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International Journal of Antimicrobial Agents 32 (2008) 308–314

Genetic characterisation of CTX-M-15-producingKlebsiella pneumoniae and Escherichia coli strainsisolated from stem cell transplant patients in Tunisia

Mohamed Salah Abbassi a, Carmen Torres b,∗, Wafa Achour a, Laura Vinué b,Yolanda Sáenz b, Daniela Costa b, Ons Bouchami a, Assia Ben Hassen a

a Laboratory of the National Bone Marrow Transplantation Centre, Bab Saadoun, Tunis, Tunisiab Área de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de La Rioja, Madre de Dios 51, 26006 Logrono, Spain

Received 6 February 2008; accepted 13 April 2008

bstract

Characterisation of extended-spectrum �-lactamase (ESBL) genes and their genetic environments as well as the presence of integronsere analysed in nine Klebsiella pneumoniae and two Escherichia coli ESBL-positive isolates recovered in the Centre of Bone Marrowransplantation of Tunisia. All strains harboured the blaCTX-M-15 gene and presented minimum inhibitory concentrations for cefotaxime andeftazidime of 256–1024 mg L−1 and 16–512 mg L−1, respectively, and eight of them showed different pulsed-field gel electrophoresis patterns.he blaOXA-1 and blaTEM-1 genes were detected in eight and ten strains, respectively. In addition, blaSHV-1, blaSHV-11 and blaSHV-27 were found inix, one and one K. pneumoniae strains, respectively. The new variant blaSHV-103 was characterised in one K. pneumoniae strain. The intI1 geneas detected in eight K. pneumoniae strains and the dfrA5 + ereA2 and aadA gene cassettes were found in one and five strains, respectively.ll strains harboured a 70 kb plasmid, and its transference in addition to blaCTX-M-15, blaTEM-1b and blaOXA-1 genes was demonstrated from

hree K. pneumoniae to E. coli. ISEcp1 and orf477 were located upstream and downstream, respectively, of the blaCTX-M-15 gene in 10 strains.he occurrence of the blaCTX-M-15 gene in unrelated strains might have originated from the dissemination of mobile genetic elements in which

SEcp1 may have played an important role.2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

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eywords: ESBL; CTX-M-15 �-lactamase; Genetic environment; SHV-10

. Introduction

Klebsiella pneumoniae and Escherichia coli have emergeds major nosocomial pathogens, especially in Intensive Carenits (ICUs), from the beginning of the 1980s. Neutropenicatients with haematological malignancies undergoing bonearrow transplantation are at high risk; indeed, intensive and

rolonged chemotherapy is often associated with mucosalamage, a phenomenon that enhances bacterial translo-ation and therefore leads to invasive infections such as

acteraemia [1]. Excessive use of antibiotics to prevent andeduce the frequency of infections has led to the selectionnd emergence of resistant bacteria. For K. pneumoniae

∗ Corresponding author. Tel.: +34 941 299 750; fax: +34 941 299 721.E-mail address: carmen.torres@unirioja.es (C. Torres).

adrMi-

924-8579/$ – see front matter © 2008 Elsevier B.V. and the International Societyoi:10.1016/j.ijantimicag.2008.04.009

iella pneumoniae; Escherichia coli

nd E. coli, �-lactam resistance associated with the produc-ion of extended-spectrum �-lactamases (ESBLs) is a causef concern in nosocomial environment and constitutes anncreasing problem in healthcare centres worldwide [2,3].

ESBLs are usually plasmid-encoded enzymes derived ini-ially from the classical TEM- and SHV-type �-lactamasesy one or more amino acid substitutions that confer differ-nt levels of resistance to ceftazidime, cefotaxime and otherroad-spectrum cephalosporins and monobactams [2,3]. Inhe past they were predominately reported in K. pneumoniaend E. coli strains. However, this pattern has now changedramatically owing to the emergence and expansion of the

elatively new CTX-M enzymes [2–6]. More than 65 CTX-

�-lactamases are recognised so far and they are clusteredn six groups based on their amino acid identities: CTX-M-1,2, -8, -9, -25 and -45 groups [6]. The CTX-M-1 group is

of Chemotherapy. All rights reserved.

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M.S. Abbassi et al. / International Journ

requently detected in Europe, however variations have beeneported from country to country, with the CTX-M-26, -15nd -3 �-lactamases being more frequently reported in theK in contrast to CTX-M-9, -10 and -14 that are more preva-

ent in Spain [5,7]. In Tunisia, CTX-M-15, -14, -16, -8 and1 enzymes have been previously reported in K. pneumoniaend E. coli strains isolated from humans or food samples ofnimal origin [8–10].

The epidemiology of CTX-M-producing strains is quiteomplex. Outbreaks of clonal strains have been reportedhroughout the world [9,11,12]; in parallel, many authorsave reported the dissemination of genetic elements includinglaCTX-M genes rather than strain dissemination [13,14].

We have observed in recent years the emergence of ESBL-roducing K. pneumoniae and E. coli isolates exhibitingCTX-M phenotype, recovered from stem cell transplant

atients hospitalised in the Tunisian Bone Marrow Transplan-ation Centre. The purpose of this study was to characterisehese isolates in order to determine the specific type of ESBLshey harbour as well as their clonal relationship.

. Materials and methods

.1. Bacterial strains

A retrospective survey of ESBL-producing K. pneumo-iae and E. coli isolates was carried out in the Tunisianone Marrow Transplantation Centre from December 1998

o September 2007. During this period, 105 K. pneumoniaend 154 E. coli strains with clinical significance were isolated.hirty-four (32.4%) and 19 (12.3%) ESBL-producing K.neumoniae and E. coli isolates, respectively, were detected.mongst them, nine (26.5%) K. pneumoniae and two (10.5%). coli isolates with a CTX-M phenotype (higher level of

esistance to cefotaxime than ceftazidime) were detectednd included in this study for further characterisation. Allhese isolates were obtained in 2003–2007 and were identi-ed using the API20E system (bioMérieux, Marcy l’Etoile,rance), and their origins were as follows (number of iso-

ates): blood (4); urine (3); sputum (2); catheter (1); and pus1).

.2. Antimicrobial susceptibility testing

Antimicrobial susceptibility was determined by the diskiffusion method according to the recommendations of thentibiogram Committee of the French Society of Microbiol-gy (CA-SFM) [15]. The double-disk synergy test betweenmoxicillin/clavulanic acid and cefotaxime or ceftazidimeas performed to determine ESBL production. The mini-um inhibitory concentrations (MICs) of cefotaxime and

eftazidime (Bio-Rad, Marnes-la-Coquette, France) wereetermined by the agar dilution method [16] and by EtestAB BIODISK, Solna, Sweden) following the manufacturer’snstructions. The CA-SFM MIC breakpoints were considered

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timicrobial Agents 32 (2008) 308–314 309

nd E. coli ATCC 25922 was used as a quality controltrain.

.3. Characterisation of β-lactamase genes, theirenetic environments and associated integrons

Genomic DNA was extracted by Wizard® GenomicNA Purification Kit (Promega, Madison, WI). The pres-

nce of blaTEM-, blaSHV-, blaOXA- and blaCTX-M-typeenes was detected by polymerase chain reaction (PCR)sing specific primers and conditions as reported previously8]. Amplified DNA fragments were sequenced on bothtrands and the nucleotide and their deduced amino acidequences were compared with those included in the Gen-ank database as well as with those deposited at the websitettp://www.lahey.org/Studies/ in order to confirm the specificype of �-lactamase gene.

The presence of intI1 and intI2 genes, encoding class 1nd 2 integrases, respectively, as well as the variable regionf the class 1 integrons were analysed by PCR as previouslyeported [17]. Characterisation of gene cassettes included inhe variable region of class 1 integrons was performed byCR mapping and/or sequencing. Positive and negative con-

rol strains from the collection of the University of La Rioja,pain, were included in each PCR experiment.

.4. Identification of the genetic environment of thelaCTX-M-15 gene

The nucleotide sequences of the upstream (ISEcp1) andownstream (orf477) regions of the blaCTX-M-15 gene in thesolates were determined by PCR using the specific primersnd conditions as previously reported [14,18].

.5. Cefotaxime resistance transfer assays

Mating experiments were performed using E. coli J53-(pro, met, RifR) as recipient strain. Overnight cultures of

onor and recipient strains grown in Mueller–Hinton broth at7 ◦C were mixed together at 1:10 (v/v) proportion and incu-ated at 37 ◦C for at least 4 h without shaking. Then, 0.1 mL ofhe mixture was spread onto the surface of Mueller–Hintongar plates containing rifampicin (200 mg L−1) plus cefo-axime (2 mg L−1). Transconjugants growing on the selectionlates were subjected to an ESBL screening test, antibi-tic susceptibility testing and PCR to confirm the possiblecquisition of bla genes. In addition, plasmid extraction waserformed to detect possible conjugative ESBL-encodinglasmids.

.6. Plasmid isolation

Plasmid DNA was extracted by the alkaline extractionethod as described by Birnboim and Doly [19] and analysed

y gel electrophoresis through 0.8% agarose gel. Plasmids of. coli V517 (53.7, 7.2, 5.4, 5, 4, 3, 2.6 and 2 kb) as well

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10 M.S. Abbassi et al. / International Journ

s E. coli containing pIP173 (126 kb) (kindly provided byrof. Jérome Etienne, Lyon, and Prof. Alain Philippon, Paris,rance, respectively) were used as molecular size markers

o estimate the molecular weight of plasmids in the studiedtrains.

.7. Molecular strain typing

The clonal relationship between the different isolates wastudied by pulsed-field gel electrophoresis (PFGE) followinghe method of Gautom [20]. Plugs were digested with 30 U ofbaI (Promega, Charbonnières-les-bains, France) and loaded

nto a 1% agarose gel. Electrophoresis was performed in aHEF-DRIII apparatus (Bio-Rad Laboratories, Richmond,A) under the following conditions: 6 V cm−1 for 23 h at4 ◦C, with initial and final pulse times of 2 s and 30 s, respec-ively. DNA banding patterns were visually compared andlonal relationships were established following the criteriaf Tenover et al. [21].

. Results

.1. β-Lactam susceptibility profile and associatedesistance

All nine K. pneumoniae and two E. coli strains includedn this study presented cefotaxime and ceftazidime resis-ance, a positive ESBL test and a resistance phenotype thatuggested a CTX-M �-lactamase. The MIC values for cefo-axime (256–1024 mg L−1) were 2–32-fold higher than foreftazidime (16–512 mg L−1) (Table 1). All strains showedesistance to aztreonam and cefepime but remained sus-eptible to imipenem. Eight of the nine K. pneumoniaetrains and one E. coli strain exhibited resistance to trimetho-rim/sulfamethoxazole. Furthermore, aminoglycoside andiprofloxacin resistance were common in these strains. Takenogether, these results showed that all the strains exhibitedesistance from three to eight non-�-lactam drugs (Table 1).

Eight of the strains (six K. pneumoniae and 2 E. coli)howed different patterns when studied by PFGE, exhibit-ng more than six bands of difference; the remaining three. pneumoniae isolates presented closely or possibly relatedatterns (three to six bands of difference) (Fig. 1).

.2. Identification of ESBL-encoding genes by specificCR and sequencing

A positive CTX-M PCR amplicon was obtained in all K.neumoniae and E. coli strains and, after sequencing, thelaCTX-M-15 gene was identified in all strains (Table 1). Inhe case of K. pneumoniae, all strains harboured blaSHV

nd blaTEM-1 genes in association with blaCTX-M-15. ThelaSHV-1 gene was detected in six K. pneumoniae strainsnd blaSHV-11 and blaSHV-27 genes in one strain each. One. pneumoniae strain harboured a new blaSHV gene, which

it

timicrobial Agents 32 (2008) 308–314

as been named blaSHV-103 by the Lahey Clinic grouphttp://www.lahey.org/studies) and has been included in theenBank database with the accession no. EU032604. Thisew blaSHV variant showed one deduced amino acid substi-ution (Lys250 → Arg) and two silent nucleotide mutationsC357 → T, A402 → G) in comparison with the blaSHV-1ucleotide sequence (GenBank accession no. AF148850).he blaTEM-1a and blaTEM-1b variants were detected in six and

hree K. pneumoniae strains, respectively. In addition, PCRnd sequence analysis revealed the presence of the blaOXA-1ene in six K. pneumoniae strains. Regarding E. coli strains,he blaCTX-M-15 gene was detected in association with thelaTEM-1b gene in one of the two E. coli strains and with thelaOXA-1 gene in both strains (Table 1).

.3. Presence and characterisation of integrons

Eight of nine K. pneumoniae strains harboured class 1ntegrons, whereas class 2 integrons were not found. Ampli-cation of the class 1 variable region was positive in six of

he K. pneumoniae strains showing sizes of ca. 800 bp and000 bp. Sequence analysis showed two gene cassette arrays:adA in five strains and dfrA5 + ereA2 in one strain. On thether hand, neither class 1 nor class 2 integrons were detectedn the E. coli strains.

.4. Genetic environment of the blaCTX-M-15 gene

PCR amplification using specific primers reported by Sal-din et al. [18] yielded an amplicon of ca. 0.8 kb in all butne K. pneumoniae strain, indicating the occurrence of thensertion sequence ISEcp1 upstream of the blaCTX-M-15 gene.rf477 was detected downstream of the blaCTX-M-15 gene inll the strains.

.5. Resistance transfer and susceptibility ofransconjugants

Cefotaxime resistance was transferred by conjugationrom three of the nine K. pneumoniae strains (Kp5305-1,p5906A and Kp5067) to E. coli J53-2, with frequen-

ies ranging from 10−4 to 10−6; however, no E. colitrains transferred the cefotaxime resistance. All the obtainedransconjugants acquired, en bloc, cefotaxime, ceftazidime,entamicin, kanamycin and tobramycin resistance. Sequencenalysis of PCR products from these transconjugants revealedhe acquisition of the blaTEM-1 and blaOXA-1 genes concomi-antly with blaCTX-M-15; however, blaSHV genes were notransferred.

.6. Plasmids content

Plasmid DNA was obtained from all the strains. All thesolates had at least two plasmids with sizes ranging from 2 kbo 70 kb (Table 1). Analysis of the transconjugants revealed

M.S.A

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32(2008)

308–314311

Table 1Phenotypic and genotypic characterisation of the nine CTX-M-15-producing Klebsiella pneumoniae strains (Kp), their transconjugants (TX) and two Escherichia coli strains (Ec) of clinical origina

Strainb Year of isolation Source MIC (mg/L) Genes encoding �-lactamases Genetic environment ofblaCTX-M-15 gene

Class 1 integrons Non-�-lactam antibioticresistance phenotype

Plasmids (kb)

CTX CAZ CTX-M SHV TEM OXA ISEcp1 orf477 intI1 Gene cassettes

Kp5249-2 2003 Urine 256 16 CTX-M-15 SHV-1 TEM-1a – + + + aadA GEN, TOB, KAN, STR, TET,CIP, SXT

70, 5, 4, 2.5

Kp5305-1 2003 Sputum 1024 128 CTX-M-15 SHV-1 TEM-1a OXA-1 + + + aadA GEN, TOB, KAN, STR, TET,CHL, CIP, SXT

70, 4, 3

Kp5906A 2004 Blood 1024 64 CTX-M-15 SHV-11 TEM-1b OXA-1 + + – – GEN, TOB, KAN 70, 6, 4, 3Kp6554A 2004 Blood 1024 512 CTX-M-15 SHV-1 TEM-1a – + + + – GEN, TOB, KAN, TET,

CHL, CIP, SXT70, 3

Kp5067 2006 Catheter 1024 512 CTX-M-15 SHV-103 TEM-1b OXA-1 + + + aadA GEN, TOB, KAN, TET,CHL, CIP, SXT

70, 4

Kp5764 2006 Sputum 1024 512 CTX-M-15 SHV-27 TEM-1b OXA-1 + + + dfrA5, ereA2 GEN, TOB, KAN, TET,CHL, CIP, SXT

70, 2

Kp6744-1 2006 Pus 1024 512 CTX-M-15 SHV-1 TEM-1a OXA-1 + + + aadA GEN, TOB, KAN, STR, TET,CHL, CIP, SXT

70, 6, 4, 3

Kp7695-1 2006 Blood 1024 256 CTX-M-15 SHV-1 TEM-1a – – + + aadA GEN, TOB, KAN, STR, TET,CIP, SXT

70, 5, 4, 3

Kp215 2007 Urine 512 256 CTX-M-15 SHV-1 TEM-1a OXA-1 + + + – GEN, TOB, KAN, TET, CIP,SXT

70, 35, 6

Ec5184 2003 Blood 1024 128 CTX-M-15 – – OXA-1 + + – – GEN, TOB, KAN, STR, CIP 70, 35Ec6615 2005 Urine 1024 32 CTX-M-15 – TEM-1b OXA-1 + + – – GEN, TOB, KAN, TET,

CHL, CIP, SXT70, 4, 2

TX-Kp5305-1 – – 1024 32 CTX-M-15 – TEM-1a OXA-1 + + NAc NA GEN, TOB, KAN 70TX-Kp5906A – – 256 32 CTX-M-15 – TEM-1b OXA-1 + + NA NA GEN, TOB, KAN 70TX-Kp5067 – – 256 32 CTX-M-15 – TEM-1b OXA-1 + + NA NA GEN, TOB, KAN 70

a CTX, cefotaxime; CAZ, ceftazidime; GEN, gentamicin; TOB, tobramycin; KAN, kanamycin; STR, streptomycin; TET, tetracycline; CHL, chloramphenicol; CIP, ciprofloxacin; SXT, trimetho-prim/sulfamethoxazole.

b Kp, Klebsiella pneumoniae; Ec, Escherichia coli; TX, transconjugant.c NA, non applicable.

312 M.S. Abbassi et al. / International Journal of Antimicrobial Agents 32 (2008) 308–314

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he presence of the largest plasmid (ca. 70 kb) harboured byach donor strain.

. Discussion

The present study represents the first molecular reportf CTX-M-producing Enterobacteriaceae strains from neu-ropenic patients in Tunisia and one of the rare studies fromncological malignancy patients [22,23]. The detected preva-ences of ESBL-producing E. coli and K. pneumoniae strains12.3% and 32.4%, respectively) are similar to previousates reported in our centre (15.5% and 37.5%, respectively)24]. According to the SENTRY surveillance programmeata (1998–2002), similar frequencies of ESBL-producing. coli and K. pneumoniae isolates were observed in Singa-ore [25], but lower frequencies were observed in Europe,ustralia, South Africa and Taiwan [25,26]. The special typef patients included in this study could account for this situ-tion.

Amongst the different groups of described ESBLs, nowa-ays the production of CTX-M enzymes is an emerginghenomenon that has been called ‘the CTX-M pandemic’4]. All our strains harboured the blaCTX-M-15 gene andost of them also contained the blaTEM-1 and blaOXA-1

enes, whose occurrence has been previously described inTX-M-15-producing E. coli and K. pneumoniae strains

7,13,27–29]. The blaCTX-M-15 gene, first described in 200130], has been found in Enterobacteriaceae in different coun-ries in the world [7,11,12,31,32]. In Tunisia, the presence ofhe blaCTX-M-15 gene was firstly described in a study of the

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harles Nicolle Hospital in the period 2000–2003 [9], andnterestingly at the end of 2003 this phenomenon emerged inur centre.

In addition, the blaSHV-1 gene was present in six of ourtudied K. pneumoniae strains, another two strains containedlaSHV-11 and blaSHV-27, respectively, and one K. pneumo-iae strain harboured a new SHV variant, named blaSHV-103.he amino acid sequences of SHV-11 (not an ESBL) andHV-27 (an ESBL) enzymes, detected in two of our K. pneu-oniae strains, differed from SHV-1 by the Gly156 → Asp

mino acid substitution in SHV-27 and Leu35 → Gln in SHV-1 (http://www.lahey.org/Studies/). The deduced amino acidequence of the novel blaSHV-103 gene is different from thosef previously described SHV-type enzymes owing to the pres-nce of the Lys250 → Arg amino acid substitution. In theHV family, ESBL activity is most frequently associated withGly238 → Ser substitution and is frequently increased by alu240 → Lys substitution; however, neither of these amino

cid changes were found in our strain.Multiresistance, a common trait in our strains, has

ften been described for ESBL- and particularly CTX-M-roducing clinical isolates [4,9,10,13,27–29]. In Enterobac-eriaceae, it has been reported that multiple antimicrobialesistance genes were located inside on highly efficientecombination and expression systems called integrons, withlasses 1 and 2 being the most frequently encountered [33].ight of the nine K. pneumoniae strains studied harboured

lass 1 integrons, which included in their variable region theadA, dfrA5 and ereA2 gene cassettes related to streptomycin,rimethoprim and erythromycin resistance, respectively. ThefrA5 + ereA2 association has been previously reported in dif-

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erent bacterial isolates such as E. coli or Vibrio [34,35] but, tour knowledge, it is the first time that this gene arrangementas been found in K. pneumoniae, although the occurrence ofreA2 alone as a gene cassette has been previously reportedn a multiresistant K. pneumoniae strain [36].

The clonal dissemination of CTX-M-15-producing Enter-bacteriaceae has been previously reported in manyeographic areas [5,9,11,12,29,31,32], whereas six of the. pneumoniae and the two E. coli strains in our studyere clonally unrelated by PFGE. This clonal diversity

lso observed in some reports [5,29] indicates either hor-zontal transfer of blaCTX-M-15 and/or mobilisation of thislement by genetic mobile elements. Detection of the ISEcp1equence upstream of the blaCTX-M-15 gene in all but onetrain suggests that ISEcp1 may have played an impor-ant role in the mobilisation of blaCTX-M-15. Indeed, thisnsertion sequence, frequently found tightly associated witheveral blaCTX-M genes [8,9,14,30,37], is able to mobiliseuch genes and provides promoter sequences enhancinglaCTX-M expression [37]. The absence of amplificationf the ISEcp1–blaCTX-M-15 region in one of our stud-ed strains might be due to the presence of a truncatedSEcp1 sequence, as was previously reported by Eckertt al. [38]. The orf477 sequence was present in all thetrains downstream of the blaCTX-M-15 gene and a similarrganisation has been reported for blaCTX-M-15 in severalnterobacteriaceae isolates in many geographical locations

8,38]. Taken together, and despite the variability of regionspstream of the blaCTX-M-15 gene, a common origin cannote excluded.

It is worth noting that all the strains harboured a plas-id of ca. 70 kb, which was transferred from three K.

neumoniae strains to E. coli J53-2 concomitantly withhe acquisition of cefotaxime, gentamicin, kanamycin andobramycin resistance. Plasmids of variable sizes harbour-ng the blaCTX-M-15 gene have been reported by manyuthors [7,9,13,27–30], therefore in our strains localisationf the blaCTX-M-15 gene on the 70 kb plasmid is quite likely.wing to a number of limitations, we could not confirm

his hypothesis by hybridisation of plasmid or chromoso-al DNA. In addition, it is noteworthy that the linkage

f CTX-M-15, TEM-1 (absent in one strain) and OXA-1found in nine strains) has been reported in Europe [13,28,29]s well as Tunisia, Central African Republic, Canada andndia [9,13,27,30], owing to the dissemination of closelyelated plasmids of variable sizes such as pC15-1a (Gen-ank AY458016) and pCTX15 [27,30]. This trait, found inur strains, also supports the hypothesis of plasmid localisa-ion.

In conclusion, CTX-M-15 is the most prevalent-lactamase detected amongst the ESBL-positive K. pneu-oniae and E. coli strains with a CTX-M phenotype in

ur hospital. The presence of the blaCTX-M-15 gene amongstnrelated strains and the unique genetic background of thelaCTX-M-15 gene (except in one strain) argued for geneticransit of mobile elements amongst unrelated strains. The

[

timicrobial Agents 32 (2008) 308–314 313

argest plasmid found in all the strains may harbour thelaCTX-M-15 gene, although this point should be investigated.

Funding: This work was supported in part by the ProjectAF2006-14207-C02 and the Tunisian Ministry of Higherducation, Scientific Research and Technology (UR99/08-4).

Competing interests: None declared.Ethical approval: Not required.

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