Thomas Graillon
email: tom.graillon@free.fr

Frederic Castinetti

Veronique Chabert-Orsini

MD Isabelle Morange

MD Thomas Cuny

PhD Frédérique Albarel

MD Thierry Brue

PhD Henry Dufour

PhD Frédéric Castinetti

PhD Véronique Chabert-Orsini

Functioning gonadotroph adenoma with severe ovarian hyperstimulation syndrome: A new emergency in pituitary adenoma surgery? Surgical considerations and literature review

Keywords: adnexal torsion, gonadotroph, ovarian hyperstimulation syndrome, pituitary adenoma, secreting adénome hypophysaire, gonadotrope, sécrétant, syndrome d'hyperstimulation ovarienne, torsion d'annexe

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Functioning gonadotroph pituitary adenomas (FGPAs) are defined according to the expression and secretion of biologically active gonadotrophins. FGPAs are rare. The first case of FGPA was reported in 1995 [START_REF] Djerassi | Gonadotroph adenoma in a premenopausal woman secreting follicle-stimulating hormone and causing ovarian hyperstimulation[END_REF]. The prevalence of FGPA is estimated to be 2.9-8.1% of all non-functioning pituitary and gonadotroph adenomas [START_REF] Caretto | Ovarian hyperstimulation syndrome due to follicle-stimulating hormone-secreting pituitary adenomas[END_REF]. FGPAs are principally symptomatic in premenopausal women and young girls. Ovarian hyperstimulation syndrome (OHSS) leads to an increase in abdominal waist circumference due to an increase in ovarian cyst volume as well as abdominal and pelvic pain, which can be either chronic or acute. In contrast, in menopausal women, ovarian insensitivity to FSH leads to FSH hypersecretion with few symptoms. In this short manuscript, we report two cases of OHSS induced by FGPA.

Methods

These two cases were selected from our database of pituitary adenomas that were surgically removed in the last 3 years. Pre-and postoperative clinical and biological data were collected.

Patients and cases description

The first case is a 36-year-old woman who underwent medically assisted procreation, conjoint infertility analysis, and ultimately, in vitro fertilization. Menses were regular without galactorrhea. Moderate increased prolactin (36 ng/ml, normal value < 20 ng/ml) led to a cerebral MRI, which revealed a 5-mm microadenoma. Cabergoline was rapidly initiated (half a tablet/week), which resulted in a normal prolactin level. An injection of a GnRH analog was performed for assisted procreation procedures (decapeptyl 3 mg IM). Two weeks after the GnRH analog injection without gonadotrophin, the patient presented with an obvious clinical and radiological OHSS. Interestingly, the FSH levels were clearly increased. The estradiol level was 22,357 pg/ml, the progesterone level was 14.86 ng/ml, and beta HCG was negative at day 15 (table 1). The following day, the estradiol level was 25,000 pg/ml, the progesterone level was 18.46 ng/ml, and the prolactin level was 6.8 ng/ml. A pelvic ultrasound revealed a large ovarian diameter of 11 cm with follicles > 30 mm. At day 24 after the GnRH analog injection, estradiol increased to 31,293 pg/ml and progesterone increased to 22.58 ng/ml. The pelvic ultrasound again showed a large ovarian diameter, which had increased to more than 15 cm. Preventive anticoagulation was introduced and cabergoline was increased to 1 tablet/week. Cerebral MRI showed a 13-mm grade II A Knosp 1 pituitary macroadenoma (Fig. 1). The volume was increased compared with that on the initial MRI. Visual examination was normal. Endoscopic transsphenoidal surgery was performed, which resulted in complete tumor removal. A neuropathologist observed anti-FSH immunostaining in 15% of the adenomatous cells, whereas chromogranin A was diffusely positive. The proliferation index was 2%, and P53 expression was absent. Postoperative levels of FSH, estradiol, and prolactin were normal (table 1). No new pituitary deficiency was observed. Mild hyponatremia was observed at day 3 and was managed with water deprivation and salt supply. Estradiol and progesterone levels were normal at 1 month. Pelvic pain was significantly decreased from the first postoperative day. Abdominal echography at postoperative day 3 showed a decrease in volume of different ovarian cysts (9 x 3 cm for the right ovary and 10 x 5 cm for the left ovary). Menses returned after 2 months. After three months, an MRI of the pituitary did not reveal any residual pituitary adenoma. After four months, abdominal ultrasound showed normal ovaries without cysts. The long-term outcome was favorable, and the in vitro fertilization procedure was performed 5 months after surgery.

The second case is a 29-year-old woman who presented with multiple ovarian cysts that were diagnosed based on abdominal and pelvic pain. A hypophysiogram revealed an increase in FSH and estradiol, a suppressed LH level, and a 2-fold increase in prolactin (table 2). An MRI of the pituitary showed a 23-mm pituitary macroadenoma (fig. 2). Treatment with cabergoline

(1/2 tablet every week) was initiated while the patient awaited surgery, which was planned for 7 days after treatment initiation. However, 3 days after the diagnosis, she was admitted for acute pelvic pain: a pelvic MRI revealed the presence of large ovarian cysts and a suspected right adnexal torsion (fig. 3). Bilateral adnexal detorsion was performed via coelioscopy, and resulted in a favorable outcome. One week later, the patient was transferred to the neurosurgery unit where she underwent endoscopic transsphenoidal surgery with complete tumor removal. The pituitary adenoma was soft, and the sella turcica was filled with abdominal fat related to peroperative CSF leak. The outcome was favorable in this case. A neuropathologist observed FSH immunostaining in 40% of adenoma cells, whereas chromogranin A was diffusely expressed. The proliferation index was 2%, and P53 expression was absent. At 3 months, no pituitary deficiency was observed, and the ovarian cysts had resolved. No residual tumor was observed on a MRI at 3 months (fig. 2). Five months later, the patient became pregnant spontaneously. The pregnancy was uneventful, and the long-term outcome was favorable.

Discussion and literature review

Clinical symptoms at diagnosis

Most FGPAs are diagnosed in premenopausal women. The most frequent clinical symptoms are menstrual irregularity (including secondary amenorrhea, oligomenorrhea, spontaneous vaginal spotting, or severe menorrhagia), infertility, galactorrhea, and OHSS, the symptoms of which include abdominal and pelvic pain, bloating, and multiple ovarian cysts. Pelvic ultrasound or MRI might be helpful for showing large multi septated cysts (> 5 cm) on both ovaries, which may have a "soap-bubble" or "wheel-spoke" appearance. OHSS is a classical complication of assisted reproductive procedures. In the first case, the OHSS was revealed and diagnosed after the injection of GnRH, which led to an exacerbation of the OHSS. OHSS induced by a GnRH agonist and the subsequent diagnosis of FGPA was previously reported [START_REF] Castelbaum | Exacerbation of ovarian hyperstimulation by leuprolide reveals a gonadotroph adenoma[END_REF][START_REF] Kihara | Ovarian hyperstimulation caused by gonadotroph cell adenoma: a case report and review of the literature[END_REF]. In 2 cases, OHSS was also diagnosed during pregnancy [START_REF] Ghayuri | Ovarian hyperstimulation syndrome caused by pituitary gonadotroph adenoma secreting follicle-stimulating hormone[END_REF][START_REF] Baba | Spontaneous ovarian hyperstimulation syndrome and pituitary adenoma: incidental pregnancy triggers a catastrophic event[END_REF]. [START_REF] Halupczok | Ovarian Hyperstimulation Caused by Gonadotroph Pituitary Adenoma--Review[END_REF]. Rare cases of OHSS with normal FSH and estradiol levels with probable low-secreting gonadotroph pituitary adenomas were also reported [START_REF] Kihara | Ovarian hyperstimulation caused by gonadotroph cell adenoma: a case report and review of the literature[END_REF][START_REF] Shimon | Ovarian hyperstimulation without elevated serum estradiol associated with pure follicle-stimulating hormone-secreting pituitary adenoma[END_REF][START_REF] Kanaya | Continuous folliclestimulating hormone exposure from pituitary adenoma causes periodic follicle recruitment and atresia, which mimics ovarian hyperstimulation syndrome[END_REF]. This biological profile is the distinguishing feature of polycystic ovary syndrome (PCOS), in which LH is usually increased relative to FSH and testosterone is increased. Finally, the size of the ovarian cysts usually remains smaller than 10 mm in PCOS vs greater than 15 mm in OHSS [START_REF] Halupczok | Ovarian Hyperstimulation Caused by Gonadotroph Pituitary Adenoma--Review[END_REF]. Hyperandrogenism is predominant in PCOS, and hyperestrogenism is predominant in

FGPA
OHSS. An LH-secreting gonadotroph pituitary adenoma was also reported [START_REF] Castelo-Branco | Ovarian hyperstimulation, hyperprolactinaemia and LH gonadotroph adenoma[END_REF]. In males, FSH elevation is associated with normal, low, or elevated LH and testosterone levels.

Decreased testosterone is related primarily to hyperprolactinemia and hypogonadism [START_REF] Cote | Functional Gonadotroph Adenomas: Case Series and Report of Literature[END_REF].

Risk of OHSS

Severe forms of OHSS could be life-threatening and may be associated with hypovolemic shock, renal failure, adult respiratory distress syndrome, and thromboembolism [START_REF] Baba | Spontaneous ovarian hyperstimulation syndrome and pituitary adenoma: incidental pregnancy triggers a catastrophic event[END_REF][START_REF] Grossman | The pathophysiology of ovarian hyperstimulation syndrome: an unrecognized compartment syndrome[END_REF][START_REF] Delvigne | Review of clinical course and treatment of ovarian hyperstimulation syndrome (OHSS)[END_REF].

Fluid shift to a third sector and ascites are also described. Adnexal torsion occurred in a case of multiple and voluminous cysts. As reported, acute abdomen related to adnexal torsion could lead to an extremely emergent situation that requires acute surgical management with coelioscopic detorsion or salpingo-oophorectomy [START_REF] Cote | Functional Gonadotroph Adenomas: Case Series and Report of Literature[END_REF]. This severe complication is unpredictable. However, multiple and voluminous cysts lead to a high short-term risk, which strongly suggests that the pituitary adenoma be removed with very little delay [START_REF] Delvigne | Review of clinical course and treatment of ovarian hyperstimulation syndrome (OHSS)[END_REF]. The data from the literature showed that adnexal torsion mainly occurs in ovarian cysts greater than 5 cm: 5 to 22 cm (9 cases) in the study by Gorkemli et al. ( 17), 9-10 cm (1 case) in the study by Gil Navarro et al. [START_REF] Navarro | Ovarian torsion and spontaneous ovarian hyperstimulation syndrome in a twin pregnancy: A case report[END_REF], and 5.7-17.5 cm (5 cases) in the study by Spitzer et al. [START_REF] Spitzer | Adnexal Torsion in Pregnancy after Assisted Reproduction -Case Study and Review of the Literature[END_REF]. The cyst diameter threshold remains difficult to establish given that the cyst diameter in OHSS induced by a gonadotroph pituitary adenoma is usually greater than 5 cm [START_REF] Halupczok | Ovarian Hyperstimulation Caused by Gonadotroph Pituitary Adenoma--Review[END_REF]. Of note, in our 2 cases, the short-term outcome of OHSS after pituitary surgery was favorable. A decrease in cyst volume was observed during the first several postoperative days, which in turn decreased the risk of adnexal torsion. The resolution of ovarian cysts and OHSS occurred mostly in the first postoperative month.

Differential diagnosis

One case of ectopic FSH secretion in a carcinoid tumor was reported [START_REF] Burgos | Ovarian hyperstimulation secondary to ectopic secretion of follicle-stimulating hormone. Literature review prompted by a case[END_REF], but the main differential diagnosis of OHSS is PCOS, as described above. However, pregnancy should always be considered.

Neuropathology

Macroadenomas are described most often, but some cases of microadenomas have also been reported. The pathological features of FGPA are similar to those of non-functioning pituitary adenomas. Usually, there is a majority of FSH-immunopositive cells, and even some cases that lack LH and FSH immunostaining have been reported. The difference in the mechanism of FSH secretion between functioning and non-functioning pituitary adenoma remains uncertain. Although our 2 FGPA cases did not show aggressive or invasive features, several cases of invasive and recurrent GPA were reported in the literature [START_REF] Roberts | Spontaneous ovarian hyperstimulation caused by a follicle-stimulating hormone-secreting pituitary adenoma[END_REF][START_REF] Benito | Gonadotroph tumor associated with multiple endocrine neoplasia type 1[END_REF][START_REF] Karapanou | Gonadotroph pituitary macroadenoma inducing ovarian hyperstimulation syndrome: successful response to octreotide therapy[END_REF][START_REF] Pentz-Vidovic | Evolution of clinical symptoms in a young woman with a recurrent gonadotroph adenoma causing ovarian hyperstimulation[END_REF].

Surgical considerations

Transsphenoidal surgery is the first-line treatment for FGPA. The outcome is particularly favorable in cases of total removal. The estradiol level usually returns to normal within the first week. Clinical symptoms of OHSS improve during the first several postoperative days.

Moreover, the cyst volume begins to decrease during the first several postoperative days. In our 2 patients, cysts were no longer visible on ultrasound 3-4 months after surgery. In these 2 cases, OHSS induced by FGPA was cured by surgery. One patient became pregnant only a few months after surgery; the pregnancy was uneventful. Cases of early spontaneous pregnancy after transsphenoidal surgery have been reported, which suggests a favorable endocrinological outcome [START_REF] Castelbaum | Exacerbation of ovarian hyperstimulation by leuprolide reveals a gonadotroph adenoma[END_REF][START_REF] Ghayuri | Ovarian hyperstimulation syndrome caused by pituitary gonadotroph adenoma secreting follicle-stimulating hormone[END_REF][START_REF] Sicilia | Multiple ovarian cysts and oligomenorrhea as the initial manifestations of a gonadotropin-secreting pituitary macroadenoma[END_REF][START_REF] Murakami | Management of ovarian hyperstimulation due to follicle-stimulating hormone-secreting gonadotroph adenoma[END_REF][START_REF] Mor | Diagnosis of pituitary gonadotroph adenomas in reproductive-aged women[END_REF][START_REF] Sugita | Successful pregnancy and delivery after removal of gonadotrope adenoma secreting follicle-stimulating hormone in a 29-yearold amenorrheic woman[END_REF]. No postoperative pituitary deficiency was observed in our 2 cases, and only transient and minor hyponatremia was seen. The operative features seem similar to those of non-functioning gonadotroph adenomas. In cases of cavernous sinus invasion, incomplete tumor removal might require complementary treatment.

Radiotherapy and radiosurgery are efficient and are therefore recommended [START_REF] Djerassi | Gonadotroph adenoma in a premenopausal woman secreting follicle-stimulating hormone and causing ovarian hyperstimulation[END_REF][START_REF] Roberts | Spontaneous ovarian hyperstimulation caused by a follicle-stimulating hormone-secreting pituitary adenoma[END_REF][START_REF] Benito | Gonadotroph tumor associated with multiple endocrine neoplasia type 1[END_REF][START_REF] Karapanou | Gonadotroph pituitary macroadenoma inducing ovarian hyperstimulation syndrome: successful response to octreotide therapy[END_REF][START_REF] Rongieres-Bertrand | A spontaneous and severe hyperstimulation of the ovaries revealing a gonadotroph adenoma[END_REF][START_REF] Tashiro | A follicle-stimulating hormone-secreting gonadotroph adenoma with ovarian enlargement in a 10-year-old girl[END_REF][START_REF] Chaidarun | Gonadotropinomas[END_REF]. As previously mentioned, our 2 cases did not exhibit any aggressive or invasive features.

Nevertheless, cases of aggressive and recurrent FGPA, which require iterative surgery and radiotherapy/radiosurgery, are reported in the literature [START_REF] Roberts | Spontaneous ovarian hyperstimulation caused by a follicle-stimulating hormone-secreting pituitary adenoma[END_REF][START_REF] Benito | Gonadotroph tumor associated with multiple endocrine neoplasia type 1[END_REF][START_REF] Karapanou | Gonadotroph pituitary macroadenoma inducing ovarian hyperstimulation syndrome: successful response to octreotide therapy[END_REF][START_REF] Pentz-Vidovic | Evolution of clinical symptoms in a young woman with a recurrent gonadotroph adenoma causing ovarian hyperstimulation[END_REF].

Timing of surgery

As reported previously, OHSS could lead to life-threatening situations. The lack of effective medical therapy suggests pituitary surgery as a first-line therapy. In cases of severe OHSS or voluminous ovarian cysts greater than 5 cm, the authors suggest not delayed pituitary surgery to avoid complications such as adnexal torsion. In the postoperative period, the decrease in the plasma estradiol concentration considerably decreases the risk of adnexal torsion within the first several postoperative days. Therefore, in cases of severe OHSS, short-term pituitary surgery should be preferred to transient dopamine agonist therapy and delayed pituitary surgery.

Medical management

No medical therapy is currently proven efficient for FGPA. D2DR expression was demonstrated in FSH-secreting FGPA, but the expression pattern of dopamine receptors remains uncertain [START_REF] Murata | Successful pregnancy after bromocriptine therapy in an anovulatory woman complicated with ovarian hyperstimulation caused by follicle-stimulating hormone-producing plurihormonal pituitary microadenoma[END_REF]. Dopamine agonists seem to be the most pertinent drugs for this indication. Treatment with DAs led to decreased FSH and estradiol levels, and in some cases, allowed pregnancy [START_REF] Murata | Successful pregnancy after bromocriptine therapy in an anovulatory woman complicated with ovarian hyperstimulation caused by follicle-stimulating hormone-producing plurihormonal pituitary microadenoma[END_REF][START_REF] Paoletti | Effectiveness of cabergoline in reducing follicle-stimulating hormone and prolactin hypersecretion from pituitary macroadenoma in an infertile woman[END_REF][START_REF] Knoepfelmacher | Effectiveness of treating ovarian hyperstimulation syndrome with cabergoline in two patients with gonadotropin-producing pituitary adenomas[END_REF][START_REF] Gryngarten | Spontaneous ovarian hyperstimulation syndrome caused by a follicle-stimulating hormone-secreting pituitary macroadenoma in an early pubertal girl[END_REF]. However, the effect is mostly transient and moderate and is

clearly not a substitute for surgical treatment [START_REF] Ambrosi | Precocious puberty in a boy with a PRL-, LH-and FSH-secreting pituitary tumour: hormonal and immunocytochemical studies[END_REF][START_REF] Clemente | Macroorchidism and panhypopituitarism: two different forms of presentation of FSH-secreting pituitary adenomas in adolescence[END_REF]. In our second case, DAs failed to control OHSS and the prevention of adnexal torsion.

GnRH agonists were demonstrated to provide a short-term increase in FSH/LH secretion and therefore an increase in estradiol secretion, and in one case, an increase in tumor volume [START_REF] Knoepfelmacher | Effectiveness of treating ovarian hyperstimulation syndrome with cabergoline in two patients with gonadotropin-producing pituitary adenomas[END_REF].

Moreover, GnRH agonists pose a risk of apoplexy [START_REF] Chanson | Pituitary apoplexy caused by GnRH-agonist treatment revealing gonadotroph adenoma[END_REF].

Although SST2-3 and SST2-3-5 expression was demonstrated in an FSH-secreting FGPA and an LH/PRL-secreting pituitary adenoma, respectively, the expression pattern of somatostatin receptors remains uncertain [START_REF] Ceccato | Gonadotropin secreting pituitary adenoma associated with erythrocytosis: case report and literature review[END_REF][START_REF] Saveanu | A luteinizing hormone-, alpha-subunit-and prolactin-secreting pituitary adenoma responsive to somatostatin analogs: in vivo and in vitro studies[END_REF]. Octreotide injection was demonstrated efficient in one case [START_REF] Karapanou | Gonadotroph pituitary macroadenoma inducing ovarian hyperstimulation syndrome: successful response to octreotide therapy[END_REF]. In another case, treatment with octreotide combined with a DA over 7 months decreased the tumor volume [START_REF] Ceccato | Gonadotropin secreting pituitary adenoma associated with erythrocytosis: case report and literature review[END_REF]. An interest in somatostatin agonists remains.

Patient outcome

In our 2 cases, total surgical removal without hormonal deficiency led to an excellent outcome: one patient was pregnant 5 months later, while the other patient was able to undergo an in vitro fertilization procedure 5 months after surgery. The data from the literature demonstrated a favorable endocrine and tumoral outcome. Some cases of aggressive and recurrent FGPA might lead to complex therapeutic situations, but those cases are rare [START_REF] Djerassi | Gonadotroph adenoma in a premenopausal woman secreting follicle-stimulating hormone and causing ovarian hyperstimulation[END_REF][START_REF] Benito | Gonadotroph tumor associated with multiple endocrine neoplasia type 1[END_REF][START_REF] Karapanou | Gonadotroph pituitary macroadenoma inducing ovarian hyperstimulation syndrome: successful response to octreotide therapy[END_REF][START_REF] Pentz-Vidovic | Evolution of clinical symptoms in a young woman with a recurrent gonadotroph adenoma causing ovarian hyperstimulation[END_REF]. Therefore, long-term follow-up with biological and imaging surveillance is required.

Conclusion

The authors reported two cases of OHSS related to FSH-secreting FGPA. OHSS was spontaneous in one case and was revealed by GnRH injection in the other case. OHSS was complicated in one case by a bilateral adnexal torsion. Dopamine agonists were non-efficient in the two cases. Surgical removal of pituitary adenoma remains the preferred treatment and should not be delayed in cases of severe OHSS in order to prevent the occurrence of OHSSrelated complications. Adnexal volume was increased (6.5 x 4.2 x 6.6 cm on the left side and 15 x 10 x 6 cm on the right side) with multiple cysts. The right adnexal pedicle presented a turn of spire, and the right adnexal parenchyma was edematous and was not enhanced by gadolinium, which strongly suggests right adnexal torsion. Peritoneal effusion was also observed. Peroperative observation concluded to bilateral adnexal torsion.

Second cas: IRM pelvienne préopératoire (séquences coronale (A-B) et axiale T2 (C)).

Les ovaires sont volumineux (6.5 x 4.2 x 6.6 cm du côté gauche et 15 x 10 x 6 cm du côté droit) et présentent de multiples kystes. Le pédicule ovarien droit présente un tour de spire avec un parenchyme ovarien oedémateux et non rehaussé par le gadolinium, suggérant fortement une torsion de l'annexe droite. Une suffusion péritonéale est également observée. En peropératoire, il a été constaté une torsion d'ovaires bilatérale.

Table Legends

Table 1: Pre and postoperative results of endocrine tests for patient presented in the first case.

Bilan endocrinien pré et post-opératoire du premier patient présenté.

Table 2: Pre and postoperative results of endocrine tests for patient presented in the second case.

Bilan endocrinien pré et post-opératoire du second patient présenté.

Figure 1 :

 1 Figure 1: First case: Preoperative cerebral MRI with coronal T2, T1 gadolinium, and sagittal T1 gadolinium sequences displayed a pituitary macroadenoma. Premier cas: IRM cérébrale preopératoire avec séquence T2 et T1 avec injection de gadolinium coronale et T1 sagittale mettant en évidence un macroadénome hypophysaire.

Figure 2 :

 2 Figure 2: Second case: Pre and postoperative cerebral MRI Coronal and sagittal T2 MRI (A, B) revealed a pituitary macroadenoma. Three-month postoperative MRI with sagittal T2 (C), coronal T1 gadolinium (D) and T1 Fat-Sat (E) sequences showed pituitary adenoma removal and intrasellar filling with abdominal fat. Second cas: IRM cérébrale pré et post-opératoire IRM T2 coronale et sagittale (A, B) mettant en évidence un macroadénome hypophysaire. IRM hypophysaire à 3 mois avec séquences T2 sagittale (C), T1 gadolinium coronale (D) et T1 fat-sat (E) mettant en évidence l'exérèse de l'adénome hypophysaire ainsi que la présence de graisse intrasellaire.

Figure 3 :

 3 Figure 3: Second case: Preoperative pelvic MRI (coronal (A-B) and axial (C) T2 sequences).

  has been rarely reported in post-menopausal women. After approximately 45 years of The variable increase in prolactin is due to pituitary stalk deconnection, hyperandrogenism and an increase in estradiol. In the literature review by Hallupzock et al., FSH was normal in 17/31 cases and was increased in 14/31 cases; LH was decreased in 27/32 patients, estradiol was elevated in 29/32 cases, and prolactin was increased in 24/27 patients

age, high FSH levels usually do not modify ovarian function and estrogen secretion. As in a non-functioning PA, mass effect and visual disturbance are the most frequent revealing symptoms.

Only rare cases have been reported in children (precocious puberty) and males (testicular enlargement or tumoral syndrome).

Biology at diagnosis

A high estrogen level and a high or paradoxical non-decreased FSH level are classically observed. The FSH level is usually increased whereas the LH level is low; this is related to negative feedback and antehypophyseal deficiency even in cases of LH-positive immunoreactivity

[START_REF] Djerassi | Gonadotroph adenoma in a premenopausal woman secreting follicle-stimulating hormone and causing ovarian hyperstimulation[END_REF][START_REF] Ghayuri | Ovarian hyperstimulation syndrome caused by pituitary gonadotroph adenoma secreting follicle-stimulating hormone[END_REF][START_REF] Sicilia | Multiple ovarian cysts and oligomenorrhea as the initial manifestations of a gonadotropin-secreting pituitary macroadenoma[END_REF][START_REF] Roberts | Spontaneous ovarian hyperstimulation caused by a follicle-stimulating hormone-secreting pituitary adenoma[END_REF][START_REF] Shimon | Ovarian hyperstimulation without elevated serum estradiol associated with pure follicle-stimulating hormone-secreting pituitary adenoma[END_REF][START_REF] Murakami | Management of ovarian hyperstimulation due to follicle-stimulating hormone-secreting gonadotroph adenoma[END_REF]

.

Disclosure

The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper


Dr Frédérique Albarel
email: frederique.albarel@ap-hm.fr

Dr Fatine Elaraki

Pr Brigitte Delemer
email: bdelemer@chu-reims.fr

Daily life, needs and expectations of patients with acromegaly in France: an on-line survey Vie quotidienne, besoins et attentes des patients atteints d'acromégalie en France : le point de vue des « e-patients »

Keywords: acromegaly, daily life, impact, e-survey, needs, expectations création de groupes de parole. Cette acromégalie, vie quotidienne, retentissement, enquête internet, besoins, attentes

Acromegaly can impair quality of life, but impact on patients' daily life, needs and expectations been little explored. Objectives: To better understand the impact of acromegaly on patients' daily life, and evaluate their needs and expectations. Patients and methods: An on-line questionnaire survey of acromegaly patient and relative community members was conducted on the Carenity website. Results: 25 patients and 3 relatives, with a mean age of 48.9 years, responded. Diagnosis of acromegaly was recent (60% within 10 years). Signs at diagnosis were mainly clinical (fatigue, headache) and physical changes (acral enlargement). Reported complications comprised morphological changes (93%), bone and joint symptoms (75%) and metabolic disorders (75%). Pain and fatigue impacted the daily life of 61% and 54% patients, respectively. Acromegaly had strong impact on mood (79%), leisure (75%) and social life (71%). Patients mostly got information from specialized websites (71%), their endocrinologist (61%) and patient associations (54%). The information sought was patient testimony (82%), practical advice (64%), and information on clinical trials (61%) and treatments (50%). Patients wished to have patient-physician discussion groups (25%), and better knowledge of acromegaly on the part of those they were in contact with (25%). Conclusion: Acromegaly has a major impact on patients' daily life and mood. Patients wished their disease to be better known, and advocated setting up discussion groups. This study should improve acromegaly education programs and help adapt the services and information provided.

Résumé

L'acromégalie peut entraîner une altération de la qualité de vie. Ses conséquences sur la vie quotidienne des patients, leurs besoins et attentes sont peu connus. Objectifs : Il s'agit de mieux comprendre l'impact de l'acromégalie sur la vie quotidienne et évaluer les besoins ainsi que les attentes des patients. Patients et méthodes : Des patients acromégales et leurs proches, membres des associations de patients, ont répondu à un questionnaire sur internet sur la plateforme Carenity. Résultats : 25 patients et 3 proches, 48,9 ans d'âge moyen, ont répondu au questionnaire. Le diagnostic d'acromégalie était récent (60 % < 10 ans avant). Les premiers signes au diagnostic étaient majoritairement des symptômes cliniques (fatigue, céphalées) et des modifications physiques (infiltrations des extrémités). Les complications rapportées étaient des modifications morphologiques (93 %), des symptômes osseux/articulaires (75 %) et des anomalies métaboliques (75 %). La douleur (61 %) et la fatigue (54 %) retentissaient sur la vie quotidienne. L'acromégalie avait un impact important sur le moral (79 %), les loisirs (75 %) et la vie sociale (71 %). Les principales sources d'information étaient les sites internet spécialisés (71 %), l'endocrinologue (61 %) et les associations de patients (54 %), avec des recherches sur des témoignages de patients acromégales (82 %), des conseils pratiques (64 %), des informations sur les essais cliniques (61 %) et les traitements (50 %). Ils souhaitaient des groupes de parole patients/médecins (25 %) et une meilleure connaissance de l'acromégalie autour d'eux (25 %). Conclusion : L'acromégalie a un impact important sur la vie et le moral au quotidien des patients. Ces derniers voudraient que l'on connaisse mieux leur maladie et la

INTRODUCTION

Acromegaly is a chronic acquired disorder characterized by excessive secretion of growth hormone (GH) and increased insulin-like growth factor 1 (IGF-1) levels, caused in most cases by a benign pituitary adenoma. Nowadays, its prevalence may vary between 28 to 137 per million people, while its annual incidence ranges between 2 and 11 cases per million inhabitants [START_REF] Lavrentaki | Epidemiology of acromegaly: review of population studies[END_REF] .

Acromegaly is characterized by the development of a dysmorphic syndrome with progressive acral and cranial enlargement 2 , accompanied by a number of comorbidities as hypertension, glucose intolerance and diabetes mellitus, sleep apnea and respiratory diseases, and heart failure. Chronic rheumatologic disorders, with joint deformations associated with pain and impotence [START_REF] Hoskuldsdottir | The incidence and prevalence of acromegaly, a nationwide study from 1955 through 2013[END_REF] are also frequent and usually persist even after control of the disease.

Acromegaly can have physical, psychological and social manifestations, which may impact quality of life (QoL), especially in patients with active disease [START_REF] Crespo | Update on quality of life in patients with acromegaly[END_REF] . The great majority of patients reported musculoskeletal and joint pain ; depressive and anxiety symptoms are commonly observed in those who have been diagnosed with an extended delay 5 .

To better understand the impact of acromegaly on patients' daily life and evaluate the needs and expectations of patients in terms of services and information, a survey was carried out online in patients or relatives registered in a social network of individuals with acromegaly.

PATIENTS AND METHODS

Design of the study

The survey was carried out by Else Care company on the Internet platform Carenity (https://www.carenity.com/), the French social network of patients suffering from chronic diseases from March 2, 2016 to April 24, 2016.

An online questionnaire was sent to the members of acromegaly communities on Carenity website. Carenity patients' community was solicited to participate in the survey by means of invitations and reminders sent by email. The members of the French patients' association "Acromégales, Pas Seulement…" [APS; Patients with Acromegaly, but not only] were also recruited through the Facebook page and invitations and reminders sent by email.

Participants

All patients with acromegaly members of Carenity community and living in France (including overseas territories), as well as their relatives, could be included in the study. Relatives were to answer in place of the patient with acromegaly.

Questionnaire

The online questionnaire was designed by Carenity and validated by Dr F. Albarel and the association APS as an expert of the disease. The questionnaire comprised 4 parts: main stages and circumstances of acromegaly diagnosis, management of treatment and acromegaly impact on DL, search of information on acromegaly, and needs and expectations of acromegalic patients (Questionnaire available as supplementary information).

RESULTS

Demographics and clinical characteristics of respondents

Of 47 patients recruited by email, 36 responded to the questionnaire, 28 responses to the questionnaire were valid.

Demographics and clinical characteristics of respondents are shown in Table 1. Most respondents were female (79%) and 89% were patients, while 11% were relatives responding for the patient. Patients were aged 48.9 years on average and more than half was 41 to 60 years old (57%). Patients reported having been diagnosed recently (≤1 year earlier) or quite recently (2-5 years earlier) for 14% and 32% of respondents, respectively, while 18% of patients were diagnosed over 20 years earlier. To the question "which kind of medication for acromegaly do you have?" 25% answered having no treatment, while the others declared taking one to 3 medications.

First signs of acromegaly and reported complications

Respondents reported in average 3 warning signs that led to diagnosis. Physical changes (n=30 responses), with frequent foot and hand (12 et 11 respectively) deformation were reported (Figure 1). Patients also noticed clinical symptoms (n=41 responses), mainly fatigue (n=7 patients), headache (n=6) but also increased sweating and weight gain (5 patients each).

Pain was also reported as a warning symptom (n=8 responses). Furthermore, respondents declared undergoing a mean of 8 symptoms they considered as "complications" related to acromegaly. In 32% of cases, they reported more than 10 symptoms, among which morphological changes, bone and joint symptoms and metabolic disorders were the most frequently reported (93%, 75% and 75%, respectively) (Figure 2).

Only one patient (4%) had no "complication".

Impact of acromegaly and its treatment on patients' daily life

Of the 21 respondents who declared being on treatment at the time of the survey, 19 (90%) had their treatment administered at home, and 2 (10%) at the nurse's office. Besides helping with the injection of the treatment, the nurse played also a role in the management of acromegaly by providing psychological support for 28% of patients (n=5/18), giving information on the prescribed treatment (n=3/18) or providing care for acromegaly-related complications (n=3/18).

To get greater autonomy in the management of their treatment, patients most often expect the availability of a new oral treatment (n=6/19), more information on their disease (n=2/19) and more attentive listening from their physician (n=2/19). However, a third of them (n=6/19) report that they do not miss anything to be more autonomous.

Regarding the main acromegaly-related difficulties cited by the patients as impacting their daily life (Figure 3), pain and fatigue were the most commonly reported (n=17/28 and n=15/28, respectively). Other difficulties impacting quality of life were reported (Table 2), such as reduced mobility (n=5/28) and impact on professional life (n=4/18). Difficulties concerning management (burden of treatment, surgery…), or of psychological nature were less frequently cited. Regarding the impact of acromegaly-related difficulties on daily life, it was considered quite important to very important on their daily mood for 79% of patients, on their hobbies (75%) and on their social life (71%). The impact of the disease on their professional and sexual life was also quite to very important in 68% and 67% of cases, respectively.

Seeking information on acromegaly

The most common source of information on their disease for patients with acromegaly was the Internet with websites specialized in orphan diseases and acromegaly (71%), but also the endocrinologist at the hospital (61%) and patients' associations (54%), while they solicit their general practitioner in only 21% of cases (Figure 4). Patients get information through several ways (mean: 3.6 sources) and in about half the cases (46%), they have 4 or more sources. The vast majority (82%) seeks testimonies from patients with acromegaly, and practical advices for a better daily life (64%), information on ongoing clinical trials (61%), treatments (50%), acromegaly-related complications (36%) and types of social assistances available (36%).

Patients were also looking for information on specialists or reference centers (29%), administrative problems related to acromegaly in general (29%), information on acromegaly in general (18%), or practical advices for medical follow-up (14%).

Among the most expected services and information, patients cited the implementation of discussion groups including patients and physicians (n=7/28), a better knowledge of their disease (from the lay audience and health professionals in particular) (n=7/28), information tools on acromegaly (n=6/28), and a multidisciplinary management of their disease (n=4/28).

DISCUSSION

This survey carried out in a population of patients with acromegaly (n=28; 3 relatives) shows that this rare disease has an important impact on patients' daily life. Furthermore, although they already have several sources of information the patients express their need to be better informed, to have dedicated services, and their wish to share about their disease in discussion groups with patients and physicians.

In this survey, patients reported having experienced a mean of 3 warning signs suggestive of acromegaly, which vary depending on the patients. The most striking signs are non-specific clinical symptoms such as headache and fatigue, but also excessive sweating and weight gain, which are also observed along with a large range of symptoms in other studies [START_REF] Fernandez | Prevalence of pituitary adenomas: a communitybased, cross-sectional study in Banbury (Oxfordshire, UK)[END_REF][START_REF] Hoskuldsdottir | The incidence and prevalence of acromegaly, a nationwide study from 1955 through 2013[END_REF][START_REF] Abreu | Challenges in the diagnosis and management of acromegaly: a focus on comorbidities[END_REF] .

Presence of concomitant physical changes, especially acral enlargement and facial changes, as reported in many series [START_REF] Fernandez | Prevalence of pituitary adenomas: a communitybased, cross-sectional study in Banbury (Oxfordshire, UK)[END_REF][START_REF] Hoskuldsdottir | The incidence and prevalence of acromegaly, a nationwide study from 1955 through 2013[END_REF][START_REF] Abreu | Challenges in the diagnosis and management of acromegaly: a focus on comorbidities[END_REF] , was very frequently noticed by the patient themselves in our study. In contrast, joint and musculoskeletal pain are less common features noticed at diagnosis, in accordance with previous studies [START_REF] Fernandez | Prevalence of pituitary adenomas: a communitybased, cross-sectional study in Banbury (Oxfordshire, UK)[END_REF][START_REF] Daly | High prevalence of pituitary adenomas: a crosssectional study in the province of Liege, Belgium[END_REF] , although arthralgia was reported in 50% of patients (n=26/52) at diagnosis in another study [START_REF] Hoskuldsdottir | The incidence and prevalence of acromegaly, a nationwide study from 1955 through 2013[END_REF] . Symptoms considered by patients as "complications" of acromegaly are multiple (8 in average per patient) and very frequent, especially morphological changes which are experienced by almost all patients (93%), but also bone and joint symptoms and metabolic disorders (75% of cases), which is in line with literature data [START_REF] Fernandez | Prevalence of pituitary adenomas: a communitybased, cross-sectional study in Banbury (Oxfordshire, UK)[END_REF][START_REF] Hoskuldsdottir | The incidence and prevalence of acromegaly, a nationwide study from 1955 through 2013[END_REF][START_REF] Abreu | Challenges in the diagnosis and management of acromegaly: a focus on comorbidities[END_REF] .

Results of our survey also show that acromegaly have a notable impact on patients' daily life, especially pain and fatigue which are by far the most often cited as being a daily burden (60% and 54% of patients). Joint and musculoskeletal pain is described in up to 90% of acromegalic patients [START_REF] Miller | Impact of musculoskeletal disease on quality of life in long-standing acromegaly[END_REF] and this condition, which may persist even after long-term remission of the disease 9, 10 , has been found to impair patients' QoL in several studies [START_REF] Crespo | Update on quality of life in patients with acromegaly[END_REF][START_REF] Miller | Impact of musculoskeletal disease on quality of life in long-standing acromegaly[END_REF][START_REF] Biermasz | Morbidity after longterm remission for acromegaly: persisting joint-related complaints cause reduced quality of life[END_REF][START_REF] Wassenaar | Clinical osteoarthritis predicts physical and psychological QoL in acromegaly patients[END_REF] . In our survey, acromegaly could also have a negative impact on QoL, as it restricts patients' access to hobbies and impact on social life of most patients. High pain scores of the spine, knee, and hip have been reported in long-term cured acromegaly patients which limit physical functioning and psychological well-being [START_REF] Miller | Impact of musculoskeletal disease on quality of life in long-standing acromegaly[END_REF][START_REF] Wassenaar | Clinical osteoarthritis predicts physical and psychological QoL in acromegaly patients[END_REF] . This effect on psychological well-being was also observed in our survey, as 79% of the respondents estimated that acromegaly, besides impairing their professional, personal, sexual and social life, has an important to very important impact on their daily mood. Some patients also reported that their body image is impaired, some have difficulties to face scrutinizing looks and feel lack of understanding from their relatives, others experience depression. According to the literature, psychological status, and especially body image impairment, is one of the most relevant factors affecting QoL 4 , even after long-term biochemical remission of acromegaly [START_REF] Roerink | Persistent self-consciousness about facial appearance, measured with the Derriford appearance scale 59, in patients after longterm biochemical remission of acromegaly[END_REF] .

Management of treatment that needs to be injected could also be a burden for the patient. In most cases, in this survey, the patient has his treatment administered at home by a nurse.

According to some patients on treatment (n=6/19), the best way to improve their autonomy in treatment management and what they expect would be a new oral treatment to be marketed.

Patients also expect more information on acromegaly and listening from health professionals.

Although they have become experts in their own disease by getting information mostly on specialized websites and from their endocrinologist, acromegalic patients expect their disease to be better known from the lay audience and health professionals. But what they mostly await is getting information on existing and developing treatments or ongoing clinical trials, and practical advices and tools to improve their daily life. Due to their daily relationship with treated patients, nurses should have a key role to play by providing such information, but in fact, their major role in acromegaly management is the psychological support they give to the patient. Most patients (80%) seek testimonies from their "peers" who could help them to better understand their own problems and to feel less isolated with their rare disease. They also often search for information about their disease in patients' associations, forums, blogs and patients' networks. Hence, services patients mostly expect to better manage their disease daily is the implementation of discussion and meeting groups including patients and physicians, probably to get better understanding of their condition, psychological support, practical advices, information tools and accurate information on acromegaly and treatments that might finally improve their QoL.

This study has several limits, first because of its design. Sometimes, questionnaire has been sent through an association of patients with acromegaly and therefore 75% of patients knew its existence, which implies a recruiting bias. Indeed, patient with the worse quality of life is more prone to look for solution and contact patient's association or website, so the result of this e-survey does represent the opinion of a subgroup of "connected" patient. Furthermore, as it is a rare disease, the sample size of the study is small; it is thus difficult to generalize these results.

However, the main value of this study is that this is the first time acromegalic patients talk about their disease through such a "e-survey". This study therefore provides the direct reflection of how the patients really live with acromegaly, the impact it has on their daily life and the burden of the disease on their mood, their social, personal and professional life. These results will be a very useful basis to further implement patient education programs, and to propose new services and information adapted to the needs of patients with acromegaly. Indeed, several pituitary education programs have been developed in France for the last few years, and this study, considering specifically acromegalic patients' needs will allow to improve and complete their contents.

In conclusion, here is the first "e-survey" evaluating daily life, needs and expectations of acromegalic patients. Symptoms and complications of acromegaly seem to have a major impact on patients' daily life, on their mood and their personal and professional activities. The main expectations of acromegalic patients are a better knowledge and better recognition of their disease, more information on current and developing treatments for acromegaly, practical advices and information tools, and finally, the possibility to talk about their disease with their peers and physicians within discussion and meeting groups. Probably evolution o existing education programs based on these data could partly meet their needs. Plusieurs réponses possibles, nombre total de réponse : 100

a N= 36 responses, as some patients could take more than 1 treatment

Figure 1 .

 1 Figure 1. Warning signs leading to diagnosis (N=28 respondents. Several possible answers, total number of responses = 79). Results are expressed in number of patients having cited the sign.

Figure 1 .

 1 Figure 1. Signes d'alerte menant au diagnostic (N=28 répondeurs. Plusieurs réponses

Figure 2 .

 2 Figure 2. Rate of acromegaly-related manifestations reported as "complications" by the respondents at the time of the survey (N=28). Several possible answers, total number of responses: n=223.

Figure 2 .Figure 3 .

 23 Figure 2. Taux de manifestation en lien avec l'acromégalie rapportée comme des

Figure 3 .Figure 4 :

 34 Figure 3. Retentissement de l'acromégalie sur la vie quotidienne des patients:

Figure 4 :

 4 Figure 4: Sources d'informations pour les patients acromégales (N=28 répondeurs).

Figure 1 .

 1 Figure 1. Warning signs leading to diagnosis (N=28 respondents. Several possible answers,

Figure 3 .

 3 Figure 3. Impact of acromegaly on patients' daily life: main experienced difficulties.

Figure 4 :

 4 Figure 4: Sources of information for patients with acromegaly (N=28 respondents).

Table 1 . Demographics and clinical characteristics of respondents (N=28) Tableau 1. Caractéristiques démographiques et cliniques des répondeurs (N=28)Table 2 . Impact of acromegaly on patients' daily life -Other difficulties

 12

	(N=28

Retentissement de l'acromégalie sur la vie quotidienne des patients -Autres difficultés

  (N=28 répondeurs, plusieurs réponses possibles, nombre total de réponses: 36)

Acknowledgments:

We thank Marielle ROMET (Santé Active Edition, France) for providing medical writing assistance. This assistance was funded by IPSEN, France. We thank the patient association "Acromégales, Pas Seulement" for their support, their reading and approval of this study.

Disclosure of interest: F.A. is a member of advisory boards, and has received conference fees from: IPSEN, Novartis and Pfizer. B.D. is a member of advisory boards, and has received conference fees from: IPSEN, Novartis and Pfizer. F.E. is an employee of IPSEN pharma.

Figure 2. Rate of acromegaly-related manifestations reported as "complications" by the respondents at the of the survey (N=28


Laurence Mathieu
email: laurence.mathieu@univ-lorraine.fr

Anaïs Keraval

Nico F Declercq

Jean-Claude Block

Laurence Orcid

Mathieu

Nico F Orcid

Declercq

Assessment of a low-frequency ultrasound device on prevention of biofilm formation and carbonate deposition in drinking water systems

Keywords: drinking water biofilm, low-frequency ultrasound, carbonate deposition, heat exchangers, water pipes

Highlights

-Low-frequency ultrasound was studied on drinking water biofilms and CaCO 3 deposits -Ultrasounds affect both biofilm and planktonic cell number and activity -US treatment did not prevent initial bacterial adhesion and CaCO 3 deposition -Low-frequency ultrasounds markedly limit biofilm formation and scaling

Introduction

Bio-fouling of any materials in contact with water results in a range of adverse issues for industrial engineered water systems such as decrease in water transport efficiency, heat exchange limitation, corrosion, increased maintenance costs, and environmental impact (as traditional cleaning strategies sometimes involve the use of environmentally unfriendly chemicals). Alternative procedures, i.e. less drastic, eco-aware, with a low water footprint and respectful of material integrity are required, particularly to reduce their impact on the environment.

In this context, ultrasonic treatments represent a very promising alternative. Indeed, ultrasonic waves have been used in many environmental, industrial and medical sectors: algal bloom control, food and beverage processing, sonochemistry, nanotechnology, mineral processing, welding, surface cleaning, medical scanning and non-destructive testing [START_REF] Mason | Sonochemistry and the environment -providing a "green" link between chemistry[END_REF][START_REF] Rokhina | Low-frequency ultrasound in biotechnology: state of the art[END_REF][START_REF] Chemat | Application of ultrasound in food technology: Processing, preservation and extraction[END_REF][START_REF] Johansson | Cavitation and non-cavitation regime for large-scale ultrasonic standing wave particle separation systems -In situ gentle cavitation threshold determination and free radical related oxidation[END_REF][START_REF] Park | Recent advances in ultrasonic treatment: challenges and field applications for controlling harmful algal blooms (HABs)[END_REF]. Likewise, ultrasonic waves (US) are proved to have 'synergistic curative' effects on deposits when combined with other disinfection/cleaning processes: eg US+UV [START_REF] Zhou | Experimental study on the disinfection efficiencies of a continuous-flow ultrasound/ultraviolet baffled reactor[END_REF], US+enzymes [START_REF] Oulahal-Lagsir | Escherichia coli-milk" biofilm removal from stainless steel surfaces: synergism between ultrasonic waves and enzymes[END_REF], US+microbubbles [START_REF] Agarwal | Removal of biofilms by intermittent low-intensity ultrasonication triggered bursting of microbubbles[END_REF], US+turbulence [START_REF] Lamminen | Mechanisms and factors influencing the ultrasonic cleaning of particle-fouled ceramic membranes[END_REF], US+antibiotics [START_REF] Pitt | Ultrasonic enhancement of antibiotic action on gram-negative bacteria[END_REF][START_REF] Peterson | The effect of frequency and power density on the ultrasonicallyenhanced killing of biofilm-sequestered Escherichia coli[END_REF][START_REF] Carmen | Ultrasonically enhanced vancomycin activity against Staphylococcus epidermidis biofilms in vivo[END_REF][START_REF] Kopel | Surface acoustic waves increase the susceptibility of Pseudomonas aeruginosa biofilms to antibiotic treatment[END_REF], US+bleach [START_REF] Duckhouse | The effect of sonication on microbial disinfection using hypochlorite[END_REF], US+ozone [START_REF] Zhao | Impacts of ultrasound and ozone disinfection of WWTPs secondary effluent[END_REF].

More recently, ultrasonic waves have been successfully applied to prevent marine macrofouling (caused by the attachment of large organisms such as barnacles, seaweed, mussels, and diatoms) on vessel hulls with devices emitting mechanical waves in the ultrasonic (>20 KHz) frequency range [START_REF] Guo | Effect of ultrasound on cyprid footprint and juvenile barnacle adhesion on a fouling release material[END_REF][START_REF] Legg | Acoustic methods for biofouling control: a review[END_REF][START_REF] Habibi | An acoustic antifouling study in sea environment for ship hulls using ultrasonic guided waves[END_REF]. Moreover a few successful experimental studies have been reported on inhibition of fouling in heat exchangers and pipes by application of a few short pulses of high power ultrasonic waves: pulses of 0.2 s with 100 s inter-pulse intervals per day [START_REF] Taylor | Ultrasonics as an alternative to biocides for controlling the growth of the colonial hybroid Garveia franciscana[END_REF], or pulses of 3 × 30 s per day [START_REF] Bott | Biofouling control with ultrasound[END_REF]. Mechanisms explaining such an effect are generally not reported but they appear to be frequency and power dependent, and they could be related to both mechanical effects and local cavitation phenomena [START_REF] Erriu | Microbial modulation by ultrasound: Current concepts and controversies[END_REF]. Additionally, experimental systems used for generating ultrasound are very diverse and difficult to compare, because of differences in the applied piezoelectric transducers, electrical discharge, ultrasound water bath, etc. Although very limited, the literature indicates that low ultrasonic frequencies could be accurate for preventing the fouling of surfaces by bacterial biofilm.

However, the demonstration of ultrasound efficiency in inhibiting biofilm formation is not an easy task as ultrasound waves should de facto prevent deposition/attachment of soft colloids such as single bacterial cells, multiplication of attached living cells or the formation of viscoelastic clusters described in young water biofilms [START_REF] Abe | Cohesiveness and hydrodynamic properties of young drinking water biofilms[END_REF]. Indeed, experimental demonstrations are needed to get a clear proof of concept of ultrasound technology under conditions relevant to engineered water systems.

In heat exchangers, precipitation fouling, ie the formation of a solid layer of deposits of inverse solubility (CaCO 3 , CaSO 4 , Ca 3 (PO 4 ) 2 , etc.), and viscous hydrated organic deposits on the side of plates in contact with coolants determine heat exchange limitation, pressure drop and eventually complete clogging of the systems [START_REF] Andritsos | Calcium carbonate scaling in a plate heat exchanger in the presence of particles[END_REF][START_REF] Müller-Steinhagen | C4 fouling of heat exchanger surfaces[END_REF]. Such phenomena are observed with river waters but also when drinking waters are used for cooling the systems, especially if they are oversaturated with respect to CaCO 3 .

In drinking water pipes, relatively thin viscoelastic biofilms are made up of highly diversified bacterial populations embedded in an exopolymer matrix [START_REF] Flemming | The biofilm matrix[END_REF][START_REF] Mathieu | Bacterial repopulation of drinking water pipe walls after chlorination[END_REF]. They represent a major reservoir for many undesirable bugs and sometimes pathogens [START_REF] Wingender | Biofilms in drinking water and their role as reservoir for pathogens[END_REF] that distribution system managers want to prevent.

Despite the above-mentioned studies documenting the inactivation effect of US waves on biofilms composed of pure bacterial strains, almost nothing is known on the effect of ultrasound on complex bacterial consortia from drinking water and scaling. Thus in this work we assessed the preventive effect on drinking water biofouling of continuous power ultrasonic waves of low frequency generated by transducers of low electric power (10 and 25 W) provided by SOFCHEM; TWIN-f® Ultrasonic system, already described by [START_REF] Techer | Contrasted effects of an anti-cyanobacterial ultrasound device on the nontarget freshwater invertebrate species Gammarus roeseli[END_REF]. The novelty of the study resides first on the dual experimental approach used. Two drinking water systems with specific geometry and operating conditions were tested: (i) a lab-scale plate heat exchanger operating in batch with continuously recycled water, i.e. planktonic bacteria were constantly exposed to ultrasonic waves over the 14-day assay period and (ii) a continuously fed flow-through pilot used for mimicking water circulation in pipes (Propella TM reactor), i.e. planktonic bacteria were exposed to ultrasonic waves for a time equal to the hydraulic residence time of the reactor, which is 48 h. Both systems were fed with drinking water of different chemical composition ('N' and 'M', respectively). The number of bacterial cells (planktonic and sessile), the saturation index of water and the calcium deposited on the materials were used as indicators of the ultrasound efficiency. Finally, the originality also belongs to the pioneering results obtained here that show for the first time that although ultrasound treatment did not prevent initial bacterial adhesion and CaCO 3 deposition, it markedly limits biofilm formation and scaling. This highlights the value of continuous application of low-frequency ultrasound waves in preventing biofilm formation and scale deposits in drinking water systems.

Materials and methods

Characteristics of the waters

Drinking waters from two origins were used for cooling the heat exchanger and feeding the Propella TM reactor. The water 'N' was a relatively soft water from the network of the city of Nancy, F and was spiked with Ca(OH) 2 (final concentration 100 mg l -1 ), and pH adjusted to 9.0 with HCl to get oversaturated water (saturation index, SI = 1.6). The water 'M' was a scaling water issued from the network of the city of Metz, F. Table 1 shows some of the physicochemical and microbiological characteristics of these two waters. The calcium carbonate equilibrium of the waters was expressed through the saturation index (SI) such as SI = pH -pH s . The pH s is obtained from the equation: pH s = pK 2 -pK s + p[Ca 2+ ] + p[HCO 3 -] + 5pfm; where K 2 is the carbonic acid dissociation constant; K s is the CaCO 3 solubility constant; [Ca 2+ ] is the concentration of calcium ions (g-moles L -1 ); [HCO 3 -] is the concentration of hydrogen carbonate ions (g-moles L -1 ); and f m is the ionic strength [START_REF]Standard methods for the examination of water and wastewater, 22nd Edition[END_REF]. The two waters were oversaturated with respect to CaCO 3 and non-corrosive (SI > 0), which should favour scaling of the materials.

Heat exchanger

Two lab-scale heat exchangers (Flexiplak GCP-012 equipped with six stainless-steel plates -length 42 cm; width 12 cm; thickness 0.06 cm) (Figure S1 in the Supplemental material) -one for the assay, the other one as control -were connected on one side to deionized warmed water (> 40°C) and on the other side to cooling drinking water 'N' (average temperature 23 ± 1 °C) spiked with Ca(OH) 2 (final concentration 100 mg l -1 ). A schematic view of the pilot setup is shown on Figure S2 in the Supplemental material. At T0, the whole system was filled with 25 L of oversaturated 'N' water. As water was continuously recycled throughout the assays (batch), the hydraulic residence time (HRT) was equivalent to the duration of the assays (14 days in most of the assays). The transducer generating ultrasonic waves provided by the Sofchem company (Rueil Malmaisons, France) (Figure S1) consisted of a single-frequency device connected to a computer-driven generator. The transducer was tightly fastened to a metal bar in contact with the six heat exchanger plates. The ultrasound frequency delivered by the transducer was selected from previous works [START_REF] Habibi | An acoustic antifouling study in sea environment for ship hulls using ultrasonic guided waves[END_REF][START_REF] Techer | Contrasted effects of an anti-cyanobacterial ultrasound device on the nontarget freshwater invertebrate species Gammarus roeseli[END_REF] and two electric powers were tested: 10 and 25 W corresponding to a theoretical intensity of 0.5 and 1.25 W cm -² of the transducer.

The good transmission of the ultrasonic waves to all plates and the control of the frequency were checked thanks to a piezometric sensor (Piezotech, Arkema, Lyon, France).

Propella reactors

Two Propella™ reactors (one assay, one control) were made up of 100 mm diameter by 500 mm long stainless-steel pipes, (Figure S3 in the Supplemental material). A schematic view of the pilot system is shown in Figure S4 in the Supplemental material. The water velocity in the reactor was controlled with a marine propeller (300 rpm), which pushed the water through an inner stainless-steel cylinder, generating a flow parallel to the pipe wall (~ 0.1 m s -1 ). These flow-through fed reactors were continuously fed with drinking water 'M' (37.5 ml h -1 ) providing a hydraulic residence time of 48 hours. Four lines of four 2 cm 2 coupons made of unpolished stainless steel (SS 316L) were immersed in the Propella™ reactors in the annular space between the pipe wall and the inner cylinder (Figure S3) in order to analyze the deposits (bacteria + scale). The ultrasound transducer provided by the Sofchem Company was tightly secured on the side of the Propella™ reactor at ~ 10 cm from the bottom (Figure S3). The supplied electrical power to the transducer was fixed at 10 W. Proper transmission of the ultrasonic waves into the pipe wall was checked thanks to a piezometric sensor (Piezotech, Arkema, Lyon, France), and the distribution of ultrasound waves inside the bulk water was measured thanks to a 0.5 mm needle hydrophone (Precision acoustics Ltd) connected to an oscilloscope (HDO4022 -200 MHz 154 High Definition 2.5 GS/s), via a booster amplifier of a minimum gain of 25 dB.

Bacterial cell counting in water and in the biofilm

The total number of cells was determined after bacterial staining with SYBR™ Green I Nucleic Acid Gel Stain (S7563, Invitrogen, France) at a final concentration 1X for 15 min in the dark. For some water analysis, quantification of the membrane-altered bacterial populations was determined using SYTOX™ Orange Nucleic Acid Stain (S11368, Invitrogen, France) at a final concentration of 0.25 µM and an incubation for 15 min in the dark). Biofilms analysis were assessed after dispersion of the attached bacteria cells from the surfaces (20 cm² surface area for heat exchanger plate; 2 cm² for the coupons immersed in Propella reactor) by gentle sonication (20 kHz; power output 10 W) for 2 × 30 s, using an immersion ultrasonic probe (Ø 9 mm; Labsonic B. Braun) placed in 10 ml distilled-bacteriafree water covering the analyzed surface. Then staining of the dispersed biofilm and counting were done as for water samples.

For the heat exchanger experimentations, both the water and dispersed-biofilm samples were analysed by flow cytometry (FCM) performed with the BD Accuri™ C6 flow cytometer (BD Biosciences, USA), equipped with two lasers (a blue one at 488 nm and a green one at 552 nm).

Commercial ultrapure water (Aqua B. Braun, Braun, France) was used as sheath fluid. Bacteria DNAstained with SYBR-I were detected using the FL2 detector (510 ± 15 nm) and bacteria DNA-stained with SYTOX-Orange by the FL1 detector (610 ± 20 nm). Events were triggered on the forward scatter parameter with a threshold 5000 and on FL1 or FL2 with a threshold 2000 according to the detector used. The data were analyzed using BD Accuri™ C6 software (BD Biosciences).

For Propella TM reactor assays, total bacteria cell counting was done by epifluorescence microscopy. Water and dispersed-biofilm stained samples were filtered through 25 mm diameter, 0.2 µm pore-size black polycarbonate membranes (Nucleopore, Whatman, VWR, France). After mounting the membrane in anti-fading mounting oil (Citifluor, Ltd., London), counting was performed with an epifluorescence microscope (BX40, Olympus, Japan) equipped with a ×100 immersion objective lens, a 470-490 nm excitation filter and a 520 nm barrier filter. Between 30 and 100 randomly chosen microscopic fields were counted for each sample. Moreover, direct in situ staining with SYBR™ Green I was also done on coupons after the biofilm dispersion procedure in order to quantify the residual biofilm cells on the surface. This constituted an indirect estimation of the adhesion strength of the biofilm onto the surface, which permitted to appreciate the role of ultrasound waves on the biofilm cohesion. Bacteria cell counting was done after staining and direct observation of the coupons under epifluorescence microscope as for counting on membrane. As residual biofilm could represent in some conditions a significant fraction of the biofilm, we expressed the total number of biofilm cells as the number of biofilm-dispersed bacteria plus the residual bacteria cells on coupon. Altogether, the number of bacteria was expressed as cells ml -1 and cells cm -² for water and biofilm, respectively.

Cultivable bacteria counting and their identification

The number of cultivable bacteria in water from heat exchangers and Propella was determined by counting colonies grown on R2A agar (Oxoïd, CM0906, Thermo Fisher Scientific, Dardilly, France) after 2-day incubation at 30 ± 1°C (to be close to the water temperature during the assays). The results were expressed as colony forming unit (CFU-2d) per milliliter. Identification of the bacterial colonies grown on R2A agar was done by MALDI-TOF-MS (Microflex LT ® , Bruker, Champs sur Marne, France). MALDI-TOF (Matrix-assisted laser desorption/ionisation-time of-flight) is a technology based on mass spectrometry, analysing the proteins of microorganisms to serve as a landmark for acute identification. Proteins are first co-crystallised in a matrix then ionised by a laser beam (MALDI). The released ions are accelerated and separated in a vacuum tube under the action of an electric field based on their mass/charge ratio, and detected individually over time by a particle detector (TOF). The entire set of molecules is therefore be displayed as a series of peaks forming a spectrum which is characteristic of an organism and is compared to a database of reference spectra, thus enabling identification [START_REF] Singhal | MALDI-TOF mass spectrometry: an emerging technology for microbial identification and diagnosis[END_REF]. (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) (Microflex LT ® , Bruker, Champs sur Marne, France). For each sample, three colonies of each morphological type were re-isolated on R2A agar and one colony per isolate was identified.

Direct spotting of bacteria cells and full protein extraction using formic acid were performed following the manufacturer's recommendations. After drying each spot at room temperature, 1 µL matrix1 HCCA (α-cyano-4-hydroxycinnamic acid in 50% acetonitrile/2.5% trifluoroacetic acid) was added before analysis. The identification criteria used were those recommended by the manufacturer. 10 2.6. Calcium carbonate analysis on the coupons For measurements on heat exchanger plate, thanks to an home-made plastic frame, deposits from a 3.80 cm² surface area were directly acidified to pH near 0 with 10 ml of ultra-pure HNO 3 (65 %) and filtered through 0.2 µm polyethersulfone filters) (Millex GP, Merck Millipore Ltd, Molsheim, France) to reach a pH near 0. The calcium was analysed by HORIBA, Longjumeau, France). The elemental analysis of calcium, which is a direct indicator of scaling, was performed by inductively coupled plasma optical emission spectrometry, ICP OES (HORIBA, Longjumeau, France) [START_REF]International Organization for Standardization, Water quality -Determination of selected elements by inductively coupled plasma optical emission spectrometry (ICP-OES)[END_REF]. For Propella TM reactor, the whole coupon (2 cm²) was immerged into 10 ml of ultra-pure HNO 3 and the dosage was performed as for heat exchanger plate.

Results

Assessment of ultrasound wave transmission in the heat exchangers and the Propella TM reactors

When a transducer is used to generate ultrasound, it is important not only that it is properly coupled to the medium into which it is supposed to emit, but also that transmission is not hampered by severe acoustic mismatches or other issues. If that medium is a solid structure and the aim is to further transmit the ultrasonic waves into the bulk of water present inside the structure, then additional concerns must be raised and checked as to how effectively sound is actually emitted into the liquid. It is with these issues in mind that measurements were taken on the side of the plates of the exchanger (not in the bulk as it was not possible to measure ultrasound distribution in the bulk water in such closed systems) and at different locations in the bulk of the liquid of the Propella TM reactor to verify that indeed sound was effectively transmitted from the generating transducer, through the structure, into the liquid.

For the heat exchanger, the electric tension (mV) measured with the piezometric sensor on the side of the plates was five times lower than that measured directly at the surface of the transducer, due to some loss of energy between the transducer and the metal bar, and the metal bar and the plates. As a consequence, the plates have been subjected to a low acoustic intensity close to 2 and 5 W/cm 2 during the assays.

For the Propella TM reactor, due to the geometrical complexity and the inherent diffraction and scattering of acoustic waves, the sound field in the liquid was expected to be complicated and absolutely not homogeneous. The distribution of ultrasound waves inside the bulk water was measured with an hydrophone connected to an oscilloscope (Figure S5). To investigate the local spectra, a discrete Fourier transform was performed using a fast Fourier transform algorithm (FFT). The signal showed a major resonance frequency at 46 KHz and its harmonic at 92 KHz. The occurrence of additional lobes in between the secondary lobes was caused by windowing of the temporal signal.

More importantly however, the intensity of the signal measured was highly dependent on the position of the hydrophone, which indicates, as may be expected, an acoustic field as the result of the complicated interaction of sound with the structure under examination, determined by scattering effects in and behind the internal cylinder.

Effect of ultrasound waves on the saturation index and planktonic biomass of waters circulating in the heat exchanger

The saturation index (SI) and the number of bacterial cells were measured in the bulk water 'N' circulating in the cooling circuit of the heat exchangers after 14 days of work (Table 2). SI was lower in the control (no ultrasound) comparatively to the pilot treated with ultrasonic waves, especially when a high power (25 W) was applied. It means that the scaling potential of the water was decreased in the control after 14 days, and that the accumulation of carbonate on the surface was limited, unlike in the ultrasound assays.

The number of total bacterial cells in the bulk water 'N' was relatively high in the control (no ultrasound) (approximately 1.4 × 10 5 ml -1 ) (Table 2), as a result of bacterial growth within the experimental system during the 14-day assay period (Figure S6 in Supplemental material). In the assays with ultrasound, the number of total bacterial cells was decreased by a factor 7 (2 × 10 4 ml -1 ) and equal to the number found in the feeding water (Table 1). This lower bacteria density in treated water indicated a bacterial growth inhibition by ultrasonic waves, evidenced after 10 days of work (Figure S6 in Supplemental material). However, at the single cell level, the mean fluorescence intensity of the stained cells was not decreased by the ultrasounds (Figure 1a), suggesting no nucleic acid alteration as SYBR-I still complex to DNA. Only the distribution of the granulosity of cells (SCC parameter) was changed from a multimodal distribution in the control without US waves to a lower and more homogeneous granulosity in the treated samples (Figure 1b).

The inhibition of bacteria growth in water was confirmed by the reduction of the number of cultivable bacteria from 18 % of the total number of cells in the control to 2 % in the assays at 10 W (Table 2).

Difference in the phenotype of cultivable bacteria was also explored as two types of colony appeared according to the treatment: white-and orange-coloured colonies were largely represented in the control, whereas in the assays only orange-coloured colonies were observed in the assays with ultrasound. Identification with MaldiMALDI TOFTof indicated that Pseudomonas stuzeri (high probability at the species level) was representative of half of the white colonies, and Blastomonas ursicola (high probability at the genus level) was representative of most of the orange colonies.

Complementary measurements done with SYTOX® Orange dye used for staining bacterial cells with compromised cytoplasmic membranes showed also two populations, one brighter than the other one (Figure 2). Only the former is representative of damaged membrane of bacteria. This total SYTOXfluorescent population (ie highly fluorescent bacteria plus slightly fluorescent bacteria) represented approximately 50 % of the total population measured by SYBR-I staining both in the control and in the assay at 10 W (Table 3). In the control, 15 % of the total bacteria were highly-fluorescent with SYTOX meaning that the cells had damaged-membranes, when 34 % of the bacteria were only slightly stained with SYTOX (low permeability to SYTOX). A relatively similar distribution was observed in the assays with US (transducer at 10 W), with 8.6 % of highly-fluorescent cells with SYTOX (damaged membranes), while 35.6 % of slightly fluorescent cells (low permeability to SYTOX). Such a similar distribution in the assay and the control (especially no increase in the highlyfluorescent population) suggested ultrasonic waves did not affect significantly the permeability of the bacterial envelopes.

Effect of ultrasound waves on the fouling (calcium deposit and biofilm) of heat exchanger plates

The calcium concentrations deposited at the surface of the heat exchangers plates were low, on average 258 µg/cm 2 in the control, and 12 µg cm -2 and 0.8 µg cm -2 for the two exchanger subjected to ultrasound (transducers at 10 W and 25 W, respectively) (Figure 3). These results completed and were consistent with our previous SI-related observations, ie the higher the SI of the waters was, the lower the calcium deposit on the plate surface was. It also evidenced that deposits (mineral and biological) occurred even during the US treatment, suggesting that the generated vibrations within the stainless steel plates did not prevent their attachment in the time course of the assay.

As reported for water, the number of bacterial cells of the biofilm accumulated on the stainless-steel surfaces of the control plates (2 to 9 × 10 5 cells cm -2 ) was decreased by a factor of 7 in the reactors treated by ultrasounds (Figure 4). This is coherent with the lower number of bacteria in 'N' water treated with US waves (transducer 10 W). Flow cytometry (FCM) was applied in order to quantify SYBR-I fluorescence in single cells to discriminate between low and high fluorescent bacteria, revealing an extinction of the fluorescence of the stressed bacteria that could be explained by alteration of the nucleic acids (ie target of SYBR-I). Whereas the bacterial number was lowered by ultrasound, the mean fluorescent intensity of biofilm bacteria subjected to ultrasound was comparable to the control (Figure 5), indicating no detectable alteration of nucleic acids took place in bacteria subjected to ultrasound.

All together these results suggest that the ultrasound irradiations mostly inactivate the bacteria activity, which results in growth reduction and cultivability decrease but did not compromised cell membrane permeability (at least to SYTOX Orange).

Effect of ultrasound waves on planktonic bacteria and biofilm accumulation in pipe reactors

Flow-through reactors mimicking water distribution pipes (Propella TM reactors) were equipped with suspended coupons and continuously fed with drinking water 'M'. In the bulk of water, the number of bacterial cells measured at pseudo-stationary state (between days 18 and 28) was systematically higher in the control than in the reactor subjected to ultrasound waves (power 10 W) (Figure 6 and Figure S7). The dynamic of the bacteria within the reactors was also interesting as differences between control and assays appeared after 7 HRT. Indeed in the Propella TM submitted to ultrasound waves the bacterial number increased in the liquid phase until day 13 and then tended to decrease to the initial values of the feeding water, whereas bacterial density continuously increased in the control reactor (Figure S7). This again suggests that a limitation of the bacterial population activity within the reactor.

On the stainless-steel coupons immersed in the Propella TM reactors, the difference in the number of biofilm cells in the assay and the control was approximately of 1 log (Figure 6 and Figure S8). It means that bacteria cells attached to the material in spite of the 'vibration' of the surface, but their accumulation or growth was reduced in the time-course of the experiment (28 days). In the reactor subjected to ultrasound waves the lower density of attached bacteria in the reactor submitted to ultrasound waves matched with the lower number of cells in the water bulk. The good reproducibility of the results was interesting although the distribution of the US wave frequency inside the Propella TM was not homogeneous (Figure S5).

Additionally, the bacterial adhesion strength onto the stainless-steel surfaces was indirectly estimated by comparing the number of bacteria remaining on the coupon after application of the lab-procedure for biofilm detachment (Table 4). The remaining biofilm was equal to 2.5 % of the total biofilm for coupons subjected to ultrasound waves compared to 10 % of the total biofilm for coupons in the Propella TM control.

Discussion

Our investigation was carried out with 'power ultrasonic waves' at low frequency (46 KHz and 92 KHz) and low intensity according to the classification of [START_REF] Martini | An overview of ultrasound[END_REF][START_REF] Erriu | Microbial modulation by ultrasound: Current concepts and controversies[END_REF]. A low intensity is interesting as it could be below the "acoustic cavitation threshold" [START_REF] Ince | Ultrasound as a catalyser of aqueous reaction systems: the state of the art and environmental applications[END_REF], which results in material damage being avoided. Ultrasound waves applied continuously improved the 'chemical stability' of the water and limited the quantity of calcium carbonate deposition. It is in good agreement with the observations of Nakagawa et al. [START_REF] Nakagawa | Removal of pipe fouling inside pipes using ultrasonic waves[END_REF] who reported an effective hard deposit exfoliation at the antinodes of vibration.

Vasyliev et al. [START_REF] Vasyliev | Ultrasonic modification of carbonate scale electrochemically deposited in tap water[END_REF] have recently confirmed the effect of ultrasounds on scale demonstrating that depending on their intensity, ultrasound can cause either a surface clean-up by cavitation [START_REF] Pečnik | Scale deposit removal by means of ultrasonic cavitation[END_REF] or a rise of CaCO 3 nucleation site density, building a protective scale layer on the surface. However understanding the mechanisms through which ultrasonic waves affect such carbonate deposition is not straightforward. On the one hand, the complexity of the CaCO 3 deposition on a flow surface arises from the numerous multicomponents of the system that include all ionic species, mass transfer and chemical reactions [START_REF] Segev | Rigorous modelling of the kinetics of calcium carbonate deposit formation[END_REF], and the synergistic effect of suspended solids [START_REF] Andritsos | Calcium carbonate scaling in a plate heat exchanger in the presence of particles[END_REF]. On the other hand, ultrasound can promote carbonate precipitation in solution rather than on the solid surface [START_REF] Al Nasser | Monitoring of aggregation and scaling of calcium carbonate in the presence of ultrasound irradiation using focused beam reflectance measurement[END_REF]. In other words, the lower deposition of calcium on the heat exchanger plate could be explained by both cavitation erosion and changes in the calcocarbonic equilibrium of the bulk.

These continuous low-intensity and low frequency ultrasound waves were affecting both the biofilm and planktonic cell number and activity. Indeed, whatever the tested systems (heat exchangers and Propella TM reactor) the number of attached cells was 1 log lower with ultrasound treatment than in the control without ultrasound treatment. The number of attached cells measured throughout the assays subjected to ultrasound was equivalent to the number of bacteria generally measured after a few hours of immersion and in the order of 10 4 cm -² [START_REF] Mathieu | Drinking water biofilm cohesiveness changes under chlorination or hydrodynamic stress[END_REF][START_REF] Mathieu | Bacterial repopulation of drinking water pipe walls after chlorination[END_REF]. It means that the initial adhesion of the drinking water bacteria on the surfaces was not significantly affected by the vibrations (as previously reported by [START_REF] Pitt | Ultrasound increases the rate of bacterial cell growth[END_REF] with S. aureus adhering on polyethylene rods), but that their activity was hindered preventing their growth. As a result, the number of cultivable bacteria exposed to ultrasound was lower. The low bacterial activity could explain the lower attachment strength of the cells to the sonicated materials as the matrix exopolymer synthesis may have also been inhibited, lowered or delayed. Our observation contrasts with that of [START_REF] Pitt | Ultrasound increases the rate of bacterial cell growth[END_REF][START_REF] Dai | Low ultrasonic stimulates fermentation of riboflavin producing strain Ecemothecium ahsbyii[END_REF] who reported about the growth stimulation of some bacteria and fungy. However our results are in line with that of Joyce et al. [START_REF] Joyce | Assessing the effect of different ultrasonic frequencies on bacterial viability using flow cytometry[END_REF][START_REF] Joyce | The development and evaluation of ultrasound for the treatment of bacterial suspensions. A study of frequency, power and sonication time on cultured Bacillus species[END_REF] and Gao et al. [START_REF] Gao | Inactivation of Enterobacter aerogenes in reconstituted skim milk by high-and low-frequency ultrasound[END_REF], who reported a loss of cultivability. Another unexpected finding from our work is that the cytoplasmic membrane permeability was not really compromised as the permeability to specific fluorochrome such as SYTOX Orange was not modified. This is contrary to previous results [START_REF] Qian | Effect of low-intensity ultrasound upon biofilm structure from confocal scanning maser microscopy observation[END_REF][START_REF] Chapman | Bacteriostatic effect of sequential hydrodynamic and ultrasound-induced stress[END_REF][START_REF] Dai | Effects of low-intensity ultrasound on the growth, cell membrane permeability and ethanol tolerance of Saccharomyces cerevisiae[END_REF] who reported a membrane permeability enhancement but for assays done at much higher frequencies (500 kHz and 1.65 MHz,), or higher power, excepted Dong et al. [START_REF] Dong | Experimental study on the influence of lowfrequency and low-intensity ultrasound on the permeability of the Mycobacterium smegmatis cytoderm and potentiation with levoflaxin[END_REF] who worked in the same rank of frequency (42 KHz) and intensity (<1 W/cm 2 ). One may also suppose that low frequency and low intensity ultrasound perturbations of the envelopes, if any, would be easily and rapidly reversible after the ultrasound irradiation stop. In this study we measured a 46 KHz ultrasound wave in the water and one harmonic of lower intensity at 92 KHz. Then, it is difficult to link the observed effect of ultrasound to one or to the other of these two wavelengths, or their combination. No other frequency, which could have been generated by the vibrations of the plates and pipe walls, was detected in the water.

An abundant literature has been produced on the effect of ultrasonic waves on planktonic and sessile algae (see the review of [START_REF] Chemat | Application of ultrasound in food technology: Processing, preservation and extraction[END_REF][START_REF] Legg | Acoustic methods for biofouling control: a review[END_REF][START_REF] Johansson | Cavitation and non-cavitation regime for large-scale ultrasonic standing wave particle separation systems -In situ gentle cavitation threshold determination and free radical related oxidation[END_REF]521,[START_REF] Legg | Acoustic methods for biofouling control: a review[END_REF][START_REF] Huang | Effects of ultrasound on microbial growth and enzyme activity[END_REF][START_REF] Park | Recent advances in ultrasonic treatment: challenges and field applications for controlling harmful algal blooms (HABs)[END_REF]). According to it, there is no universal optimal frequency as alteration of algal cells was obtained in the range of 20 to 1,320 KHz depending on the ultrasound generator and the algal species. Despite evidences supporting the efficiency of low-power ultrasonic waves on algae and invertebrates such as barnacles, few tests have been conducted on bacteria and carbonate deposition under drinking water conditions. A priori, the use of low-frequency ultrasound waves for disturbing/inactivating bacterial cells, which behave as 0.2 µm soft colloids, is very challenging. Indeed, in water the ultrasound velocity is of 1,500 m s -1 , and the wavelength of the ultrasound applied in this work is close to 3×10 -2 meter. It means that the small planktonic cells should not be directly affected by such a wavelength/frequency. The same can be said for 100 µm viscoelastic young biofilm clusters, which should absorb ultrasound wave energy with reduced impact on the structure. Indeed, the absorption of sound in water-like materials at 46 kHz is in the order of 0.8 dB/km [START_REF] Ainslie | A simplified formula for viscous and chemical absorption in sea water[END_REF] and capsular material has been identified as a protective structure [START_REF] Gao | Inactivation of microorganisms by lowfrequency high power ultrasound: 1. Effect of growth phase and capsule properties of the bacteria[END_REF].

Ultrasound-induced inhibition of bacterial growth could be explained by some other mechanisms such as cavitation and pressure variations. First, microbubble cavitation (especially 'asymmetric collapse' close to the surfaces) generating locally reactive oxidative species (ROS), and the fast flow rate of water (up to 100 µm/s) generated by microstreaming [START_REF] Lamminen | Mechanisms and factors influencing the ultrasonic cleaning of particle-fouled ceramic membranes[END_REF][START_REF] Chahine | Modeling of surface cleaning by cavitation bubble dynamics and collapse[END_REF][START_REF] Johansson | Cavitation and non-cavitation regime for large-scale ultrasonic standing wave particle separation systems -In situ gentle cavitation threshold determination and free radical related oxidation[END_REF][START_REF] Yusof | Physical and chemical effects of acoustic cavitation in selected ultrasonic applications[END_REF] have a major effect on bacteria and biofilms [START_REF] Gao | Inactivation of bacteria and yeast using high-frequency ultrasound treatment[END_REF][START_REF] Kirzhner | Removal of microbial biofilm on water hyacint plants roots by ultrasonic treatment[END_REF]. Cavitation even in the case of low-power, low-frequency treatment represents a possibility that cannot be excluded as such a phenomenon is dependent on many parameters such as temperature, quantity of dissolved gazes and physicochemical heterogeneities on surfaces or in the bulk (particles). Second, rapid pressure variations from positive to negative values (= 46,000 times per second) may also damage some cell structures such as the membrane and prevent adaptation or repair. Indeed, many bacteria do not adapt so easily to rapid hydrostatic pressure changes [START_REF] Kamar | Pressure and temperature dependence of growth and morphology of Escherichia coli: Experiments and stochastic model[END_REF][START_REF] Marietou | Effects of high hydrostatic pressure on coastal bacterial community abundance and diversity[END_REF].

Conclusion

Mitigating (bio)-fouling with a low-frequency and low-intensity ultrasound device was tested under conditions relevant to engineered drinking water systems. The ultrasound devices were tightly fastened outside the systems allowing an ultrasound application without stopping the water system work. Continuous power ultrasonic waves of low frequency (major resonance frequency at 46 KHz and its harmonic at 92 KHz) generated by transducers of low electric power (10 and 25 W) did limit drastically drinking water biofilm accumulation in the two systems tested in this study (a heat exchanger with recycling water loop, and a continuously fed flow-through reactor equivalent to a distribution system pipes). In the two reactors, biofilm bacterial density was limited to approximately 10 4 to 10 5 cells cm -2 equivalent to the initial attachment step without any bacterial attachment to the walls was not prevented (approximately 10 4 to 10 5 cells cm -2 cells stick in a few hours onto the material surfaces) but the activity of the cells was limited such as no further multiplication of the attached cells and no biofilm growth were observed. Moreover the attachment strength of the biofilm bacterial cells in the assay with subjected to ultrasound waves was weaker than in the control, promising an easier further cleaning of the heat exchanger and pipes if needed. Additionally, waterborne planktonic bacteria submitted to the ultrasound waves transmitted throughout the walls behave the same way as the attached cells (no growth, low cultivability on nutritive medium). At such a low frequency and low intensity, cell membrane damaging did not appear as the first cause of no growth arrest. Lastly, calcium concentrations deposited at the surface of the heat exchanger were lower thanks to the ultrasound treatment, which should participate to a lower fouling and clogging of heat exchangers.

Low-frequency and low-intensity ultrasound technology sounds very promising in drinking water biofilm mitigation. Such an effect was not predictable as preventing attachment of bacteria, which behave as soft colloids, was expected to be very difficult and the thin viscoelastic drinking water biofilms were expected to be insensitive to such waves. Future works should contribute to a better understanding of ultrasound mechanistic effects on bacterial cells and optimizing industrial application (different modes of application e.g. continuous versus discontinuous). and both different modes of application (e.g. continuous versus discontinuous) and cellular mechanisms should be further explored.

Figure 1 :

 1 Figure 1: Distribution of the number of SYBR-I stained planktonic bacteria (FCM measurement) in water 'N' at day 14 of experiment in the heat exchanger. (a) Fluorescence intensity (FL2 detector) and (b) granulosity (SCC parameter). Blue curves correspond to control without treatment (n=4), red curves correspond to the assay with ultrasound (transducer at 10 W) (n=4), green curves correspond to assay with ultrasound (transducer at 25 W) (n=2).

Figure 2 :

 2 Figure 2: Distribution of the number of planktonic bacteria stained with SYTOX Orange in water 'N' at day 14 of experiment in the heat exchanger. Blue curves represent the control without ultrasounds and red curves correspond to the assays with ultrasounds at 10 W. Four independent assays were performed.

Figure 3 :

 3 Figure 3: Calcium (µg cm -2 ) accumulated in 14 days on the surface of the heat exchanger stainless steel plates fed with water 'N' (n= 3 to 4).

Figure 4 :

 4 Figure 4: Number of SYBR-I stained bacterial cells measured by FCM in the biofilm accumulated in 14 days on the surface of the heat exchanger plates fed with water 'N'. (n=4).

Figure 5 :

 5 Figure 5: Distribution of Fluorescence intensity (FL2 detector) of the SYBR-I stained biofilm bacteria (FCM measurement) at day 14 of experiment in the heat exchanger fed with water 'N'. Blue curves correspond to control without treatment (n=4), red curves correspond to the assay with ultrasound (transducer at 10 W) (n=4), green curves correspond to assay with ultrasound (transducer at 25 W) (n=2).

Figure 6 :

 6 Figure 6: Number of bacterial cells after SYBR-I staining at pseudo-steady state (between days 18 to 28 of kinetics) in the water phase (planktonic cells) (A) and within the biofilms (attached cells) formed on stainless-steel coupon immersed in the flow-through Propella reactors fed with water 'M' (control = no ultrasound; assay = ultrasound -power 10 W). Histograms correspond to averages of 4 values and the error bar represents the SD values.

(± 3 . 5 ×Table 3 :Table 4 :

 3534 10 3 ) not measured Mean proportions (%) of non-fluorescent, slightly fluorescent ,and highly fluorescent cells after staining with SYTOX Orange in the 'N' water sampled at day 14 in the heat exchangers (control and assay). Data are average values on 4 independent assays for control and the assays with ultrasound. = SYBR-I staining of the cells measured by FCM (b) = fraction of SYBR-I stained cells not stained by SYTOX Orange on the same sample. (c) = fraction of the bacteria cells with low fluorescence stained by SYTOX Orange within the total SYBR-I stained cells (d) = fraction of SYTOX Orange stained cells with high fluorescence compared to total cells stained by SYBR-I Number of total bacterial cells cm -2 (SYBR-I staining) on the coupons immersed for 14 days in the Propella reactor (water 'M') and the fraction remaining after the lab-procedure detachment for biofilm analysis (n=3).

Table 1 :

 1 Physicochemical and microbiological characteristics of water 'N' (after spiking with Ca(OH) 2 and pH adjustment) and water 'M' (n = 2 to 4).

	Parameters	Drinking water 'N'	Drinking water 'M'
	Bacteria (cells mL -1 )	2.2 × 10 4	2.6 × 10 4
	pH	9.0	8.3
	Conductivity (µS cm -1 )	1,190	1,031
	Chlorine (mg Cl 2 L -1 )	< 0.05	< 0.05
	Calcium (mg L -1 )	54	97
	Complete alkalimetric title (CAT) (°f )	29	24
	Total hardness (TH) (°F)	9	29
	Saturation index (SI)	1.6	1.4

Table 2 :

 2 Saturation index and number of bacterial cells after SYBR-I staining and FCM quantification at day 14 in the cooling water 'N' circulating in the heat exchangers (n=2 to 4). The values between brackets represent the standard deviations.

		Saturation index	Bacteria	Bacteria
			(cells mL -1 )	(CFU mL -1 )
			1.4×10 5
	Control	0.11 (± 0.02)	(± 6.9 × 10 4 )	3.6×10 4
			2.0×10 4
	Assay (10 W)	0.23 (± 0.03)	(± 2.6 × 10 3 )	3.8×10 2
			2.7×10 4
	Assay (25 W)	0.47 (± 0.01)

Acknowledgements: This work was supported by the EU (FEDER-FSE Lorraine and Massif des

Vosges) and the Région Grand Est (DPR-NT n°2016/5618). Thanks to J.-P. Moulin and Z. Fechtali Moute for their continuous technical support throughout the entire project. A special thank to J.

Ducourneau for stimulating and critical discussions of the results.

Disclosure statement:

The authors acknowledge any financial interest or benefit they have arising from the direct applications of their research.

ORCID Laurence Mathieu: 0000-0001-8747-1347 ORCID Nico F. Declercq: 0000-0002-8721-8664