Invest Clin 64(3): 296 - 307, 2023 https://doi.org/10.54817/IC.v64n3a3

Corresponding author: Elba Guerrero. Laboratorio de Genética Molecular, Centro de Microbiología y Biología

Celular, Instituto Venezolano de Investigaciones Científicas (IVIC), Caracas, Venezuela. E-mail: elbaguerrero@

hotmail.com

Phenotypic and genotypic study of

antibiotic-resistant Escherichia coli isolates

from a wastewater treatment plant in Zulia

state, Venezuela.

Elba Guerrero

, Lizeth Caraballo

, Howard Takiff

, Dana García

and Marynes Montiel

3,4

Laboratorio de Genética Molecular, Centro de Microbiología y Biología Celular,

Instituto Venezolano de Investigaciones Científicas (IVIC), Caracas, Venezuela.

Centro de Investigación del Agua, Universidad del Zulia, Maracaibo, Venezuela.

Facultad Experimental de Ciencias. Universidad del Zulia, Maracaibo, Venezuela.

Escuela Superior Politécnica del Litoral, Facultad de Ciencias de la Vida, Guayaquil,

Ecuador.

Keywords: antibiotic re sistance; E. coli; wastewater; phylogroups.

Abstract. Antibiotic-resistance in bacteria is a global health problem, and

wastewater treatment plants can play a role in their dissemination. In this work,

we used PCR and plasmid transformation to characterize antibiotic-resistance

and the phylogenetic groups of Escherichia coli isolated from a treatment plant

in Zulia, a state in western Venezuela. Thirty-six bacteria isolates were analyzed,

of which 27 resulted resistant by disc diffusion primarily to tetracycline and

sulfisoxazole but also to trimethoprim, chloramphenicol, ampicillin, and cip-

rofloxacin. The tetA, sul2, floR, and blaTEM resistance genes were frequently

present and, in most cases, transferable. dfrA12, tetB, sul3, sul1, and aadA2

genes also were detected. The integrase gene intI1 was common in multidrug-

resistant isolates. These results suggest that E. coli from the treatment plant

is a reservoir of antibiotic-resistance genes, which signify a potential health

threat. Additionally, the phylogroup C was predominant, which is unusual and

may represent an adaptation of this group to environmental conditions or per-

haps the most frequent phylogroup entering from the influent.

Study of antibiotic-resistant E. coli from a wastewater plant 297

Vol. 64(3): 296 - 307, 2023

Estudio fenotípico y genotípico de aislados de Escherichia coli

resistentes a antibióticos de una planta de tratamiento

de aguas residuales del estado Zulia, Venezuela.

Invest Clin 2023; 64 (3): 296 – 307

Palabras clave: genes de resistencia; E. coli; aguas residuales; filogrupos.

Resumen. La resistencia bacteriana a antibióticos es un problema de salud

global y las plantas de tratamiento pueden jugar un papel en su diseminación.

En este trabajo caracterizamos, mediante PCR y transformación de plásmidos,

la resistencia a antibióticos y los grupos filogenéticos de Escherichia coli ais-

lada de una planta de tratamiento en el estado Zulia, Venezuela. Se analizaron

36 aislados bacterianos, de los cuales 27 resultaron resistentes por difusión en

disco principalmente a tetraciclina y sulfisoxazol, pero también a trimetoprim,

cloranfenicol y ampicilina. Los genes tetA, sul2, floR y blaTEM se encontraron

comúnmente en los aislados resistentes y fueron en la mayoría de los casos

transferibles; adicionalmente se detectaron los genes dfrA12, tetB, sul3, sul1 y

aadA2. El gen de integrasa intI1 se detectó en la mayoría de los aislados multi-

resistentes. Estos resultados sugieren que E. coli en la planta de tratamiento es

un reservorio de genes de resistencia a antibióticos, lo que significa una amena-

za potencial para la salud. Adicionalmente predominó el filogrupo C, lo que es

inusual y podría deberse a una adaptación de este a las condiciones ambientales

o podría ser el mayoritario en el influente.

Received: 28-09-2022 Accepted: 23-03-2023

INTRODUCTION

The indiscriminate use of antibiotics

in human and animal medicine, as well as

for prophylaxis and growth promotion in

animal husbandry, threatens to reduce the

effectiveness of these fundamental drugs.

Antibiotic-resistant bacteria, although oc-

curring naturally, are also released into the

environment, where they may outcompete

sensitive bacteria due to the presence of an-

tibiotics and other chemical contaminants

that are also released into the environment.

The resistance genes in these bacteria can

then be transferred to other pathogenic and

non-pathogenic bacteria, thereby increasing

the environmental reservoir of resistant bac-

teria and genetic resistance determinants.

Escherichia coli is a commensal bacte-

rium that inhabits the intestines of humans

and other animals and is often used to in-

dicate environmental fecal contamination.

Some E. coli are pathogens that can cause

urinary tract, gastrointestinal or nosocomial

infections. Antibiotic-resistance in environ-

mental E. coli has also been proposed as an

indicator to monitor the extent of antibiotic

resistance in the environment

The bacterial load of wastewater dis-

charged into natural water bodies is signifi-

cantly reduced by treatment plants. However,

these plants may also promote the spread of

antibiotic-resistant bacteria and resistance

genes by providing favorable conditions for

increasing the relative abundance of resis-

tant bacteria and the horizontal transfer of

298 Guerrero et al.

Investigación Clínica 64(3): 2023

the genes conferring this resistance

. Al-

though there are few treatment plants in

Venezuela and much of the wastewater is

discharged directly into the environment,

there is a wastewater treatment plant in the

state capital Maracaibo, located in the “El

Tablazo” Petrochemical Complex of the Mi-

randa municipality of Zulia state. The plant

was designed so that the petrochemical in-

dustry could reuse some of its effluent water

while the rest would be discharged into the

giant Lake Maracaibo.There have been very

few studies on antibiotic-resistance in bac-

teria isolated from raw or treated wastewa-

ter in Venezuela, but such studies represent

essential surveillance measures to assess

the extent of antibiotic-resistance in the

environment and plan corrective strategies.

Accordingly, we set out to perform a pheno-

typic and molecular study of antibiotic resis-

tance in E. coli isolates from the wastewater

treatment plant mentioned above.

MATERIALS AND METHODS

E. coli was isolated from water samples

of the “El Tablazo” treatment plant (Miran-

da Municipality, Zulia State) collected from

May to October 2012 for a microbiological

quality evaluation. The system includes a

pre-treatment to remove solids followed by

absorption, biological oxidation, and a first

chlorine injection. The water is then trans-

ported to the plant at “El Tablazo” and sub-

jected to physical and biological treatment

based on reactors where dissolved organic

matter is removed, followed by a secondary

settling. Then a first effluent is discharged

into Lake Maracaibo. Another portion of the

water to be used by the petrochemical com-

plex is treated with a flocculant and chlorine.

Sampling sites were four different sec-

tions of the treatment plant: pre-treated

influent (Site1); after physical processing

(Site 2); after biological processing (efflu-

ent to the Maracaibo lake, Site 3); and the

chlorine disinfection point (Site 4). There

were six water samples from Site 1 and Site

4, four from Site 2, and five from Site 3.

The water samples were collected in

sterile bottles and processed according

to the procedures described in the Stan-

dard Methods for examination of Water and

Wastewater to determine coliform by the

fermentation technique

. Samples showing

growth in EC broth were streaked onto EMB

agar to select typical E. coli colonies, which

were sub-cultured in nutrient agar tubes for

transport. Re-isolation was performed on

McConkey agar, and colonies were cultured

in LB broth and then stored in 20% glycerol

at -80°C. All assays were performed on the

bacteria regrown from the frozen stocks.

Biochemical identification and antibiotic

susceptibility testing

Bacterial isolates were first identified

with the following biochemical tests: TSI,

indole-motility, methyl red, Voges Proskauer,

citrate, and urea.

Resistance was assessed with the Kirby

Bauer disc diffusion method, using commer-

cial discs with the following antibiotics: tet-

racycline 30 µg (TE), ampicillin 10 µg (AMP),

ampicillin-sulbactam 10/10 µg (SAM), sulfi-

soxazole 250 µg (SF), chloramphenicol 30 µg

(C), trimethoprim 5 µg (W), trimethoprim-

sulfamethoxazole 1.25 µg /23.75 µg (SXT),

ciprofloxacin 5 µg (CIP), aztreonam 30 µg

(ATM) and imipenem 10 µg. E. coli ATCC

25922 was used as an antibiotic-susceptible

control strain. The results were interpreted

according to CLSI guidelines

PCR amplification

PCR was used to confirm the bacteria

as E. coli, determine phylogroups, and de-

tect the presence of resistance genes and

the integrase gene intI1. All PCR reactions

were performed on boiled bacterial lysates,

using Taq DNA polymerase with ThermoPol

buffer (NEB), following the manufacturer’s

instructions, using previously reported spe-

cific primers, some of which were modified

Study of antibiotic-resistant E. coli from a wastewater plant 299

Vol. 64(3): 296 - 307, 2023

as indicated below. PCR was performed to

amplify genes conferring resistance to tet-

racycline (tetA and tetB)

, sulfisoxazole

(sul1, sul2, and sul3)

6-8

, chloramphenicol

(floR and cat)

, ampicillin (blaTEM)

and

trimethoprim (dfrA12 and dfrA7&17)

10,

. The detection of the intI1 integrase was

with primers described by Moura

. In con-

trast while the phylogroup identification was

performed using the quadruplex plus group

C specific PCR described by Clermont et al.

Negative controls without template DNA

were included in each PCR assay. A subset

of the PCR products were confirmed by DNA

sequencing (Macrogen, Korea) and used as

positive controls for the detection of resis-

tance genes and intI1, as well as the deter-

mination of phylogroups.

The reactions were performed with

previously reported primers and conditions

or with the following variations: Sul1-R.

5’-TGATCTAACCCTCGGTCTCT-3’ tem-

perature of annealing (Ta) 56°C, blaTEM-F

5’-GCATACACTATTCTCAGAATGA-3’ bla-

TEM-R 5’-CTCACCGGCTCCAGATTTAT-3’

Ta 56°C, dfr7&17-F 5’-CATTTGACTCTC-

TATGGGTGTTC TT-3’ Ta 58°C.

To avoid analyzing duplicate resistant

isolates, REP-PCR was performed on isolates

showing the same phenotype and genotype,

using the REP1 and REP2 primers as previ-

ously described

. E. coli-specific PCR was

performed using primers to amplify rrs (75F

and 619R) or gad

15, 16

, with E. coli XL1-blue

as a positive control.

Transformation and conjugation assays

Transferability of the detected antibi-

otic resistance genes was assessed by heat

shock transformation using transformation

competent E. coli DH5α as the recipient

strain and plasmid DNA obtained by alkaline

lysis from all isolates resistant to tetracy-

cline, ampicillin, chloramphenicol, or trim-

ethoprim in which PCR had detected a re-

sistance gene. Transformants were selected

on LB agar plates containing either carbeni-

cillin (50 µg/mL), ampicillin (32 µg/mL),

tetracycline (30 µg/mL), chloramphenicol

(30 µg/mL), or trimethoprim (20 µg/mL)

as appropriate. Phenotypic resistance was

confirmed for each transformant, and the

presence of plasmid DNA and the relevant

resistance genes were verified.

In some cases, the capacity for conjuga-

tion was assessed in liquid medium using E.

coli J62-2 as the recipient strain. Selection

was performed on LB agar plates supple-

mented with tetracycline (30 µg/mL) and ri-

fampicin (50 µg/mL). Transconjugants were

confirmed by phenotypic resistance, amplifi-

cation of the same resistance gene detected

in the donor, and ERIC-PCR with primers de-

scribed by Versalovic et al.

RESULTS

Here, we characterized thirty-six iso-

lates identified as E. coli with biochemical

tests and identified as E. coli with biochemi-

cal tests and PCR amplification of rrs or

gad. These isolates originated from the four

sampling sites: 13 from Site 1, four from Site

2, nine from Site 3, and ten from Site 4.

Resistance phenotypes

As shown in Fig. 1, the highest fre-

quency of resistance was to tetracycline and

the lowest to ciprofloxacin and ampicillin-

sulbactam, with intermediate prevalences

of resistance to the other antibiotics tested,

including ampicillin.

Twenty-four isolates (66.6%) were fully

resistant to at least one antibiotic, corre-

sponding to 7/13, 2/4, 7/9, and 8/10 iso-

lates from sampling points 1 to 4, respective-

ly (Table 1). Five of these 24 isolates (20.8%)

also showed intermediate resistance to one

or two additional antibiotics (two AMP-CIP,

one CIP, and two SAM). Among the 12 re-

maining isolates, three had only intermediate

resistance (two AMP and one TE), and nine

(9/36, 25%) were fully sensitive to all an-

tibiotics tested. There were also seven iso-

lates (7/24, 29.1%) fully resistant to three

or more antibiotics of different classes and

300 Guerrero et al.

Investigación Clínica 64(3): 2023

Table 1

Phenotypic and genotypic profiles of the bacterial isolates.

Sampling site Resistance phenotype (FR/IR) Resistance genotype Phylogroup

1 TE tetB B1

1 TE-SF-W-TS tetA-sul2 C

1 TE-SF-W-TS/amp-cip tetA-sul2 C

1 TE-SF- TS-C/amp-cip tetA-sul2-floR A

1 TE-SF-C/ cip tetA-sul2-floR A

1 TE-C tetA-floR A

1 amp nd B1

1 S na C

1 S na B2

2 TE tetA C

2 TE-SF-W-TS-AMP-C/sam tetA-sul1-blaTEM-floR-dfrA12-intI1 C

2 S na A

3 TE tetA B1

3 TE-W tetA C

3 TE-SF-W- TS-AMP-C-CIP/sam tetA-sul3-blaTEM-intI1 C

3 TE-SF-W-TS- AMP-SAM tetA-sul3-blaTEM-dfrA12-intI1 C

3 TE-AMP tetB-blaTEM C

3 AMP-C blaTEM- floR B1

3 AMP-W blaTEM C

3 te nd C

3 S na A

4 TE tetA C

4 TE tetA A

4 TE-SF tetA B1

4 TE-SF tetA-sul2 A

4 TE-SF tetA-sul2 C

4 TE-SF tetA-sul2 B1

4 TE-SF-W-TS-C tetA-sul3-intI1 C

4 TE- W-C tetA- tetB- flor-intI1 A

4 amp nd C

4 S na C

FR: Fully resistant, IR: intermediate resistance (lowercase), S: sensitive, nd: not determined, na: not apply. The

abbreviations for the antibiotics are the same as in Fig. 1.

Study of antibiotic-resistant E. coli from a wastewater plant 301

Vol. 64(3): 296 - 307, 2023

thus multi-drug resistant or MDR. Two of

these originated from each of sampling Sites

1, 3, and 4, and one isolate was from Site 2

(see Table 1). All isolates were sensitive to

aztreonam and imipenem.

Resistance genes

The genotypic resistance profiles and

genes detected are described in Table 1. The

most frequently detected genes were tetA

(20/22) and sul2 (8/12), while all isolates

fully resistant to ampicillin contained bla-

TEM (Fig. 1). The floR gene was detected in

most (6/8) of the chloramphenicol-resistant

isolates, none of which contained the cat

gene. The only amplified determinant as-

sociated with trimethoprim resistance was

dfrA12, which was detected in just two of the

ten trimethoprim-resistant isolates.

Of the seven MDR-resistant isolates, the

intIl gene was amplified from 5, representing

21% of the resistant isolates and 13.9% of

total isolates (Table 1). In one of the isolates

in which the intIl gene was detected, amplifi-

cation and sequencing with primers specific

for conserved segments of class 1 integrons

detected dfrA12 and aadA2, which encodes

an aminoglycoside adenyl transferase con-

ferring streptomycin resistance.

Transfer of resistance determinants

Transformants were obtained from

the isolated plasmid DNA of 20/24 resis-

tant isolates. Most of the transformants

(18/20) were recovered on media with tet-

racycline, while only 1/20 were recovered

on media with chloramphenicol and 1/20

on media with trimethoprim. However, the

major part of the genes conferring resis-

tance to sulfisoxazole and chlorampheni-

col were co-transferred with the tetA gene

(Table 2). Despite repeated attempts, no

transformants were obtained from plasmid

DNA isolated from the remaining four re-

sistant isolates.

The tetA gene was transferred from al-

most all isolates in which it was detected

(19/20). The blaTEM gene was transferred

Fig. 1. Phenotypic and genotypic resistance per antibiotic. The numbers of phenotypically resistant isolates

with corresponding genotypes (for full resistance) are indicated. Resistance genotypes are numbered

from 1 to 4 according to the detection frequency from highest to lowest. 1: tetA (TE); sul2 (SF and

TS); dfrA12 (W); floR (C); or blaTEM (AMP and SAM). 2: tetB (TE); or sul3 (SF and TS). 3: tetA-tetB

(TE); sul1 (SF); sul1-dfrA12(TS). 4: sul3-dfrA12 (TS). nd: Resistance genotype not determined, IR:

intermediate resistance. TE, tetracycline; SF, Sulfisoxazole; W, trimethoprim; C, Chloramphenicol;

TS, Trimethoprim-sulfamethoxazole; AMP, ampicillin; SAM, ampicillin-sulbactam; CIP, ciprofloxacin.

302 Guerrero et al.

Investigación Clínica 64(3): 2023

from 1/6 of the isolates in which it was de-

tected, the sul genes were transferred from

10/12 isolates (8 sul2 and 2 sul3), floR from

4/6 and dfrA12 from 1/2 isolates contain-

ing this gene. The tetB gene was not trans-

ferred under the conditions employed. The

resistance genes most frequently detected in

our isolates, tetA and sul2, hybridized with

plasmid DNA isolated from most (18/20), or

all isolates (8/8), respectively, in which the

genes had been detected by PCR, confirm-

ing that they were carried on plasmids (not

shown).

The transfer capability of the tetA gene

was tested by conjugation experiments with

five isolates in which the tetA gene was

detected. Transconjugants were obtained

from two isolates at the high frequencies of

7.5 x 10

-3

y 1.95 x 10

-2

, demonstrating that

the tetA gene was carried on conjugative

plasmids at least in these two isolates. One

of these five isolates with the tetA gene gen-

erated neither transformants nor transcon-

jugants; nevertheless, only scant plasmid

DNA could be obtained from this isolate,

perhaps because its plasmid was either very

large or present at a very low copy numbers.

E. coli phylogroup analysis

Of the 36 bacterial isolates studied, 20

belonged to group phylogenetic C, nine to

group A, six to group B1, and one to group

B2 (Table 1).

DISCUSSION

In the present work, we analyzed phe-

notypic antibiotic-resistance, detected cor-

responding antibiotic-resistance genes,

evaluated their transferability, and deter-

mined the phylogroups of 36 E. coli isolates

obtained from a wastewater treatment plant.

Similar to other studies of resistant E. coli

isolates from wastewater and treatment

plants

17-21

the most frequent resistance en-

countered was to tetracycline, sulfonamide,

trimethoprim, and ampicillin. In contrast,

resistance to the carbapenems and cipro-

floxacin was infrequent or not detected. Our

results differ from a previous phenotypic

study of E. coli isolates from stabilization

ponds in Maracaibo, Venezuela, that found

a higher proportion of resistance for ampi-

cillin (81.25%) followed by tetracycline and

trimethoprim

. In comparison, a study of

E. coli isolates from a treatment plant in Cu-

maná (Venezuela)

found 73.3% and 23.3% of

ampicillin and tetracycline resistance, re-

spectively. These discrepancies may be due

to differences in the treatment systems.

Although tetracycline and sulfonamide,

to which we found medium to high frequen-

cies of resistance (22% to 64%), have been,

for the most part, replaced by newer agents

in human medicine, they are still classified

as highly important by the World Health Or-

ganization.

Ampicillin (28% resistance) is

Table 2

Antibiotic resistance profiles of transformants and transconjugants.

Genotype Phenotype Number of

transformants (N=20)

Number of

transconjugants (N=2)

tetA-sul2 TE-SF 8 -

tetA-sul3 TE-SF 1 -

tetA TE 6 2

tetA-floR TE-C 3 -

tetA- sul3- dfrA12 TE-SF-W 1 -

flor- blaTEM C-AMP 1 -

Study of antibiotic-resistant E. coli from a wastewater plant 303

Vol. 64(3): 296 - 307, 2023

still frequently used and considered critical-

ly important in human medicine, as are the

antibiotics for which we found less frequent

resistance (8% to 11%).

The antibiotic resistance we observed is

common in E. coli isolated from healthy hu-

mans in low and middle-income countries

and the antibiotic-resistance in human iso-

lates of E. coli has been correlated with the

resistance in E. coli isolated from the local

wastewater

. The resistance patterns may

also be affected by selection or adaptation

to the specific characteristics of the treat-

ment plant

.A high prevalence of the tetA,

sul2, and blaTEM genes has been previously

observed in E. coli isolated from other treat-

ment plants.

17, 27

However, previous studies

have predominantly found the cat gene in

chloramphenicol-resistant isolates

17, 25

, but

most of our chloramphenicol resistant iso-

lates carried the floR gene. In contrast, the

cat gene was not detected.

We found the intI1 gene in 13.9% of

total isolates, similar to a study by Figueira

et al. that found the gene in 22.3 % of E.

coli isolates from treated wastewater

. In-

tegrons contribute to the spread of multi-

drug resistance, and class I integrons are the

most important in clinical isolates

Horizontal transmission of plasmids car-

rying resistance genes is a crucial route for

disseminating resistance. Similar to our re-

sults, a previous study of E. coli and other fe-

cal coliforms isolated from treatment plants

found that most plasmid transformants were

resistant to tetracycline, followed by chlor-

amphenicol and trimethoprim

. The resis-

tance genes tetA, blaTEM, tetB, sul, floR, and

dfrA12 can be found on chromosomes, but

as we observed, they are frequently carried

on plasmids

30-35

E. coli isolates can be classified into

seven main phylogenetic groups: A, B1, B2,

C, D, E, and F. Intestinal pathogenic E. coli

strains have been associated with groups

A, B1, and E, while extra-intestinal strains

mainly belong to groups B2 and D

Howev-

er, Group C can also include human patho-

genic strains, as shown in a study of human

isolates from the USA and Europe, in which

phylogroup C was associated with uropatho-

genic E. coli, although B2 and D predomi-

nated

. Jafari et al. found that group C was

the most common (21.3%) among Shiga

toxin-producing E. coli (STEC) patient iso-

lates

. It has been suggested that some E.

coli phylogroups, particularly groups A and

B1 (or non-B2), are more prone to develop

antibiotic resistance to traditional antibiot-

ics and fluoroquinolones

39, 40

.Most previous

studies assigning E. coli phylogroups have

used the triple PCR method

that identifies

only four groups: A, B1, B2, and D. Several

studies have found group A to predominate

in wastewater, followed by D or B1

. Group

A is also the phylogroup most frequently

observed in human commensal isolates, fol-

lowed by B2 group

. Researchers employ-

ing the quadruplex method plus the group C

specific PCR, or in silico typing, have found

that phylogroup C is less frequent than other

phylogroups in samples from birds, humans,

non-human mammals, domestic animals,

wild animals, river and lake water

43-45

. Two

studies on strains from wastewater found

that group A or B2 predominated, while

none or only 1% belonged to group C

17, 46

Therefore, the presence of group C in

most of our isolates differs from the findings

in similar studies and could be due to geo-

graphical location or climate differences, as

these factors may influence the distribution of

phylogroups

. It is also possible that previous

studies that used only the triple PCR method

classified C group isolates as group A.

In conclusion, we observed E. coli iso-

lates resistant to diverse antibiotics, includ-

ing some clinically essential agents and found

that many were associated with transferable

genetic determinants and class 1 integrons.

Also, in contrast to other studies, many of

our isolates belonged to the phylogroup C.

The characteristics of the isolates we stud-

ied may have been determined by the influ-

ent water, the nature of the treatment plant,

and environmental conditions, but an evalu-

304 Guerrero et al.

Investigación Clínica 64(3): 2023

ation of the contribution of each of these as-

pects would require a much larger study with

many more isolates. Similar studies should

be repeated in the same treatment plant

and undertaken in other treatment plants in

Venezuela, and these studies should be ex-

tended to include untreated wastewater and

focus on resistance to the newer, currently

more commonly used antibiotics.

Conflict of interest

The authors declare that they have no

conflict of interest.

Funding

The Instituto Venezolano de Investiga-

ciones Científicas supported this research.

ORCID numbers

• Elba Guerrero (EG):

0000-0002-3936-9556

• Lizeth Caraballo (LC):

0000-0003-2043-8731

• Howard Takiff (HT):

0000-0002-0480-0860

• Dana García (DG):

0009-0007-7020-5529

• Marynes Montiel (MM):

0000-0002-6249-0362

Author contributions

Methodology: EG, LC, DG, and MM.

Data analysis and original draft preparation:

EG. Supervision, writing – review, and edit-

ing: HT. All authors contributed to the study

conception, commented on previous versions,

and read and approved the final manuscript.

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