Bacterial diversity of loggerhead and green turtle eggs from two major nesting beaches from the Turkish coast of the Mediterranean

Abstract

This study was conducted during the 2018 nesting season at the Sugözü Beaches (Adana-Turkey) and Göksu Delta (MersinTurkey). Egg samples (n = 63) from loggerhead and green turtle nests (n = 43) were collected. Isolated bacteria were initially identified by phenotypic methods and then by MALDI-TOF MS. The bacterial mass spectra were analyzed using Principal Component Analysis. Bacterial isolation was performed for 55 isolates belonging to 12 genera from two major nesting sites. In Sugözü Beaches 62.2% of the bacteria species belonged to Enterobacteriaceae and in Göksu Delta 44.4% of the bacteria species belonged to Morganellaceae. Klebsiella oxytoca and Staphylococcus haemolyticus had not previously been detected in any sea turtle nests. This is the first MALDI-TOF MS study conducted for determination of bacterial variability in loggerhead turtle eggs in Turkey and serves as a reference study for the assessment of bacterial threat in sea turtle nests, enabling the establishment of suitable conservation measures and treatment processes for both sea turtles and nesting sites.

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Archives of Microbiology (2022) 204:682
https://doi.org/10.1007/s00203-022-03292-z
ORIGINAL PAPER
Bacterial diversity ofloggerhead andgreen turtle eggs fromtwo
major nesting beaches fromtheTurkish coast oftheMediterranean
EsraDenizCandan1 · OnurCandan2 · YaseminNumanoğluÇevik3
Received: 9 March 2022 / Accepted: 18 October 2022
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022
Abstract
This study was conducted during the 2018 nesting season at the Sugözü Beaches (Adana-Turkey) and Göksu Delta (Mersin-
Turkey). Egg samples (n = 63) from loggerhead and green turtle nests (n = 43) were collected. Isolated bacteria were initially
identified by phenotypic methods and then by MALDI-TOF MS. The bacterial mass spectra were analyzed using Principal
Component Analysis. Bacterial isolation was performed for 55 isolates belonging to 12 genera from two major nesting
sites. In Sugözü Beaches 62.2% of the bacteria species belonged to Enterobacteriaceae and in Göksu Delta 44.4% of the
bacteria species belonged to Morganellaceae. Klebsiella oxytoca and Staphylococcus haemolyticus had not previously been
detected in any sea turtle nests. This is the first MALDI-TOF MS study conducted for determination of bacterial variability
in loggerhead turtle eggs in Turkey and serves as a reference study for the assessment of bacterial threat in sea turtle nests,
enabling the establishment of suitable conservation measures and treatment processes for both sea turtles and nesting sites.
Keywords Bacteria· Beaches· Caretta caretta· Chelonia mydas· Göksu Delta· MALDI-TOF MS· Microbial
contamination· Sugözü
Introduction
Mediterranean coasts are under severe anthropological pres-
sure due to the high population density, touristic, industrial,
and agricultural activities. However, the beaches on these
coasts are rich in terms of biodiversity (Noroozi etal. 2019).
Turkey's Mediterranean beaches contain the most important
nesting sites of sea turtles, which are among the umbrella
species in conservation biology (Casale etal. 2018). Today,
seven sea turtle species live in the world’s seas. In the Red
List published by the International Union for Conservation
of Nature (IUCN), one species is in the data deficiency (DD)
category, while the remaining six species are in different
threatened categories ranging from “Critically Endangered
(CR) to Vulnerable (VU)” on a global scale. Turkey’s Medi-
terranean beaches include 21 officially designated nesting
sites, which are used by Caretta caretta (Loggerhead Turtle)
and Chelonia mydas (Green Turtle). Among these sites, the
Sugözü Beaches are an important nesting site for the green
turtle (Candan and Candan 2020) and Göksu Delta is an
important nesting area for the loggerhead turtle (Candan
2018).
Marine ecosystems are among the most economically
and ecologically valuable systems in the world (Barbier
2017). However, many of these ecosystems are naturally
and anthropogenically threatened (Boonstra etal. 2015). To
manage and mitigate these threats, it is necessary to inves-
tigate factors influencing the ecosystem. The most common
of these studies is monitoring of indicator species that are
thought to reflect the health of the ecosystem (Aguirre and
Tabor 2004). Sea turtles are regarded as bioindicator spe-
cies due to their ecological and physiological characteristics,
such as their long lifespan, site fidelity and delayed sexual
maturity period (Owens etal. 2005; Foti etal. 2009). In
addition, sea turtles are highly sensitive to biological and
Communicated by Erko Stackebrandt.
* Esra Deniz Candan
esradenizcandan@gmail.com
1 Department ofMedical Services andTechniques,
Giresun University Vocational School ofHealth Services,
28100Giresun, Türkiye
2 Faculty ofArts andSciences Department ofMolecular
Biology andGenetics, Ordu University, 52200Ordu, Türkiye
3 Microbiology andReference Laboratory andBiological
Products Department, General Directorate ofPublic Health,
Ministery ofHealth, 06430Ankara, Türkiye

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chemical changes in the environment, and are characterized
by the bioaccumulation of pollutants, toxins, and pathogens
(Al-Rawahy etal. 2007; Al-Bahry etal. 2009, 2012; Foti
etal. 2009). In this context, especially in studies conducted
to determine bacterial flora, various antibiotic-resistant and
environmental pollution-related bacterial species have been
identified from oral, cloaca, sand, and egg samples from
sea turtle nests (Al-Bahry etal. 2009, 2011; Foti etal. 2009;
Pace etal. 2019; Candan and Candan 2020). In addition, the
bacteria defined within the scope of these studies are seri-
ous infectious agents that cause embryonic deaths (Wyneken
etal. 1988; Craven etal. 2007; Al-Bahry etal. 2011; Honar-
var etal. 2011). The bacterial flora of sea turtles, which
are in contact with both terrestrial and marine ecosystems,
can affect the spread and severity of diseases in other liv-
ing organisms in the ecosystem or in humans (Daszak etal.
2001). For this reason, it is important to identify the bacte-
rial flora associated with sea turtles in studies conducted to
protect ecosystem health.
Phenotypic tests based on the combination of biochemical
characteristics, such as Gram stain, carbohydrate metabo-
lism and the presence of specific enzymes, etc., and genetic
tests including PCR-based techniques are generally used in
the identification of microorganisms (Fournier etal. 2013).
Although the cost of morphological and phenotypic tests
are quite low, these tests take time. The tests used in genetic
identification are quite expensive and the interpretation of
the results of these tests requires a high level of expertise. As
a consequence, these tests are not suitable for routine identi-
fication. Therefore, researchers are in search of alternatives
for fast, low-cost, and reliable methods (Santos etal. 2016).
Matrix-assisted laser desorption ionization time-of-flight
mass spectrometry (MALDI-TOF MS) analysis presents a
microorganism-specific spectral profile. This spectral profile
is defined by comparing it with the reference spectra in the
database. These protein mass spectra can be used to iden-
tify bacteria at the genus, species and even subspecies level
(Fournier etal. 2013; Schumann and Maier 2014). Although
MALDI-TOF MS has a high installation cost, bacteria can
be identified in few minutes and consumable costs with this
method compared to phenotyping methods (Fournier etal.
2013; Santos etal. 2016). MALDI-TOF MS can yield results
similar to both biochemical methods and identification meth-
ods, such as 16S rRNA gene sequencing (Rahi and Vaisham-
payan 2020). This method, which is widely used in the clinic
for the identification of bacteria, has been reliably applied in
the identification of environmental bacteria in recent years
(Santos etal. 2016; Ashfaq etal. 2019; Bermanec etal.
2021; Caliskan etal. 2021; Çevik and Ogutcu 2020).
In this study, bacterial flora of infected eggs from nests
obtained from Sugözü Beaches and the Göksu Delta, which
are important sea turtle nesting sites, were identified with
MALDI-TOF MS. At the same time, bacterial differences
among nesting sites and sea turtle species were revealed.
This study is unique in terms of application of method used
for identification of bacterial flora in sea turtle nests.
Materials andmethods
Study site
This study was conducted at sea turtle nesting sites of the
Sugözü Beaches (Adana-Turkey) (36°48.677' N-35°51.068'
E, 36°52.795' N-35° 56.017' E) and Göksu Delta (Mer-
sin-Turkey) (36°18.592' N, 33°54.258' E, 36°24.045' N,
34°04.702' E) in the 2018 nesting season (Fig. S1). The
total coastal length of the Sugözü Beaches is 3.8km, and it
is an important nesting area for green turtles (Candan and
Candan 2020; Candan etal. 2021b). This area, located in the
north of the Iskenderun Bay, is heavily exposed to indus-
trial, domestic, and agricultural pollution (Gündoğdu and
Cevik 2019; Duysak and Uğurlu 2020). In contrast, Göksu
Delta was declared a special protected area 30 years ago by
the decision of the Council of Ministers of the Republic of
Turkey (18.01.1990 dated and 90/77 numbered decree), and
there is no industrial activity in the region. Göksu Delta is
the westernmost nesting site of the green turtle on the east-
ern Mediterranean coast of Turkey and is also an important
nesting area for the loggerhead turtle, with a total coastline
of approximately 35km (Candan 2018).
Sample collection
Both nesting sites were monitored daily (between May and
September). The nests were marked and protected against
predation with cages until the hatchlings emerged. After the
incubation period, the nests were uncovered to determine
the condition of the eggs and embryos. Random samples
were taken from failed eggs during uncovering the nests for
control. Failed eggs were checked externally to detect the
presence of embryos. In the study, egg samples containing
dead embryos and exhibiting color changes were collected
from both loggerhead and green turtle nests (Fig.1). After
the surface of the egg samples was wiped with 70% etha-
nol, 100 μL of egg content was taken using sterile syringe
and added to 1mL of sterile Luria–Bertani Broth (HiMedia,
Miller, India) medium. All samples were transferred to the
laboratory in sterile packages in an icebox.
Isolation ofbacterial strains andphenotypic
identification
Bacterial samples were inoculated into Nutrient Agar
(Merck1.05450) medium. Bacteria were phenotypically
identified according to Candan and Candan (2020) using

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selective and differential media after inoculation. Iso-
lated bacterial strains were stored in Trypticase Soy Broth
containing 15% glycerol at -80ºC until MALDI-TOF MS
analysis.
Analysis ofall bacterial strains using theMALDI‑TOF
MS method
A total of 55 isolates and three external standard strains
(Bacillus cereus ATCC 10876, E. coli ATCC 25922 and
Staphylococcus aureus ATCC 29213) were analyzed by
MALDI-TOF MS simultaneously.
A MALDI-TOF MS device (Bruker Microflex LT, Ger-
many) and Flex Control 3.0 software were used for bacte-
rial strain identification. In addition, HPLC grade chemi-
cal substances; HCCA (α-cyano-4-hydroxycinnamic acid,
Bruker), ACN (acetonitrile, Sigma-Aldrich), TFA (trif-
luoroacetic acid, Sigma-Aldrich), ultra-pure water with a
0.1µm filter without DNAse and RNAse (Sigma-Aldrich)
and a Bruker bacterial test solution (BTS) containing E. coli,
RNAase and myoglobin protein profiles were also employed.
For microbial biomass analysis, the culture from a single
colony sampled with a sterile toothpick tip was applied to a
special steel 96 micro scout plate (MSP). This culture was
spread onto the wells in the plate in the form of a thin film.
After drying, 1.0µl of HCCA matrix solution (12.5mg/
ml HCCA in a mixture of 50% ACN and 2.5% TFA) was
added and allowed to dry completely at room temperature
(Bruker Daltonics; the direct transfer method). Before start-
ing the analysis, the internal quality control of MALDI-TOF
MS spectra in general bacteriology was performed using a
Bruker BTS, consisting of an extract of E. coli proteins for
mass calibration of the instrument. Then, the MALDI-TOF
MS 96 MSP was loaded onto the MALDI-TOF MS device.
The system was operated in linear positive ion mode and the
method optimized for the identification of microorganisms in
the mass range of 2000–20,000Da. A 60Hz nitrogen laser
at 337nm was used as an ion source. To obtain the spectra,
laser pulses consisting of 40 packets of 240 were performed
in the measurement of each sample. Three studies were per-
formed for each sample and the highest score was taken into
consideration by repeated reading.
Quality checks ofMALDI‑TOF MS spectra
For internal quality checks of MALDI-TOF MS analysis, all
targets were calibrated using the Bruker Bacterial Test Solu-
tion (Bruker BTS) (BrukerDaltonics Inc., Germany), consist-
ing of Escherichia coli DH5 alpha. Mass spectra calibration
in this study was successfully completed with seven peaks
(m/z; 5096.38131Da, 5381.26948Da, 6255.88537Da,
7274.97890Da, 10,299.98287Da, 13,682.31800Da, and
16,952.85611Da) assigned with a standard deviation of
59.52ppm and maximum peak error of 72.19ppm. To con-
firm that target cleaning was effective and no residual bacte-
rial material from a previous run remained, one spot on each
target contained only the matrix with no bacterial sample.
Identification criteria ofbacterial strains using
theMALDI‑TOF MS method
The identified bacteria results were reported as numeric
scores based on similarity with the reference spectra based
on a proprietary algorithm of MALDI-TOF Biotyper soft-
ware (version 3.1) comparing the presence and symmetry
of peaks in the mass spectra of the unknown strain and the
database strain entries. The log scale from 0.000 to 3.000
defines the identification matching level with the database.
Identification scores were interpreted following the manu-
facturer's recommendations as follows: scores between 2.300
and 3.000 are designated as “highly probable species iden-
tification”, scores between 2.000 and 2.299 as “probable
species identification”, scores of 1.700–1.999 as “probable
genus identification”, and scores below 1.699 are reported
Fig. 1 Deterioration of failed turtle eggs

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as “non-reliable genus identification”. The closest matches
were listed in order of these values, and the highest one indi-
cated the highest similarity in the mass spectra (Cameron
etal. 2017; Sauget etal. 2017; Horvath etal. 2020). MALDI-
TOF MS identification elicits the characteristic mass and
peak density distribution of ribosomal 16S proteins in the
sample. Since this mass spectrum is species-specific for
many microorganisms, it also represents a “molecular fin-
gerprint” (Cheng etal. 2016; Sauget etal. 2017; Shah etal.
2021).
MALDI‑TOF MS mass spectrum profile ofall isolates
andprincipal component analysis
All the identified bacteria mass spectrums were analyzed
using the Principal Component Analysis (PCA) method sup-
ported by external MATLAB software integrated into the
MALDI-TOF Biotyper software (version 3.1). The phylo-
proteomic PCA was performed to decrease the dimension-
ality of the data set and maintain the original information.
The PCA was based on the peaks acquired from MALDI-
TOF MS to find the patterns and unique peaks of individual
strains. This allows the formation of clustered groups of
spectra having similar variation characteristics and the visu-
alization of the differences between them. The data were rep-
resented in a 3D coordinate system. Optimized preliminary
procedures were applied for each spectrum to accelerate the
analysis and reduce the data mass dimension. Cluster analy-
sis was undertaken by performing PCA dendrograms which
represent the relationship and proximity of each spectrum
to one another (Cheng etal. 2016; Taban and Numanoglu
Cevik 2021).
All the analyses (PCA and dendrograms) were carried
out as per the standard operating procedure for the instru-
ment and built-in software. The raw mass spectra were pre-
processed by MALDI-TOF Biotyper software (version 3.1)
before further analysis based on the Biotyper pre-process-
ing standard method (smoothing method: Savitski-Golay;
baseline subtraction method: multi-polygon; normaliza-
tion method) (Jeong etal. 2013). Any individual spectrum
with poor quality (having background noise or too high/low
intensities) was excluded. MSP dendrograms were generated
using the Biotyper MSP dendrogram creation standard meth-
ods. A PCA-based approach was used to increase the share
of peaks, separating, and reducing the weighting of the com-
mon peak in the data sets from closely related organisms.
Multivariate statistical methods are generally used in
PCA analysis of mass spectra produced from MALDI-TOF
MS (Samad etal. 2020). The composite correlation index
(CCI) was used to statistically determine the proximity rela-
tionship between the strains from the obtained spectra. In
addition, the virtual gel view represented all the peaks found
in a spectral file and was used to identify common peaks
between different spectra of all strains used in this study.
Results
In the 2018 nesting season, 92 sea turtle nests were detected
in Sugözü Beaches and 86 sea turtle nests were detected in
Göksu Delta. A total of 63 eggs (43 green turtle eggs, and
20 loggerhead turtle eggs) which contains dead embryos
and exhibit color change were collected from 43 (24% of all
nests) nests (24 green turtle nest, 19 loggerhead turtle nest)
on both sites within the scope of the study. A total of 40
egg samples from 21 green turtle nests (23% of all nests) on
Sugözü Beaches and a total of 23 egg samples from 22 (19
loggerhead turtle nest, 3 green turtle nest) sea turtle nests
(26% of all nests) from Göksu Delta were included in the
study. Bacterial isolation was performed on 37 of 40 egg
samples collected from Sugözü Beaches and 20 of 23 egg
samples collected from Göksu Delta.
The total number of eggs, the condition of the eggs and
the mortality rates according to embryonic stages of 43
nests included in the study are presented in Table1. The
hatching success rate at the Sugözü Beaches was found to
be significantly higher than the hatching success rate in the
Göksu Delta. In both sites, it was found that embryonic
deaths occurred frequently at the early stage embryonic
development.
In total 55 isolates from two nesting sites (Sugözü
Beaches; SG and Göksu Delta; GD) were identified (two iso-
lates were not identified by the MALDI-TOF MS), then the
results were matched to the Main Spectrum Profile (MSP)
reference database library (MALDI-TOF Biotyper database)
(TableS1). Cluster localizations in dendrograms and PCA
scatter profiles of standard strains of B. cereus ATCC 10,876
(30-BC), E. coli ATCC 2592 (31 EC) and 40-SA S. aureus
ATCC 29,213 (40-SA), and similar bacteria which were
identified to the genus and species level were verified. In
addition, CCI values were calculated, and it was revealed
that the proximity and distance values between species were
compatible with their spectral similarities.
Thirty-seven bacterial species belonging to 7 genera were
identified by MALDI-TOF MS from isolates taken from egg
samples in Sugözü Beaches. These were, respectively, Cit-
robacter spp. (37.8%), B. cereus (18.9%), E. coli (18.9%),
Lysinibacillus spp., (16.2%), E. cloacae (2.7%), K. oxytoca
(2.7%) and S. haemolyticus (2.7%) (Table1).
A dendrogram profile was created by PCA analysis of
the bacteria identified by MALDI-TOF MS. The placement
of these seven genera and their subspecies in PCA clusters
was compatible with each other. Bacteria belonging to the
Enterobacteriaceae family (Citrobacter spp., E. coli and
Enterobacter sp.) were located close to each other in the

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dendrogram, branching from the same root as would be
expected. (Fig.2A).
At an arbitrary distance level of 10 (maximum dissimilar-
ity), MSP dendrogram clustered the four main groups: the
first group is the green line cluster, the second one is red,
the third is light blue and the fourth is purple, containing
Citrobacter spp., E. coli, B. cereus and Lysinibacillus spp.,
respectively. The samples of the number 30-BC (B. cereus
ATCC 10,876), 31-EC (E. coli ATCC 2592) and 40-SA (S.
aureus ATCC 29,213) are the external standard strains. They
were used for guidance on the location of their presentative
genus in clusters. (Fig.2A).
As a result of variance analysis, it was observed that
bacteria outside the Enterobacteriaceae family (B. cereus,
Lysinibacillus spp., S. haemolyticus) differed by 20%, while
bacteria belonging to the Enterobacteriaceae family differed
by less than 5% (Fig.2B). A 3D scatter profile was created
from the spectra of all bacteria and three standard strains
detected on Sugözü Beach (SG). Each point in this profile
represents the spectrum of each species. When Fig.3C
is examined in detail, it is apparent that seven genera are
clearly clustered separate from each other, except that S.
haemolyticus is very close to the E. coli cluster (Fig.2C).
A total of 14 strains, mostly belonging to the Citrobac-
ter genus, were identified in these nesting sites. When the
Composite Correlation Index (CCI) values of the Citrobac-
ter spp. members (SG-2,4, 6,13,16,18,19,20,21,26,27,32,3
3) were calculated according to the first member of the first
cluster (SG-15), it was determined that the CCI values are
over 60% (from 60% to 92%). Common biomarker proteins
in the mass spectra of randomly selected C. braakii (SG-2)
and C. freundii (SG-21) and one identified C. amalonaticus
(SG-33) are detailed in Fig. S2.
The spectra of these three randomly selected Citrobac-
teriae were compared to each other (Fig. S2). Macroscopic
examination revealed both common and differential peaks
in the spectra of different strains. The protein peaks (m/z;
4184; 4761; 5411; 6257; 7263; 8266; 9523Da) appeared to
be consistent with the spectra of three Citrobacter strains
(Fig. S2). It is clearly seen in the gel image of all Citro-
bacter strains (n = 14) that the projections of these protein
peaks are compatible with the distribution of protein peaks
of fourteen isolates. In addition, the placement of the species
in the dendrogram profile in clusters is compatible (Fig.2A),
and according to CCI calculations, it was determined that
C. amalonaticus showed an average of 70% similarity to C.
braakii, while less (45%) similarity to C. freundii identified
as Citrobacter genus by MALDI-TOF MS (Fig. S2).
Two strains of bacteria (SG-34-K. oxytoca and SG-22-S.
haemolyticus) were isolated for the first time in this nesting
site, which had not been detected in any turtle egg before.
The MALDI-TOF MS spectra of the standard strain (40-SA)
and SG-34 and SG-22 (Fig. S3) are given, respectively.
Table 1 Descriptive values for various nest parameters
EED Early stage embryonic death, MED Middle-stage embryonic death, LED Late-stage embryonic death
Site
Sugözü Beaches Göksu Delta Total
Hatched egg number (n) 1891 1351 3242
Unhatched egg number
(n)
300 537 837
Total egg number (n) 2191 1888 4079
Mean ± SE Min–Max Mean ± SE Min–Max Mean ± SE Min–Max
Clutch size 104.3 ± 2.621 76–126 85.8 ± 4.063 60–124 94.9 ± 2.80 60–126
Hatching success (%) 0.863 ± 0.015 0.74–0.98 0.726 ± 0.044 0.12–0.95 0.795 ± 0.026 0.12–0.98
EED (%) 0.118 ± 0.014 0.019–0.253 0.155 ± 0.027 0.023–0.598 0.137 ± 0.016 0.019–0.598
MED (%) 0.001 ± 0.001 0–0.010 0.039 ± 0.030 0–0.677 0.021 ± 0.016 0–0.677
LED (%) 0.015 ± 0.015 0–0.081 0.079 ± 0.022 0–0.405 0.048 ± 0.012 0–0.405

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Fig. 2 MALDI-TOF MS Biotyper PCA analysis of bacterial strains isolated from Sugözü Beaches (SG) and three standard strains (30-BC: B. cereus ATCC 10,876; 31-EC: E. coli ATCC
25,922; 40-SA: S. aureus ATCC 29,213). A The dendrogram profile and B the explanation of variance of all strains with three standard strains. C 3D Scatter profile of all strains with three
standard strains. Each spot represents one spectrum, and the plots are generated by three PCAs

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Six different genera composed of Providencia rettgeri
(27.8%), Alcaligenes faecalis, (22.2%), Bacillus cereus
(22.2), Proteus spp. (16.7%), Micrococcus luteus (5.6%),
and Paenibacillus glucanolyticus (5.6%) have been iden-
tified in egg samples from the Göksu Delta (Table1)
(Fig.3). MALDI-TOF MS (Biotyper) PCA analyzes of
all identified isolates were performed and the dendrogram
profiles, 3D scattering, and variance distributions are
presented in Fig.3. In general, the distribution of bacte-
ria into clusters according to their species is compatible
with each other in both the dendrogram profile and the 3D
scatterplot.
In these nesting sites, mass spectra and gel images of P.
rettgeri strains from the Morganellaceae family identified in
five isolates are given in Fig. S4. The noteworthy point is that
the protein peaks (m/z; 4761, 8369, 9522) found in the highest
abundance in Morganellaceae family members are also pre-
sent in P. rettgeri strains belonging to the same family.
Bacteria identified as P. rettgeri and Proteus spp. (except
GD-2) which were members of the Morganellaceae family,
yielded high score values (2.274–2.563). The cluster locali-
zation of these bacteria which were identified at the species
level in the dendrogram profiles and scatterplots, are highly
concordant. When the spectra of the five identified P. rettgeri's
are evaluated, it is seen that the projections of the biomarker
proteins (m/z; 4761, 8369, 9522) on the gel profiles match
without interruption.
Fig. 3 MALDI-TOF MS (Biotyper) PCA analysis of all the bacterial
strains isolated from Göksu Delta (GD). A The dendrogram profile
and B the list of identified microorganisms matched with dendrogram
numbers. C 3D Scatter profile. Each point represents one spectrum,
and the plots were generated by three PCAs and D the explanation of
variance of all strains

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Discussion
This study, in which the bacterial flora in sea turtle nests
was determined for the first time using the MALDI-TOF
MS method, presents new approaches for the application of
this method. A total of 55 bacteria belonging to 12 genera
were isolated from egg samples taken from nesting sites,
and these strains were identified using the Bruker MALDI-
TOF MS method. The environmental bacterial strains which
were isolated in this study were mostly defined by quite high
values (2.000–2.563). While 31 of 37 bacteria from Sugözü
Beaches (SG) were identified to the species level, and 5 of
37 bacteria identified at the genus level, only one bacterium
remained under a reliable identification threshold value with
a score of 1.427 (SG-14). This isolate was placed in the clus-
ter of genera belonging to Lysinibacillus according to PCA
analysis. With CCI calculation, the similarity of this strain to
other strains belongs to the Lysinibacillus became definitive.
Enterobacteriaceae is dominant in the Sugözü Beaches
and some species belong to this family such as Citrobacter
spp., E. cloacae, E.coli, and K. oxytoca are defined as a
potential secondary invader in infections observed in sea
animals, as well as a disease agent of serious diseases in
immune suppressed patients in the World (Alhashem etal.
2017; Trotta etal. 2021). Bacteria belonging to the Provi-
dencia and Proteus genera of the Morganellaceae family
were found to be dominant in the Göksu Delta and are
among Gram negative opportunistic pathogens which can
be isolated from various mediums and organisms includ-
ing humans and animals (Pathirana etal. 2018; Yuan etal.
2020).
A Bacillus cereus strain was also detected at both of the
study sites. These strains constitute 20% of the isolated
bacteria found in this study. The genus Bacillus is preva-
lent in the environment (Radhakrishnan etal. 2017), and
is among the strains which were frequently observed in
egg, cloaca and soil samples collected from nesting sites.
Bacillus spp. is dominant in cloacal samples (72.1%) of
green turtle (Al-Bahry etal. 2011) and in sand samples
(46.5%) of green turtle nests (Candan and Candan 2020).
Bacillus spp. was also found in 71.1% and 96.3% of the
nasal cavity samples of olive ridley turtles (Lepidochelys
olivacea) and leatherback turtles (Dermochelys coriacea),
respectively (Santoro etal. 2006b, 2008).
Other strains isolated from both of the nesting sites were
found to be highly diverse. Citrobacter (37.8%) in the Sug-
özü Beaches and Providencia (27.8%) in the Göksu Delta
constituted the dominant genera of the respective nesting
sites. Variability of diversity and intensity of the strains
could be a result of different turtle species and nesting sites
as stated previously (Al-Bahry etal. 2011; Honarvar etal.
2011; Candan and Candan; 2020; Candan etal. 2021a).
In our study, a total of seven bacteria species were
identified from green turtle nests of the Sugözü Beaches
with MALDI-TOF MS. Among the species identified by
MALDI-TOF MS, Citrobacter spp. (37.8%), B. cereus
(18.9%), and E. coli (18.9%) were most common strains.
In studies conducted on oral, cloacal and egg samples of
loggerhead and green turtles, Citrobacter spp. were fre-
quently isolated (Foti etal. 2009; Al-Bahry etal. 2012;
Goldberg etal. 2019; Candan and Candan; 2020).
Klebsiella oxytoca and S. haemolyticus identified in
samples from the Sugözü Beaches were isolated both from
the nesting site and sea turtle eggs for the first time. Staph-
ylococcus spp. from nasal and cloacal samples of green
turtle were reported by various researchers (Santoro etal.
2006b; Al-Bahry etal. 2011). Besides, S. lentus was found
to be potentially responsible for the symptoms of ulcera-
tive stomatitis in the green turtle (Vega-Manriquez etal.
2018). It is also reported that S. haemolyticus is an oppor-
tunistic pathogen that causes septicemia, peritonitis, otitis,
urinary tract infections and eye infections (Cristianawati
etal. 2017). Klebsiella sp. is among the Gram-negative
opportunistic bacteria associated with various diseases
in both humans and animals (Yang etal. 2019). Among
all Klebsiella species, besides K. pneumoniae, K. oxytoca
is also among the clinically important species (Paterson
etal. 2014). Strains belonging to the Klebsiella genus,
especially K. pneumoniae, are frequently isolated from
sea turtles. The first and only report of the isolation of K.
oxytoca was obtained from nasal and cloacal specimens of
the green turtle nesting at the Tortuguero National Park,
Costa Rica (Santoro etal. 2006a).
The related genera Providencia and Proteus were identi-
fied using MALDI-TOF MS from turtle nests in the Göksu
Delta. Proteus mirabilis and Proteus vulgaris strains, which
cause clinically important diseases (Chart 2012) have been
isolated from nasal, cloacal, and buccal cavity samples of
loggerhead and green turtle (Santoro etal. 2006a; Foti etal.
2007, 2009; Al-Bahry etal. 2011; Pace etal. 2019). Similar
to the findings of our study, Providencia rettgeri was identi-
fied in loggerhead cloacal and buccal cavity samples (Foti
etal. 2007 and Foti etal. 2009) and Proteus vulgaris was
identified in loggerhead egg samples (Awong‐Taylor etal.
2008).
Bacteria have a very important role in sea turtle diseases
as primary pathogens as well as secondary invaders when
the host’s immune system is compromised (Alfaro etal.
2008). According to the results obtained from our study and
the literature, various bacterial species with pathogenic and
non-pathogenic characteristics are found in sea turtle nest-
ing sites. The relation of sea turtles with bacteria is critical,
since the habitats of sea turtles along their migration routes
are possibly affected by these bacteria. Moreover, such infec-
tious agents are a potential danger to humans and animals.

Archives of Microbiology (2022) 204:682
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Page 9 of 11 682
Studies conducted on bacterial (Al Bahry etal. 2009,
Hanovar etal. 2011; Candan and Candan 2020) and fungal
flora (Sarmiento-Ramirez etal. 2017; Candan 2018; Candan
etal. 2021a) in sea turtle nest and egg contents have been
increasing in recent years. In these studies, the species rich-
ness of the microbial flora, the relative abundance of species,
the relationship of the flora with various nest parameters and
its effect on hatching success were examined (Awong-Taylor
etal. 2008; Hanovar etal. 2011; Candan 2018; Candan and
Candan 2020; Candan etal. 2021b). Embryonic deaths dif-
fer between embryonic stages and nesting seasons (Acker-
man 1996, Santidrián Tomillo etal. 2009). According to the
results in both nesting sites and species, embryonic deaths
were observed at different levels. Among the embryonic
stages, early embryonic deaths were quite high.
Conclusion
It has been suggested that microbial abundance can be
increased due to failed eggs (Cornelius 1986; Cornelius
etal. 1991), and infection of other eggs in the nest could
be prevented by removal of failed eggs from the nest thus
increasing hatching success (Cornelius 1986). Microbial
abundance, richness, diversity, and nest density may vary
in beach zones (Hanovar etal. 2011). Bacterial flora also
differs in studies conducted for the same beach. The varia-
tion in embryonic mortality rates between seasons may be
related to the variation in bacterial flora. For this reason,
detection of beach-specific bacterial flora should be included
in conservation-monitoring studies. Thus, it is possible to
have information about the identity of bacteria causing the
pathogenesis of sea turtles and the spread of these bacteria
in the environment. The identification method, the effec-
tiveness of which has been demonstrated in this study, is
predicted to bring innovation to wildlife studies. The results
obtained from our study reveal the necessity of wide surveil-
lance studies, such as biological monitoring of pollution in
sea turtle habitats in the Mediterranean.
Supplementary Information The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s00203- 022- 03292-z.
Acknowledgements We thank Matthew Haworth, PhD. for linguistic
advice and criticism, and Şafak Kalındamar, PhD., for comments on
the earlier version of this manuscript and volunteers of Sea Turtle Con-
servation Projects Sugözü Beaches supported by BIL (BOTAŞ Inter-
national Limited Co., Turkey) and Göksu Delta supported by General
Directorate for Protection of Natural Assets (Minister of Environment,
Urbanisation and Climate Change). We also thank Seaturtle.org team
for Maptool.
Author contributions All authors contributed to the writing—origi-
nal draft, review and editing. Conceptualization were performed by
EDC and OC, methodology and investigation performed by EDC,
sampling and field study were performed by OC, formal analysis and
data curation were performed by YNÇ. All authors read and approved
the final manuscript.
Funding The authors declare that no funds, grants, or other support
were received during the preparation of this manuscript.
Data availability The data sets generated during and/or analyzed dur-
ing the current study are available from the corresponding author on
reasonable request.
Declarations
Competing interests The authors declare no competing interests.
Conflict of interest The authors declare that they have no conflict of
interest. The authors have no relevant financial or non-financial inter-
ests to disclose.
Ethical approval None of the experiments involved here sacrifice ani-
mals, and therefore, an approval from an institutional animal research
ethics committee is not required.
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