed four groups of
variables by combining age and sex. The groups are respectively adult male,
adult female immature male and the finally immature female. We performed the
RDA analysis in XLSTAT (version 2021.2.2, Addinsoft).
Results
Mediterranean Pond Turtle
population
A total of 24 individuals were
captured, including six females and 18 males. 10 individuals were recaptured at
least once. There were no juveniles.
Descriptive statistics for the
morphological features of turtles are summarised in
the table below. Average length of the carapace is 163 mm. The posterior width
of the carapace varies 90.5–122.6 mm with an average of 111.6 mm. The weight
shows a very high standard deviation (211.7).
While comparing morphological features
between turtle groups the only significant difference (α = 0.03) was observed
in the distance between the cloaca and the carapace (immature males and
immature females). Other comparison showed no significant difference (Table 3).
Bacterial carriage
Bacterial growth was assessed on
74 bacterial cultures. Identification methods resulted in the identification of
70 strains. Four (4) strains could not be identified by the tests used in our
study due to the absence of reaction after subculturing.
After elimination of strains corresponding to contaminants, we obtained a pure
60 bacterial strains. A total of 11 bacterial genera were identified (Table 4,
Image 1). Salmonella was the most frequent isolated genus with a percentage of
21.66 (13/60) of the total isolates, followed by Escherichia and Enterobacter.
(Figure 1). These are in reality the most abundant bacterial genera in the
cloacal flora of the studied population, with prevalences
of 54% and 42%, respectively. The prevalence of Salmonella spp. and Klebsiella spp. is significantly higher in
females than in males. Vibrio, Listeria, Yersinia, and Lactobacillus
were only found in males, while Proteus is present only in females
(Table 4).
Turtle–Bacterial genera
relationships
We obtained a logistic regression
model for six bacterial genera, namely: Lactobacillus, Pseudomonas,
Enterobacter, Vibrio, Salmonella, and Escherichia.
Models were invalid for the other bacterial genera (Table 5).
The assessment of models’ fit was
based on Nalgelkerke’s R2. Like the R2
for multiple regression, the higher the value of this coefficient, the better
the model fit to data. Table 5 shows that models for Pseudomonas and Vibrio
genera give a perfect prediction with a probability of one.
In order to identify variables
that most predict presence probability of bacterial genera, we used the Wald
statistic. For the Enterobacter genus model (Table 5) the ‘Age’ and ‘H’
parameters positively influenced (Exp(B) >1) the
probability of presence of this genus at the threshold α ≤0.05, whereas there
was a negative effect (Exp(B) <1) of the ‘CL’ (α
≤0.05). The Pseudomonas and Vibrio models did not show any
significant variables.
The ‘CL’ variable had a negative
effect (Exp(B) <1; α ≤0.05) on the presence of Salmonella
and Escherichia. However, ‘TL’ had a positive effect (Exp(B) >1; α ≤0.05) on the presence of Escherichia.
Results of RDA indicate a
statistically significant association (p-value <0.01) between morphological
features and bacterial genera frequency. The first two axes of the RDA together
carry 83.36% of the constrained inertia. F1 axis carry 56% of the constrained
inertia and F2 axis carry 27.36% (Figure 2).
MI and FA individuals contributed
significantly to the construction of F1 axis and are well represented on this
axis. Male individuals contributed to the construction of F2 axis and are also
well represented on this axis (Table 6, Figure 2).
The bacterial genera Lactobacillus,
Kleibsiella, Listeria, and Proteus
contributed significantly to the construction of F1 axis and are also well
represented on it. The genera Aeromonas, Enterobacter, Vibrio,
and Yersinia contributed significantly to the construction of F2 axis
(Table 6, Figure 2).
Genera Kleibsiella
and Proteus are positively correlated with female individuals. Genera Vibrio,
Aeromonas, Yersinia, and Lactobacillus are positively
correlated with adult males, while genera Listeria and Escherichia
are more associated with immature males (Figure 2).
Discussion
The duration (March–May 2023) and
the main objective of the study, which required laboratory work to isolate and characterise bacterial carriage, were the main cause for
sampling effort reduction at one site.
It is interesting to note,
however, that sex ratio favour males, supporting that
described in a study carried out on the same population by Bakhouche
et al. (2019).
The Mediterranean Pond Turtle
population of lake Reghaïa exhibits high variability
in individual weight. This may influence microbiota diversity, particularly due
to variation in body spaces, diets, physiological conditions, and social
behaviors. Heavier individuals, often having more resources and space for their
microbes, may support a more diverse microbiota than smaller individuals (Youngblut et al. 2019; Budd et al. 2020).
Bacteriological analysis led to
the identification of 11 bacterial genera (Lactobacillus, Listeria,
Aeromonas, Pseudomonas, Vibrio, Kleibsiella,
Enterobacter, Salmonella, Escherichia, Proteus, Yersinia).
These results correspond to a basic reptilian microbiota (Colston 2017). The
predominance of Pseudomonadota could provide
information on the diet of Mediterranean Pond Turtle population of lake Réghaïa, as they are associated with a predominantly
carnivorous diet.
The growth of two gram+ bacterial
genera (Lactobacillus and Listeria) on Chapman culture media
(Salt Mannitol Agar) was made possible by their high tolerance of the medium’s
salinity. In fact, these genera tolerate concentrations of 12% and 20%,
respectively (Cole et al. 1990; Osek et al. 2022),
far exceeding the concentration of salt (selective agent) present in the
culture media used (7.5%), which enabled these strains to be isolated. The
mobility of the Yersinia strains isolated excludes the possibility that
they belong to the species Yersinia pestis, which is an immobile
bacterium (Jorgensen et al. 2015). This implies that our strains would belong
to the species Yersinia enterolitica or Yersinia
pseudotuberculosis.
The cloacal bacterial carriage
corresponds qualitatively and in proportions to those highlighted in several
previous studies of freshwater turtles, in particular Emys
orbituclaris, which is commonly recognised as syntopic for
Mediterranean Pond Turtle (Hacioglu et al. 2012;
Marin et al. 2013; Nowakiewicz et al. 2015; Ruzauskas et al. 2016).
The high prevalence of Salmonella
and Klebsiella genera, along with the
detection of Listeria, Vibrio, and Yersinia in our study,
combined with the known role of freshwater turtles and reptiles as reservoirs
of various zoonotic bacterial pathogens (Gaertner et al. 2008; Back et al.
2016; Hernandez et al. 2021), underscores the need to implement a surveillance
system at the Reghaïa Lake, which is frequently
visited by the public.
The presence of Pseudomonas
spp. which are bacteria that inhabit soil and water, can also cause disease in
plants, animals as well as immunocompromised humans (Wu et al. 2015). It is
commonly found in the oral cavity and intestinal tract of reptiles and can
cause a number of diseases such as ulcerative stomatitis, pneumonia, dermatitis
and septicaemia (Campa et
al. 1993; Warwick et al. 2013). In the Silene Nature
Park (Latvia), it was the most common bacterium found in all skin areas
(cloaca, mouth, and feet) of Emys
orbicularis individuals (Umbrasko et al. 2020).
Presence of Enterobacter
genus in Mediterranean Pond Turtle was positively influenced by age and
carapace height parameters, which are associated with larger individuals. In Emys orbicularis, this genus was more
abundant in juveniles in the study conducted by Nowakiewicz
et al. (2015).
The presence of Salmonella
in Mediterranean Pond Turtle is negatively influenced by carapace size. It is
therefore associated with small turtles. This poses an even greater risk to
children who acquire them as pets. In fact, children run a high risk of contracting
salmonellosis associated with small turtles, as they can be easily handled and
placed in the mouth (CDC 2008). Two cases of turtle-associated salmonellosis in
children were reported in Japan in 2007 and 2008 (Kuroki et al. 2015).
RDA is a proven multivariate
analysis technique for processing species-environment data (Legendre &
Legendre 1998). It was used to analyse the
determinism of biometric parameters on the presence of bacterial genera. The
first group identified comprises the genera isolated only from adult male
Mediterranean Pond Turtle, which are generally isolated from freshwater (Vibrio
and Aeromonas), plants and soil (Yersinia). Cloacal transmission
during copulation could also be a means of acquiring new microbial species
(Hidalgo-Vila et al. 2007; Nowakiewicz et al. 2015).
The second group includes the
genus Proteus and Klebsiella specific
to adult females in captured Mediterranean Pond Turtle. These bacteria of Proteus
genus are part of pathogenic or normal microflora and can be symbiotic or
change from neutral/commensal to parasitic (Drzewiecka
2016). They can also be interpreted as an indicator of pollution (Al-Bahry et al. 2012). In our case, the individuals captured
were apparently in very good health, which suggests that the presence of the Proteus
genus is due more to environmental pollution, which is well established in the
lake. However, the risk remains high, as studies (Oros
et al. 2005; Awong-Taylor et al. 2008) have reported a low hatching success
rate in Loggerhead Turtles linked to Proteus spp. and mortality in
marine turtles in the Canary Islands.
The diversity of genera isolated
from juveniles is relatively low compared with adults. The same observation has
been made in Emys orbicularis in Poland
(Nowakiewicz et al. 2015). This phenomenon may be
linked to a number of nutritional and/or behavioural
factors.
These findings highlight the
presence of bacterial genera that include species known to be potentially
pathogenic to humans, suggesting the need for further investigation into their
pathogenicity and potential implications for public health. People living near
the Réghaïa wetland, visitors and traders who sell
turtles should be more careful when handling them.
Despite its status as a protected
species under national regulations (Executive Decree No. 12-235 of 24 May 2012
establishing the list of protected non-domestic animal species in Algeria),
Mediterranean Pond Turtle is still one of the illegally traded species
(personal unpublished data). Tighter controls should minimise
the risk of diseases being transmitted by turtles. A ban on small turtles’ sale
(shells less than four inches long) prevented around 100,000 cases of
turtle-associated salmonellosis in children in 1980 which constitutes a good
example of biodiversity health related conservation (CDC 2008).
This data highlights the
importance of setting up an epidemiological and microbiological surveillance system, and strengthens the need to implement environmental
protection programmes. In addition, an
awareness-raising programme needs to be carried out
to raise awareness of significant health risks associated with pet turtle
ownership.
Table 1.
Appropriate biochemical tests for bacterial identification.
|
Test |
Test principle |
Reading |
|
Vogues-Proskauer
(VP) |
Reveals the
capacity of bacteria to produce acetone during glucose fermentation. The test
is carried out on Clark and Lubs broth already
seeded, then incubated at 37 0C for 18 to 24 hours, to which VP1
and VP2 reagents are added respectively, with 10-minute intervals in between
(Varghese & Joy 2014). |
Red/pink Coloration (VP +) Incolore (VP -) |
|
Rouge de méthyle (RM) |
Reveals the ability
of bacteria to oxidize glucose with the production of acid derivatives. This
test is carried out on Clark and Lubs broth, which
has already been seeded and then steamed at 37°C for 18 to 24 hours, to which
methyl red reagent is added (Varghese & Joy 2014). |
Red Coloration (RM +) Yellow Coloration (RM -) |
|
Triple sugar iron
(TSI) |
Highlights the
bacteria's ability to ferment the three sugars present in the medium
(Glucose, sucrose and lactose). Inoculation is performed by a central prick
in the tube and slope inoculation, followed by incubation at 37°C for 24
hours (Varghese & Joy 2014). |
Acidification of the slope (fermentation of sucrose,
lactose or both) Pellet acidification (glucose fermentation) Black precipitates (H2S production and glucose
fermentation) |
|
Citrate |
Highlights the
ability of bacteria to use citrate as their sole carbon source. This test is
performed by plating a slant agar on Simmons citrate tube incubated at 37°C
for 24–48 hours (Varghese & Joy 2014). |
Blue Coloration (Citrate +) Green coloration (Citrate -) |
|
Mannitol motility |
Used to detect
bacterial mobility and mannitol fermentation. It is performed by central
pricking in Mannitol mobility agar incubated at 37 0C for 24–48
hours (Varghese & Joy 2014). |
Yellow Coloration (Mannitol +) Red Coloration (Mannitol -) Growth by creating a disturbance from the central
prickle (Mobility +) Growth all along the central sting (Mobility -) |
|
Urease |
Demonstrates the
ability of bacteria to degrade urea to ammonia using urease. Inoculation of
urea indole medium and incubation at 37 0C for 24 hours (Varghese
& Joy 2014). |
Pink coloration (Urease +) No colour change (Urease
-) |
|
Indole production |
Demonstrates the
ability of bacteria to degrade tryptophan to indole. Test performed on urea
indole medium after seeding and incubation for 24 hours with the addition of
KOVAC's reagent (Varghese & Joy 2014). |
Formation of a red ring on the surface of the medium
(Indole +) No red ring formation (Indole -) |
Table 2.
Descriptive statistics of morphological features of the Mediterranean Pond
Turtle.
|
Parameters (unit) |
Code |
Min. |
Max. |
Mean |
Standard deviation |
|
Carapace length (mm) |
CL |
126.75 |
185.00 |
163.69 |
26..22 |
|
Anterior width of carapace (mm) |
AWC |
79.25 |
109.33 |
97.98 |
13.34 |
|
Posterior width of carapace
(mm) |
PWC |
90.50 |
122.57 |
111.58 |
14.91 |
|
Carapace height (mm) |
H |
41.75 |
58.71 |
52.71 |
7.76 |
|
Tail length (mm) |
TL |
75.25 |
87.67 |
82.07 |
5.14 |
|
Distance carapace–cloaque (mm) |
CC |
32.00 |
39.23 |
36.41 |
3.15 |
|
Weight (g) |
W |
268.50 |
727.46 |
567.65 |
211.69 |
Table 3.
Summary of the Mann-Whitney U test.
|
Male Adult (MA) – Female Adult (FA) |
|||||||
|
|
CL |
AWC |
PWC |
H |
TL |
CC |
W |
|
Mann-Whitney |
25.5 |
14.5 |
21.5 |
22.00 |
20.5 |
27.0 |
22.0 |
|
Wilcoxon |
130.5 |
119.5 |
126.5 |
127.0 |
125.5 |
132.0 |
127.0 |
|
Test statistics |
25.50 |
14.50 |
21.50 |
22.00 |
20.50 |
27.00 |
22.0 |
|
SD |
7.93 |
7.90 |
7.89 |
7.90 |
7.86 |
7.88 |
7.94 |
|
Standardised test statistics |
0.57 |
-0.82 |
0.06 |
0.13 |
-0.06 |
0.76 |
0.13 |
|
Bilateral test |
0.57 |
0.41 |
0.95 |
0.90 |
0.95 |
0.45 |
0.90 |
|
Male Immature (MI) – Female
Immature (FI) |
|||||||
|
|
CL |
AWC |
PWC |
H |
TL |
CC |
W |
|
Mann-Whitney |
7,000 |
6.00 |
7.50 |
5.00 |
9.00 |
12.00 |
6.00 |
|
Wilcoxon |
17,000 |
16.00 |
17.50 |
15.00 |
19.00 |
22.00 |
16.00 |
|
Test statistics |
7,000 |
6.00 |
7.50 |
5.00 |
9.00 |
12.00 |
6.00 |
|
SD |
2,828 |
2.83 |
2.78 |
2.83 |
2.80 |
2.83 |
2.83 |
|
Standardised test statistics |
,354 |
0.00 |
0.54 |
-0.35 |
1.07 |
2.12 |
0.00 |
|
Bilateral test |
,724 |
1.00 |
0.59 |
0.72 |
0.28 |
0.03 |
1.00 |
Table 4.
Bacterial genera identified and their prevalence in the Mediterranean Pond
Turtle.
|
Turtles |
Identified bacterial genus |
||||||||||||
|
ID |
Sex |
Maturity |
Lactobacillus |
Listeria |
Aeromonas |
Pseudomonas |
Vibrio |
Kleibsiella |
Enterobacter |
Salmonella |
Escherichia |
Proteus |
Yersinia |
|
1 |
F |
A |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
1 |
0 |
|
2 |
F |
A |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
1 |
1 |
0 |
|
3 |
F |
A |
0 |
0 |
0 |
0 |
0 |
1 |
1 |
1 |
0 |
0 |
0 |
|
4 |
F |
I |
0 |
0 |
0 |
1 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
|
5 |
F |
I |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
1 |
0 |
0 |
|
6 |
F |
I |
0 |
0 |
0 |
0 |
0 |
1 |
1 |
1 |
0 |
0 |
0 |
|
7 |
M |
A |
1 |
0 |
1 |
0 |
0 |
1 |
1 |
0 |
0 |
0 |
0 |
|
8 |
M |
A |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
|
9 |
M |
A |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
1 |
|
10 |
M |
A |
0 |
1 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
|
11 |
M |
A |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
1 |
1 |
0 |
0 |
|
12 |
M |
A |
0 |
0 |
1 |
1 |
0 |
0 |
1 |
0 |
1 |
0 |
0 |
|
13 |
M |
A |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
14 |
M |
A |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
15 |
M |
A |
0 |
0 |
1 |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
|
16 |
M |
A |
1 |
0 |
0 |
0 |
0 |
0 |
1 |
1 |
0 |
0 |
0 |
|
17 |
M |
A |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
1 |
1 |
0 |
0 |
|
18 |
M |
A |
1 |
0 |
0 |
0 |
0 |
0 |
1 |
1 |
0 |
0 |
0 |
|
19 |
M |
A |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
1 |
0 |
0 |
|
20 |
M |
A |
1 |
0 |
0 |
0 |
1 |
0 |
1 |
1 |
0 |
0 |
0 |
|
21 |
M |
I |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
1 |
0 |
0 |
|
22 |
M |
I |
0 |
1 |
0 |
0 |
0 |
0 |
1 |
0 |
1 |
0 |
0 |
|
23 |
M |
I |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
1 |
0 |
0 |
|
24 |
M |
I |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
|
Prevalence (%) |
25 |
8.33 |
20.83 |
12.5 |
4.17 |
20.83 |
41.67 |
54.17 |
41.67 |
8.33 |
12.5 |
||
Table 5.
Logistic regression models of bacterial genera in the Mediterranean Pond
Turtle.
|
|
B |
E.S |
Wald |
ddl |
Sig. |
Exp(B) |
R2 de Nagelkerke |
|
|
Lactobacillus |
Sex |
-20.51 |
16408.71 |
0.00 |
1 |
0.999 |
0.000 |
0.231 |
|
Constant |
-0.69 |
0.50 |
1.92 |
1 |
0.166 |
0.500 |
||
|
Pseudomonas |
Age |
4225.05 |
87867.62 |
0.00 |
1 |
0.962 |
|
1.000 |
|
CL |
-112.58 |
2356.29 |
0.00 |
1 |
0.962 |
0.000 |
||
|
AWC |
161.06 |
3308.13 |
0.00 |
1 |
0.961 |
8.87E+69 |
||
|
PWC |
-130.33 |
2688.50 |
0.00 |
1 |
0.961 |
0.000 |
||
|
H |
84.24 |
1814.76 |
0.00 |
1 |
0.963 |
3.86E+36 |
||
|
TL |
-100.06 |
2066.50 |
0.00 |
1 |
0.961 |
0.000 |
||
|
CC |
135.82 |
2822.41 |
0.00 |
1 |
0.962 |
9.70E+58 |
||
|
Constant |
12401.99 |
257536.43 |
0.00 |
1 |
0.962 |
|
||
|
Enterobacter |
Sex |
-2.25 |
1.48 |
2.32 |
1 |
0.128 |
0.105 |
0.392 |
|
Age |
7.58 |
3.67 |
4.27 |
1 |
0.039 |
1956.076 |
||
|
CL |
-0.29 |
0.13 |
5.07 |
1 |
0.024 |
0.748 |
||
|
H |
0.66 |
0.32 |
4.29 |
1 |
0.038 |
1.939 |
||
|
Constant |
7.76 |
5.95 |
1.70 |
1 |
0.192 |
2343.684 |
||
|
Vibrio |
Age |
227.97 |
42188.59 |
0.00 |
1 |
0.996 |
1.01E+99 |
1.000 |
|
CL |
-4.96 |
1330.69 |
0.00 |
1 |
0.997 |
0.007 |
||
|
PWC |
-2.13 |
1513.71 |
0.00 |
1 |
0.999 |
0.118 |
||
|
H |
10.85 |
2657.93 |
0.00 |
1 |
0.997 |
51704.704 |
||
|
Constant |
269.77 |
94378.88 |
0.00 |
1 |
0.998 |
1.44E+117 |
||
|
Salmonella |
CL |
-0.20 |
0.10 |
3.97 |
1 |
0.046 |
0.820 |
0.348 |
|
W |
0.02 |
0.01 |
3.59 |
1 |
0.058 |
1.025 |
||
|
Constant |
18.98 |
9.29 |
4.18 |
1 |
0.041 |
175005394.480 |
||
|
Escherichia |
Sex |
-2.99 |
1.66 |
3.27 |
1 |
0.071 |
0.050 |
0.495 |
|
CL |
-0.30 |
0.15 |
3.88 |
1 |
0.049 |
0.740 |
||
|
PWC |
0.46 |
0.25 |
3.51 |
1 |
0.061 |
1.585 |
||
|
TL |
0.37 |
0.18 |
4.41 |
1 |
0.036 |
1.446 |
||
|
CC |
-0.35 |
0.19 |
3.42 |
1 |
0.064 |
0.703 |
||
|
Constant |
-19.51 |
10.40 |
3.52 |
1 |
0.061 |
0.000 |
||
Table 6.
Results of the redundancy analysis (RDA).
|
Observations |
||||||
|
|
Scores |
Contributions |
Square cosines |
|||
|
|
F1 |
F2 |
F1 |
F2 |
F1 |
F2 |
|
Obs1 (MA) |
-0,446 |
0,759 |
0,168 |
0,488 |
0,224 |
0,650 |
|
Obs2 (MI) |
-0,601 |
-0,724 |
0,305 |
0,443 |
0,407 |
0,591 |
|
Obs3 (FA) |
0,717 |
-0,219 |
0,435 |
0,041 |
0,580 |
0,054 |
|
Obs4 (FI) |
0,330 |
0,183 |
0,092 |
0,028 |
0,122 |
0,038 |
|
Response variables |
||||||
|
|
Scores |
Contributions |
Square cosines |
|||
|
|
F1 |
F2 |
F1 |
F2 |
F1 |
F2 |
|
Lactobacillus |
-0,262 |
0,076 |
0,103 |
0,018 |
0,827 |
0,070 |
|
Listeria |
-0,154 |
-0,107 |
0,036 |
0,036 |
0,671 |
0,325 |
|
Aeromonas |
-0,135 |
0,229 |
0,027 |
0,163 |
0,224 |
0,650 |
|
Pseudomonas |
0,039 |
0,143 |
0,002 |
0,064 |
0,024 |
0,325 |
|
Vibrio |
-0,027 |
0,046 |
0,001 |
0,007 |
0,224 |
0,650 |
|
Kleibsiella |
0,563 |
0,026 |
0,479 |
0,002 |
0,936 |
0,002 |
|
Enterobacter |
0,107 |
0,311 |
0,017 |
0,299 |
0,102 |
0,868 |
|
Salmonella |
0,047 |
-0,204 |
0,003 |
0,129 |
0,046 |
0,853 |
|
Escherichia |
-0,221 |
-0,240 |
0,074 |
0,178 |
0,458 |
0,541 |
|
Proteus |
0,404 |
-0,123 |
0,247 |
0,047 |
0,580 |
0,054 |
|
Yersinia |
-0,081 |
0,138 |
0,010 |
0,059 |
0,224 |
0,650 |
For
figures & images – click here for full PDF
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