MD Julien Bonenfant
email: julien.bonenfant@chu-rennes.fr

MD Sophie Drapier

PhD Jean François Houvenaghel

MD, PhD Florian Naudet

MD, PhD Claire Haegelen

MD, PhD Paul Sauleau

MD, PhD Marc Verin

Pallidal stimulation in Parkinson's patients with contraindications to subthalamic target: a 3 years follow-up

Keywords: Parkinson's disease, deep brain stimulation, Pallidal stimulation, neuropsychological outcome, axial motor symptoms

Introduction: Over a 3-year period, we monitored the efficacy and safety of deep-brain stimulation of the globus pallidus pars interna in patients with advanced Parkinson's disease whose cognitive, psychiatric impairment and/or dopa-resistant axial motor signs made them ineligible for surgery targeting the subthalamic nucleus.

Methods: A total of 25 patients were assessed before surgery, 1 year and 3 years after surgery, on the UPDRS and a neuropsychological battery.

Results:

We noted a significant improvement of 65.9% in the Clinical global self-perceived Improvement by Visual Analog Scale and an improvement of 20.6% in the total UPDRS-III motor score at 3 years in the off-dopa condition compared to before surgery. There was an improvement in the treatment's motor complications, as measured by the UPDRS-IV, with a particularly marked reduction of 50% in the Dyskinesia subscore. Cognitive performances remained stable at 1 year but had fallen by the third year. We interpreted this deterioration as due to disease progression.

Conclusion:

Bilateral pallidal stimulation in patients with contraindications to subthalamic surgery therefore seems to be effective over the long term in treating motor symptoms, especially dyskinesias, with good neuropsychological safety.

Highlights

Efficacy at 3 years of GPi DBS in patients deemed ineligible for STN DBS.

No adverse impact of GPi DBS on axial signs and neuropsychological parameters.

INTRODUCTION

Functional neurosurgery for deep-brain stimulation (DBS) has been validated as an effective treatment of choice in patients with Parkinson's disease (PD) refractory to medical treatment. [START_REF] Limousin | Electrical stimulation of the subthalamic nucleus in advanced Parkinson's disease[END_REF][START_REF]The Deep-Brain Stimulation for Parkinson's Disease Study Group. Deep-brain stimulation of the subthalamic nucleus or the pars interna of the globus pallidus in Parkinson's disease[END_REF] The optimal target is still debated, some studies seems to show a superiority of the subthalamic nucleus (STN) over the globus pallidus pars interna (GPi), [START_REF] Krack | Subthalamic nucleus or internal pallidal stimulation in young onset Parkinson's disease[END_REF][START_REF] Odekerken | NSTAPS study group. GPi vs STN deep brain stimulation for Parkinson disease: Three-year follow up[END_REF] but some don't. [START_REF] Follett | CSP 468 Study Group. Pallidal versus subthalamic deep-brain stimulation for Parkinson's disease[END_REF][START_REF] Katayama | Double blinded evaluation of the effects of pallidal and subthalamic nucleus stimulation on daytime activity in advanced Parkinson's disease[END_REF] However, STN DBS has been shown to carry a risk of disabling side effects, including cognitive decline, [START_REF] Massano | Deep brain stimulation and cognitive decline in Parkinson's disease: a clinical review[END_REF][START_REF] Parson | Cognitive sequelae of subthalamic nucleus deep brain stimulation in Parkinson's disease: a meta-analysis[END_REF] limbic effect [START_REF] Drapier | Does subthalamic nucleus stimulation induce apathy in Parkinson's disease?[END_REF] and the worsening of dopa-resistant axial motor signs. [START_REF] Allert | Effects of bilateral pallidal or subthalamic stimulation on gait in advanced Parkinson's disease[END_REF] In the light of these side effects, we can now identify patients who should not undergo subthalamic surgery. [START_REF] Welter | Clinical predictive factors of subthalamic stimulation in Parkinson's disease[END_REF] As GPi DBS appears to entail less cognitive deterioration and fewer axial signs, [START_REF] Rodriguez-Oroz | Bilateral deep brain stimulation in Parkinson's disease: a multicentre study with 4 years follow-up[END_REF] several centers have started offering this treatment to patients contraindicated to STN DBS. Its efficacy and safety have already been studied at 6 months post-surgery [START_REF] Rouaud | Pallidal stimulation in advanced Parkinson's patients with contraindications for subthalamic stimulation[END_REF] in one such group of patients, with a motor benefit, no worsening of axial symptoms and no cognitive impairment. In the present study, we monitored its efficacy and safety in patients receiving bilateral pallidal stimulation over a 3-year period. All these patients had advanced PD refractory to all the usual medical treatments, with contraindications to STN DBS.

MATERIAL AND METHODS

Patients

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 3

During the 2004-2011 periods, 87 patients were selected for DBS surgery with idiopathic PD at Rennes University Hospital. The decision to operate had been taken because all the usual medical treatments had failed to adequately control the symptoms of the disease, and patients had a levodopa (L-dopa) responsiveness of 50%, as measured by the motor score of the United Parkinson's Disease Rating Scale (UPDRS).

Along those 87 patients, 2 refused the surgical procedure, 60 underwent STN-DBS and we studied the 25 patients who had undergone GPi DBS surgery, corresponding to the patients who had been rejected for STN surgery.

The contraindication for STN surgery was, general cognitive impairment (Mattis Dementia Rating Scale (MDRS) score < 130), [START_REF] Silberstein | Deep brain stimulation for Parkinson's disease: Australian referral guidelines[END_REF] impaired executive functions (impaired scores at almost three tests among the battery of tests assessing executive functions, according to the normal values expected in regard of age and educational level) before surgery and/or dopa-resistant axial signs (preoperative UPDRS III axial score 3 in the on-dopa condition). [START_REF] Jarraya | Parkinson's disease, subthalamic stimulation, and selection of candidates: A a pathological study[END_REF][START_REF] Charles | Predictors of effective bilateral subthalamic nucleus stimulation for PD[END_REF] Table 1 set out the patients' characteristics immediately prior to surgery.

At 3 years, one patient could not be assessed at all, owing to an advanced dementia syndrome, and two patients did not undergo the neuropsychological tests due to cognitive impairment.

Surgical procedure

Quadripolar DBS electrodes (Medtronic, Minneapolis, MN) were implanted bilaterally in the posteroventral part of the GPi under general anesthesia in a single operating session. All dopaminergic medication had been withdrawn the day before, and patients were briefly woken during the procedure to test the clinical effect of stimulation on rigidity. The ventral contact was kept above the optical tract, as evidenced by the induction of visual flashes by stimulation. There was no Intraoperative microelectrode recording during the procedure.

The pulse generators (Soletra, Medtronic) were implanted 2-3 days later. The exact location of the two selected electrode contacts (one on the left and one on the right) was determined using stereotactic coordinates derived from a 3D CT scan performed a few days after surgery. The stimulation parameters were set and adjusted during appointments with the consultant and regular follow-up hospitalizations, based solely on clinical efficacy criteria.

Assessments

The patients were assessed before surgery (baseline), 1 year (M12) and 3 years (Y3) after surgery.

The clinical assessment included the UPDRS parts I (mental, behavior and mood), II (activities of daily living), III (motor performance) and IV (complications of therapy in the past week). The off-dopa condition of the UPDRS-III was at least 8 hours after all medication had been halted, and the on-dopa condition was performed after patients had received a dose of L-dopa in the form of dispersible levodopa-benserazide, corresponding to their usual morning dose of dopaminergic medication plus 50 mg.

We defined 4 subscores from the UPDRS III, the "axial score" (out of 20) defined as the sum of items 18 (speech), 27 (rising from chair), 28 (posture), 29 (gait), and 30 (posture stability); the "speech

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 4
score" (out of 4) defined as item 18 (speech); the "akinesia score" (out of 32) defined as the sum of items 23 (finger taps), 24 (hand movements), 25 (rapid alternating movements of hands), and 26 (leg agility); the "Tremor score" (out of 28) defined as the sum of items 20 (tremor at rest) and 21 (action or postural tremor of hands).

We also defined a "dyskinesia score" (out of 13) corresponding to the sum of items 32, 33, 34 and 35 and a "motor fluctuations score" (out of 7) corresponding to the sum of items 36, 37, 38 and 39 of the The Hoehn and Yahr (H&Y) and Schwab and England (S&E) scores were also collected, along with two self-rated quality of life (QoL) scores (SF-36 and PDQ-39).

Daily dopaminergic treatment was defined as the L-dopa equivalent dose (LED), calculated using method of Deuschl et al. [START_REF] Deuschl | Neurostimulation Section[END_REF] A global self-perceived Improvement was measured with a single-item Visual Analog Scale (VAS-I)

ranging from 0% (no change) to 100% (best improvement). [START_REF] De Boer | Is a single-item visual analog scale as valid, reliable and responsive as multi-item sacles in measuring quality of life?[END_REF] The question was worded: "compared to the time before your operation, how would you judge your global improvement at the moment?"

Statistical analysis

We compared the different variables using the Wilcoxon signed-rank test, with the significance

RESULTS

Motor results

The VAS-I score was 64 ± 23.9% at M12, and remained unchanged at Y3 (65.9 ± 15.6%; p = 1).

When the patients were stimulated (on stim), but without their dopaminergic medication (off dopa), there was a significant 19.8% improvement in the total motor score of the UPDRS-III at M12 (p = 0.04), which was maintained at Y3, with an improvement of 20.6% compared with baseline (p = 0.04) (Table 2). An improvement in the Tremor subscore tended toward significance. We did not find any change in the Axial, Akinesia or Speech subscores, which remained stable at M12 and Y3. The UPDRS-II, the H&Y and the S&E scores measuring activities of daily living also remained stable at M12 and Y3.

In the on-stim/on-dopa conditions, patients' total motor score was stable at M12 (p = 0.13) and remained so at Y3 (p = 0.27) (Table 3). The Akinesia, Tremor and Axial subscores were also stable, with no significant deterioration at either M12 or Y3. There was, however, a significant worsening of the Speech subscore by M12 (p = 0.002). This was still present at Y3, but there had been no further

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

decline. There was a significant deterioration in the UPDRS-II score at Y3 compared with baseline (p=0.002). The S&E and H&Y scores remained unchanged.

Motor complications of medical therapy

There was an improvement of 28.5% (p = 0.009) of the UPDRS-IV at M12 and 33% at Y3 (p = 0.02) compared with baseline (Table 3), especially for the Dyskinesias subscore, which improved by 50% at M12 (p = 0.009) and 52% at Y3 (p = 0.007) (Table 3). No significant improvement in the Motor Fluctuations subscore was found.

Cognitive and neuropsychological results

At M12, all the cognitive and psychiatric abilities, as assessed with the UPDRS-I and the neuropsychological tests, did not change. By contrast, at Y3, we found significant deteriorations in the MDRS score (p = 0.049), the Stroop color-word (p = 0.01) and interference (p= 0.01) scores, and the TMT B-A (p = 0.01) score (Table 4). There was also a trend toward a worsening of the UPDRS-I score. Semantic and phonemic verbal fluency remained stable, as did WCST scores.

Quality-of-life scales

The SF-36 QoL score showed a trend toward deterioration between baseline and Y3 (48.8 ± 18 vs.

41.1 ± 14.7; p = 0.06), whereas the PDQ-39 scale remained stable over this period (38.21 ± 9 vs. 39.5 ± 11.8; p = 0.83).

Safety

One patient's electrode malfunctioned by Y3, following a fall with minor concussion, and one patient had to have an electrode removed at 2 years, after developing an infection (which responded favorably to treatment) without replacement. None of the patients died during follow-up.

Stimulation parameters and dopaminergic medication

The stimulation parameters (mean ± SD) at Y3 for the 25 patients were 2.96 ± 0.36 V, with a pulse width of 84.1 ± 21.6 μs and a frequency of 133.5 ± 14.9 Hz. At Y3, nine patients were stimulated using two contacts on each side, two patients were unilaterally stimulated via two contacts, while 14 patients were bilaterally stimulated via one contact on each side. None of the patients needed to have their neurostimulator changed during the 3 years of the study. The stimulation did not bring about any reduction in the LED, which was 1263 ± 513 mg at baseline, 1431 ± 438 mg at M12, and 1375 ± 617 mg at Y3.

DISCUSSION

For patients with advanced Parkinson's disease refractory to medical treatment, the existence of doparesistant axial motor signs or cognitive impairment, leads to contraindication for surgical option. We

M A N U S C R I P T A C C E P T E D

believe that these contraindications are specific to STN DBS and cannot be applied to GPI DBS, because pallidal surgery appears to entail less cognitive deterioration and fewer axial signs. [START_REF] Rodriguez-Oroz | Bilateral deep brain stimulation in Parkinson's disease: a multicentre study with 4 years follow-up[END_REF][START_REF] Rouaud | Pallidal stimulation in advanced Parkinson's patients with contraindications for subthalamic stimulation[END_REF] Our results shows a motor benefits in the off-dopa condition that is still present 3 years after surgery, corresponding to the effect of the stimulation without any medication. In the on-dopa condition, we did not find any modification in the total motor score and no worsening of the Motor Fluctuations subscore, despite the expected disease progression, the motor benefit could be interpreted as low, but cannot be compared to the benefits in other studies on GPI DBS, because our patients due to the advanced PD and dopa resistant symptoms are systematically contraindicated for surgery in those studies.

We also find a major antidyskinetic effect, with a reduction at Y3 of more than 50% in the baseline Dyskinesias score, even though there was no postoperative reduction in dopaminergic medication.

This result confirms the specific antidyskinetic effect of the pallidal target, an effect that was maintained over time. The major reduction on dyskinesias is constantly found in the published data on GPI DBS, confirming the interest of this target on dyskinesias. [START_REF] Fasano | Treatment of motor and non-motor features of Parkinson's disease with deep brain stimulation[END_REF][START_REF] Oyama | GPi and STN deep brain stimulation can suppress dyskinesia in Parkinson's disease[END_REF] We confirm the tolerance of this target with no worsening of dopa-resistant axial motor signs present prior to surgery, even though the patients selected were an at-risk population. This suggest that the stimulation had no adverse effect on axial signs at M12 and Y3, in contrast to previous reports. [START_REF] Deuschl | Deep brain stimulation: Postoperative issues[END_REF] Although there have been suggestions that GPi DBS leads to a worsening of akinetic symptoms, we did not observe any deterioration in the Akinesia subscore during the 3-year follow-up period. We did not evaluated adverse effects such as fatigue, apathy, mobility/falls and ICD and it will be useful to evaluate them with specific scales in further studies.

Dysarthria however was the only symptom that significantly worsened, with an early decline in the first year, but none thereafter. This early deterioration suggest that it was an effect of the surgery, rather than of disease progression. We therefore believe it is important to make this particular risk known and take it into account when discussing the risks and benefits with patients who are potentially suitable for GPi surgery.

On cognitive assessments, at M12, the neuropsychological parameters remained unchanged, without early worsening, attributed to the stimulation, such as has been reported in some studies of STN DBS. [START_REF] Saint-Cyr | Neuropsychological consequences of chronic bilateral stimulation of the subthalamic nucleus in Parkinson's disease[END_REF]29] At Y3, however, some scores underwent a belated decline. In our patients, with advanced PD with cognitive impairment prior to surgery, we believe that this deterioration reflect the effect of disease progression, rather than a harmful effect of the surgery, because of the absence of early deterioration, such as suggested in other studies reporting the long-term follow-up of DBS. [START_REF] Krack | Five-year follow-up of bilateral stimulation of the subthalamic nucleus in advanced Parkinson's disease[END_REF] There was a major self-estimated global clinical improvement of 65% still present at 3 years after the surgery. By contrast, there was no improvement in QoL and even a worsening of the UPDRS II at 3 years. We think that this discrepancy can be explained by the scales we used to measure this variable, as they are not particularly sensitive to change. We could also incriminate the cognitive impairment, responsible of a worsening of quality of life independently of the motor benefits, but this point on the effectiveness of the GPi DBS should be assessed in further studies.

Our study had however some obvious limitations as the size of the population, the observational design and the absence of control group. A comparison to a nonsurgical control population over the

CONCLUSION

GPi DBS in patients deemed ineligible for STN stimulation, owing to cognitive impairment or doparesistant axial signs, effectively reduces motor symptoms, and the benefits last at least 3 years.

Pallidal stimulation also considerably lessens treatment-induced motor complications, especially dyskinesias. It did not bring about any increase in akinesia, and had no adverse impact on axial signs and neuropsychological parameters. By contrast, there was a significant deterioration in speech, which will need to be taken into account when deciding on the most appropriate course of action. These results suggest that GPi DBS is a viable treatment for advanced idiopathic PD in patients ineligible for STN DBS, especially if they have acute dyskinetic complications, but those results are far from being definitive and should be assessed in further prospective studies.

  UPDRS IV. Patients underwent a neuropsychological battery, comprising the MDRS, Trail Making Test (TMT), Nelson's modified Wisconsin Card Sorting Test (WCST), Stroop test, and semantic and phonemic verbal fluency test.

  threshold set at p = 0.05. All the data are shown as means ± standard deviation (SD). Analyses were performed using R [R Development Core Team: R: A Language and Environment for Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing; 2009.].

7 same

 7 period of time would help to assess the long term effectiveness and cognitive tolerance of the stimulation.

  (on stim) at 1 year and 3 years. The comparisons were made between baseline and 1 year (M12vs.B), baseline and 3 years (Y3vs.B), and 1 year and 3 years (Y3vs.M12). Statistically significant improvement is denoted by *. Statistically significant worsening is denoted by †.A N U S C R I P TA C C E P T E DACCEPTED MANUSCRIPT

Table 1 .

 1

	Characteristics of the 25 patients prior to surgery
		Value
	Male	13 (52%)
	Mean age at surgery (years)	60 ± 7.5
	Mean disease duration at surgery (years)	12.5 ± 6.25
	Mean levodopa dose (mg/d)	1263 ± 513

Table 2

 2 Effects of stimulation in the off-dopa/on-stim condition All values are shown as means ± standard deviation. UPDRS = United Parkinson's Disease Rating Scale. H&Y = Hoehn and Yahr. S&E = Schwab and England. A reduction in these scores (apart from H&Y and S&E) represented a functional improvement. Patients' status was assessed off dopaminergic medication (off dopa), but with stimulation (on stim) at 1 year and 3 years. Comparisons were made between baseline and 1 year (M12vs.B), baseline and 3 years (Y3vs.B), and 1 year and 3 years (A3vs.M12). Statistically significant improvement is denoted by *.

		Baseline	M12	Y3
		Off-dopa	Off-dopa/on-stim	Off-dopa/on-stim		P-value
		n=25	n=25	n=24	M12vs.B Y3vs.B Y3vs.M12
	UPDRS-II (/52)	20.7 ± 7.8	20.8 ± 8.1	20.3 ± 10.2	0.88	0.69	0.8
	UPDRS-III (/108)	37.3 ± 15.3	29.9 ± 11.2	29.6 ± 18.3	0.04*	0.04*	0.42
	Akinesia score (/32)	14.9 ± 6.9	12.4 ± 6.6	14.7 ± 8.7	0.15	0.69	0.63
	Tremor score (/28)	4.7 ± 5.3	3.2 ± 3.7	2.5 ± 2.9	0.16	0.09	0.26
	Axial motor score (/20)	8.1 ± 3.6	6.6 ± 3.3	7.3 ± 4.9	0.11	0.17	0.72
	Speech score (/4)	1.5 ± 0.9	1.7 ± 0.9	1.9 ± 1.1	0.52	0.14	0.26
	H&Y (/5)	3.2 ± 1.1	3.2 ± 1.2	3.1 ± 1	0.69	0.81	0.75
	S&E (/100%)	54.4 ± 23.6	62.2 ± 18.3	58.3 ± 1	0.21	0.81	0.42

Table 3

 3 Effects of stimulation in the on-dopa/on-stim condition All values are shown as means ± standard deviation. UPDRS = United Parkinson's Disease Rating Scale. H&Y = Hoehn and Yahr. S&E = Schwab and England. A reduction in these scores (apart from H&Y and S&E) represented a functional improvement. Patients' status was assessed with dopaminergic medication (on dopa), and with electrical stimulation

		Baseline	M12	Y3
		On-dopa	On-dopa/on-stim	On-dopa/on-stim		p-value
		n=25	n=25	n=24	M12vs.B	Y3vs.B	Y3vs.M12
	UPDRS-II (/52)	8.1 ± 6	10.8 ± 6.8	13.2 ± 7.5	0.15	0.002 †	0.01 †
	UPDRS-III (/108)	11.9 ± 5.4	14.3 ± 6.6	15 ± 10.1	0.13	0.27	1
	Akinesia score (/32)	4.9 ± 2.5	5.9 ± 4.1	7.3 ± 6.9	0.41	0.10	0.77
	Tremor score (/28)	0.8 ± 1.4	0.7 ± 0.8	0.7 ± 0.9	0.96	0.90	1
	Axial score (/20)	3.2 ± 2.3	4 ± 2.5	4.6 ± 3.5	0.27	0.11	0.74
	Speech score (/4)	0.9 ± 0.7	1.6 ± 1	1.4 ± 1.2	0.002 †	0.08	0.5
	H&Y (/5)	1.9 ± 1	1.8 ± 0.8	2.3 ± 1.1	0.75	0.13	0.09
	S&E (/100%)	83.2 ± 14.3	84.8 ± 15.9	77.9 ± 16.7	0.68	0.18	0.02 †
	UPDRS-IV (/24)	8.8 ± 3.7	6.3 ± 3.3	5.9 ± 2.9	0.009*	0.02*	0.6
	Dyskinesia score (/13)	4.6 ± 2.9	2.3 ± 2.7	2.2 ± 2.1	0.009*	0.007*	0.78
	Motor Fluctuations score (/7)	3.3 ± 1.5	2.9 ± 1.6	2.9 ± 1.3	0.32	0.55	0.98

Table 4 Cognitive

 4 All values are shown as means ± standard deviation. UPDRS = United Parkinson's Disease Rating Scale; MDRS = Mattis Dementia Rating Scale; C/W = color word; IS = interference score; TMT = Trail Making Test; WCST = Wisconsin Card Sorting Test; The situations at 1 year (M12) and 3 years (A3) are compared with the preoperative situation (baseline). Statistically significant improvement is denoted by *.

			and neuropsychological results
		Baseline	M12		Y3
		Mean	Mean	p-value	Mean	p-value
		n=25	n=25		n=22
	UPDRS I	2.5 ± 1.9	2.7 ± 2.7	0.98	3.59 ± 2.5	0.054
	MDRS	133.1 ± 6.7	134 ± 6.4	0.34	129.4 ± 9.5	0.049*
	Stroop
	Word	87.7 ± 19.8	83.8 ± 16.9	0.59	82 ± 20.9	0.14
	Color	57.4 ± 11.8	62.7 ± 19.3	0.22	55.2 ± 13.8	0.33
	C/W	32.6 ± 12	31.8 ± 12.4	0.93	28.1 ± 11.2	0.01*
	IS	-1.8 ± 8.2	-3.6 ± 7.8	0.37	-4.8 ± 6.6	0.01*
	TMT
	TMT A	74.9 ± 45.9	69.3 ± 51.8	0.41	73.7 ± 44.8	0.9
	TMT B	172.2 ± 63.6	172.5 ± 87.4	0.73	198.9 ± 87.5	0.1
	TMT B-A	110 ± 52.6	98.3 ± 90.3	0.98	146.6 ± 79.2	0.01*
	Verbal fluency
	Semantic	20.8 ± 9.3	20.6 ± 7.9	0.72	21.6 ± 8.9	0.88
	Phonemic	15.8 ± 7.1	14.2 ± 7.7	0.64	14.3 ± 8.1	0.2
	WCST
	Categories	4 ± 1.9	4.2 ± 2	0.28	3.6 ± 2.1	0.3
	Errors	10.9 ± 7.4	9.7 ± 7.4	0.49	13.2 ± 8.6	0.27
	Perseveration	4.1 ± 3.6	2.9 ± 2.9	0.17	4.8 ± 4.6	0.69

Acknowledgment: We like to thanks Mr Pierre Vérin for collecting the data and Mrs Elisabeth Portier-Wiles for preparing the manuscript.

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Author Roles: Julien Bonenfant was involved in the conception, organization, and execution of research project; design, execution, and review and critique of statistical analysis; writing of the first draft of manuscript.

Sophie Drapier was involved in the execution of research project; review and critique of statistical analysis; review and critique of manuscript.

Jean-François Houvenaghel was involved in the neuropsychological assessments, review and critique of manuscript.

Florian Naudet was involved in the design and execution of statistical analysis; review and critique of manuscript.

Claire Haegelen was involved in the review and critique of manuscript.

Paul Sauleau was involved in the review and critique of manuscript.

Marc Vérin was involved in the conception and organization of research project; design and review and critique of statistical analysis; review and critique of manuscript.


Claire Valotteau

Christophe Calers

Sandra Casale

Jan Berton

Christian V Stevens

Florence Babonneau

Claire-Marie Pradier

Vincent Humblot
email: vincent.humblot@upmc.fr

Niki Baccile
email: niki.baccile@upmc.fr

Biocidal Properties of a Glycosylated Surface: Sophorolipids on Au(111)

Keywords: sophorolipid, glycolipid, surface functionalisation, self-assembled monolayer, antibacterial coating, killing-by-contact, biocidal mechanism

Classical antibacterial surfaces usually involve antiadhesive and/or biocidal strategies. Glycosylated surfaces are usually used to prevent biofilm formation via antiadhesive mechanisms. We report here the first example of a glycosylated surface with biocidal properties created by the covalent grafting of sophorolipids (a sophorose unit linked by a glycosidic bond to an oleic acid) through a self-assembled monolayer (SAM) of short aminothiols on gold (111) surfaces. The biocidal effect of such surfaces on Gram+ bacteria was assessed by a wide combination of techniques including microscopy observations, fluorescent staining and bacterial growth tests. About 50% of the bacteria are killed via alteration of the cell envelope. In addition, the role of the sophorose unit and aliphatic chain configuration are highlighted by the lack of activity of substrates modified respectively with sophorose-free oleic acid and sophorolipid-derivative having a saturated aliphatic chain. This system

demonstrates thus the direct implication of a carbohydrate in the destabilization and disruption of the bacterial cell envelope.

Introduction

Biological contamination of surfaces is a major concern in medical and pharmaceutical industries [START_REF] Hall-Stoodley | Bacterial Biofilms: From the Natural Environment to Infectious Diseases[END_REF] , food-processing installations, water pipes, naval ships as well as in monuments and cultural heritage materials. Bacteria, fungi or algae are inclined to adhere and grow onto solid surfaces, leading to the formation of biofilms [START_REF] Costerton | Microbial Biofilms[END_REF] , that may lead to undesired phenomena like loss of adhesion properties, degradation or infection. Surface contamination has an important socio-economical impact. For instance, nosocomial infections in the U.S.A. were estimated to 1.7 million in 2002 and leading to slightly less than 100,000 death cases [START_REF] Klevens | Estimating Health Care-Associated Infections and Deaths in U.S. Hospitals[END_REF] . Moreover, bacteria in biofilms are generally more resistant [START_REF] Lewis | Multidrug Tolerance of Biofilms and Persister Cells[END_REF] , thus making the antibiotics ineffective in the long run. To prevent these problems, a large amount of research is carried out to develop preventive antimicrobial coatings on surfaces, including either antiadhesive or biocidal strategies. In the latter, one mainly finds either a killing-by-contact [START_REF] Tiller | Designing Surfaces That Kill Bacteria on Contact[END_REF] or a killing-by-release [START_REF] Cado | Self-Defensive Biomaterial Coating against Bacteria and Yeasts: Polysaccharide Multilayer Film with Embedded Antimicrobial Peptide[END_REF] approach, where the active compound can be antibiotic [START_REF] Aumsuwan | The Effectiveness of Antibiotic Activity of Penicillin Attached to Expanded Poly(Tetrafluoroethylene) (Eptfe) Surfaces: A Quantitative Assessment[END_REF] , quaternary ammonium derivatives [START_REF] Tiller | Designing Surfaces That Kill Bacteria on Contact[END_REF] , nanoparticles [START_REF] Lee | Antibacterial Properties of Ag Nanoparticle Loaded Multilayers and Formation of Magnetically Directed Antibacterial Microparticles[END_REF] , etc., either blended into or deposited onto a substrate [START_REF] Rocha | Microbial Pathogens and Strategies for Combating Them: Science, Technology and Education[END_REF] , or chemically grafted onto surfaces. Nevertheless, the ecotoxicity of most of these compounds, the increasing resistance of microorganisms, or the complexity of the grafting strategy restrict their practical use at large scales. Bio-derived compounds, e.g. enzymes, molecules able to interfere with quorum-sensing phenomena or antimicrobial peptides, appear as appealing alternatives for their broad range of action, their efficiency at very low concentration and the absence of bacterial resistance. However, their high cost of synthesis and purification, pH sensitivity, susceptibility to proteolysis, as well as potential local toxicity and allergy after repeated applications, may restrict their use [START_REF] Glinel | Antibacterial Surfaces Developed from Bio-Inspired Approaches[END_REF] .

In this context, carbohydrates, one of the most common compounds in living systems, are of increased interest for a large number of medical and pharmalogical-related applications, like vaccines and anticancer drugs [START_REF] Osborn | Carbohydrate-Based Therapeutics[END_REF] , fabrication of glycoarrays for antibody recognition and cell adhesion 12 , antibiofilm and antimicrobial formulations [START_REF] Cortes-Sanchez | Biological Activity of Glycolipids Produced by Microorganisms: New Trends and Possible Therapeutic Alternatives[END_REF] , just to cite some. Even if carbohydrates alone are a source of nutrition to microorganisms rather than biocidal compounds, some reports relate the antimicrobial effects of sugar-containing molecules, such as bacterial exopolysaccharides, extracted from Escherichia coli, [START_REF] Valle | Broad-Spectrum Biofilm Inhibition by a Secreted Bacterial Polysaccharide[END_REF] and few glycoconjugates, carbohydrates covalently linked to an organic moiety (from simple alkyl chain to more complex structures), of both synthetic and natural origins.

For instance, amphotericin B, a complex glycolipid extracted from Streptomyces nodosus, is used to treat fungal infections [START_REF] Brajtburg | Amphotericin B: Current Understanding of Mechanisms of Action[END_REF] and other compounds display interesting antimicrobial properties [START_REF] Cortes-Sanchez | Biological Activity of Glycolipids Produced by Microorganisms: New Trends and Possible Therapeutic Alternatives[END_REF] . Until now, only two forms of antimicrobial actions of carbohydrate derivatives have been described: 1) a biocidal action via cellular lysis promoted by glycolipids in solution [START_REF] Cortes-Sanchez | Biological Activity of Glycolipids Produced by Microorganisms: New Trends and Possible Therapeutic Alternatives[END_REF] ; 2) an antiadhesive effect, commonly observed for polysaccharides and glycolipids either immobilized on a substrate (glycoarray) or free in solution [START_REF] Cortes-Sanchez | Biological Activity of Glycolipids Produced by Microorganisms: New Trends and Possible Therapeutic Alternatives[END_REF][START_REF] Bernal | Promising Biotechnological Applications of Antibiofilm Exopolysaccharides[END_REF][START_REF] Fessele | Effect of Aminophenyl and Aminothiahexyl Α-D-Glycosides of the Manno-, Gluco-, and Galacto-Series on Type 1 Fimbriae-Mediated Adhesion of Escherichia Coli[END_REF][START_REF] Hartmann | A Kit for the Investigation of Live Escherichia Coli Cell Adhesion to Glycosylated Surfaces[END_REF][START_REF] Kesel | Carbohydrate Coating Reduces Adhesion of Biofilm-Forming Bacillus Subtilis to Gold Surfaces[END_REF][START_REF] Wehner | Are Multivalent Cluster Glycosides a Means of Controlling Ligand Density of Glycoarrays?[END_REF] . Even though the mechanisms of action are still largely debated, the biocidal action is likely due to the amphiphilic nature of the glycolipids, which penetrate into the plasma membrane, causing lysis and possible leakage of cytoplasm material [START_REF] Chapman | Biocide Susceptibility and Intracellular Glutathione in Escherichia Coli[END_REF][START_REF] Glover | An Epr Investigation of Surfactant Action on Bacterial Membranes[END_REF][START_REF] Lang | Antimicrobial Effects of Biosurfactants[END_REF] . Since neither pure carbohydrates, nor most common lipids, like fatty acids [START_REF] Kabara | Fatty Acids and Derivatives as Antimicrobial Agents[END_REF] , have specific biocidal properties, one generally refers to as a "surfactant effect", where the role in the membrane lysis cannot be attributed to a specific part of the molecule, but rather to its physico-chemical properties in solution 25, 26 . In terms of the antiadhesive effect, this is believed to depend on competing protein-sugar interactions between the pathogen and the substrate, where proteins refer to carbohydrate-binding proteins like lectins and adhesins present in the cell envelope. Playing with the nature of a carbohydrate coating (type, mobility, number of sugars), one can either increase or reduce the affinity of cells for a surface, thus promoting, or discouraging, cell adhesion phenomena [START_REF] Bernal | Promising Biotechnological Applications of Antibiofilm Exopolysaccharides[END_REF][START_REF] Fessele | Effect of Aminophenyl and Aminothiahexyl Α-D-Glycosides of the Manno-, Gluco-, and Galacto-Series on Type 1 Fimbriae-Mediated Adhesion of Escherichia Coli[END_REF][START_REF] Hartmann | A Kit for the Investigation of Live Escherichia Coli Cell Adhesion to Glycosylated Surfaces[END_REF] .

In the context of carbohydrate-driven antimicrobial activity, we show here the first example of a glycosylated surface with biocidal properties. To do so, we use sophorolipids (SL), a family of bolaform glycolipids characterized by a sophorose unit (glucose β(1-2)), linked through a glycosidic bond to oleic acid (Figure 1), and having a COOH group free of access for chemical grafting at the opposite side of the carbohydrate. Sophorolipids are characterized by a low toxicity, high biodegradability, an environmental compatibility and possess both antifungal and/or antimicrobial properties in solution [START_REF] Cortes-Sanchez | Biological Activity of Glycolipids Produced by Microorganisms: New Trends and Possible Therapeutic Alternatives[END_REF][START_REF] Baek | Mitigation of Harmful Algal Blooms by Sophorolipid[END_REF][START_REF] Kim | Characteristics of Sophorolipid as an Antimicrobial Agent[END_REF] . They are produced in large amounts by the yeast Starmerella bombicola 29 , they are already commercialized worldwide (e.g., Soliance, Ecover, IntoBio, Saraya) [START_REF] Mukherjee | Towards Commercial Production of Microbial Surfactants[END_REF] and, probably the most interesting feature, they are ready as-such, needing no specific chemical synthesis nor complex protection/deprotection modification. They constitute an ideal platform for large-scale antimicrobial surface applications, if compared to competitors like antimicrobial peptides or customed glyconjugates. In addition, our approach demonstrates the direct involvement in the cell envelope-destabilization/disruption of a carbohydrate, the role of which is largely debated in the biocidal properties of glycolipids in solution [START_REF] Nobmann | The Antimicrobial Efficacy and Structure Activity Relationship of Novel Carbohydrate Fatty Acid Derivatives against Listeria Spp. And Food Spoilage Microorganisms[END_REF] but also in the more general context of carbohydrate/lipidic membrane interactions [START_REF] Moiset | Disaccharides Impact the Lateral Organization of Lipid Membranes[END_REF] .

The open acidic form of sophorolipids (SL) will be grafted on model flat gold (111) surfaces using short thiolamines as primers (cysteamine, cys). The modified gold surfaces are characterized step by step by Polarization-Modulation Reflection Absorption Infrared Spectroscopy (PM-RAIRS) and X-ray Photoelectron Spectroscopy (XPS). Antimicrobial activity is tested against Listeria ivanovii, a Gram positive bacterium, surrounded by a thick peptidoglycan layer without outer membrane, since sophorolipids appears more effective in solution against bacteria exhibiting such cell envelope.

Antimicrobial properties are qualitatively and quantitatively evaluated through a combination of Atomic Force Microscopy (AFM) and Scanning Electron Microscopy with Field Emission Gun (SEM-FEG), fluorescent staining and bacterial growth tests and compared to blank samples constituted by the gold surface alone and the cysteamine-modified gold surface. In order to elucidate the role of the sophorose group in the biocidal action, we develop and graft on control gold surfaces three SLrelated molecules, either bearing the same sugar but a different chain, or playing with the chain without sugar (Figure 1) : 1) oleic acid (OA), which mimics only the aliphatic chain of sophorolipids, 2) a sophorolipid-derivative having a fully-saturated aliphatic chain (SL0), important to observe the possible role of the molecular conformation at the gold surface due to the presence of a C=C double bond and 3) stearic acid (SA), which mimics the aliphatic chain of the fully-saturated sophorolipidderivative.

OA

Materials and Methods

Cysteamine (cys), N-hydroxysuccinimide (NHS), 1-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), oleic acid (OA), stearic acid (SA), formaldehyde, dimethyl sulfoxide and sodium chloride (NaCl) were obtained from Sigma Aldrich (Saint Quentin Fallavier, France). Sophorolipids (SL)

were derived from a commercial acidic and lactone mixture of sophorolipids purchased from Soliance (France) (Sopholiance S, batch number: 11103A, dry content: 60 ± 6 %). To obtain a high purity (> ~92 mol%, by 1 H NMR) form of the non-acetylated acidic sophorolipids only, we employed a classical hydrolysis route using 5 M NaOH followed by acidification by HCl and pentanol extraction (please refer to ref. [START_REF] Baccile | Practical Methods to Reduce Impurities for Gram-Scale Amounts of Acidic Sophorolipid Biosurfactants[END_REF] for more details) of the sophorolipid only. The fully-saturated sophorolipid compound (SL0) was derived from the previous one using a Pd-catalyzed hydrogenation reaction described in ref. [START_REF] Cuvier | Ph-Triggered Formation of Nanoribbons from Yeast-Derived Glycolipid Biosurfactants[END_REF] . All solvents were reagent-grade and were used without any further purification.

Water was purified with a milliQ system (Millipore, resistivity > 18 MΩ.cm -1 ) from EMD Millipore Corp. (Billerica, MA, USA). Glass substrates, coated successively with a 50 Å thick layer of chromium and a 200 nm thick layer of gold were purchased from Arrandee (Werther, Germany).

Surface preparation

The substrates have been prepared using a standard protocol [START_REF] Yala | Elaboration of Antibiofilm Materials by Chemical Grafting of an Antimicrobial Peptide[END_REF] . Prior to use, the gold-coated substrates were annealed in a butane flame to ensure a good crystallinity of the top layer, after what they were exposed to UV-ozone during 15 minutes and rinsed successively in a bath of ultrapure water and in a bath of absolute ethanol during 10 minutes. First, the substrates were immersed in an ethanolic solution of Cys at 10 mM. After 3 hours, the substrates were sonicated in ultrapure ethanol to desorb the non-grafted molecules and thoroughly rinsed in ethanol and then in ultrapure water before being dried under a flow of dried air. Next, to promote the grafting of the SL on the thiolamine, the carboxylic acid termination of SL (50 mg.L -1 ) was activated by succinimide ester using a mixture of EDC (77 mg.L -1 ) and NHS (23 mg.L -1 ) in water. After 1 hour under stirring, the cys-modified gold substrates were immersed for 3 hours in this solution. Successive rinsing in ultrapure water and ethanol were performed to remove non-covalently grafted reactants before drying under a flow of dried air. Quartz Crystal Microbalance was used to verify that the rinsing procedure efficiently eliminated the excess of unreacted SL. OA, SA and SL0 were grafted onto gold following a similar protocol, using ethanol as solvent. The as-obtained surfaces are depicted in Figure 1. All samples were characterized by PM-RAIRS and XPS after each step of functionalization.

Characterization Techniques

Polarized Modulated Reflexion Absorption InfraRed Spectroscopy (PM-RAIRS)

PM-RAIRS measurements were performed using a Nicolet Nexus 5700 FT-IR spectrometer equipped with a nitrogen-cooled HgCdTe wide band detector. Infrared spectra were recorded at 8 cm -1 resolution, by co-addition of 128 scans. A ZnSe grid polarizer and a ZnSe photoelastic modulator were placed prior to the sample in order to modulate the incident beam between p and s polarizations (HINDS Instruments, PM90, modulation frequency = 36 kHz). The sum and difference interferograms were processed and underwent Fourier-transformation to yield the PM-RAIRS signal which is the differential reflectivity (ΔR/R°) = (Rp-Rs)/(Rp+Rs), where Rp is the signal parallel to the incident plane while Rs is the perpendicular contribution. The measurements were done at two different voltages applied to the modulator ZnSe crystal to optimize the sensitivity.

InfraRed Attenuated Total Reflexion (IR-ATR)

IR-ATR measurements were recorded using a Nicolet 5700 FT-IR spectrometer equipped with a nitrogen-cooled MCT detector. Infrared spectra were obtained by co-adding 256 scans at 8 cm -1 resolution and referencing against the background of air.

Atomic Force Microscopy Imaging (AFM)

AFM images of dried surfaces were recorded using a Caliber AFM microscope from Bruker Instruments Inc. Topographic images were taken in the tapping mode. Silicon nitride tips (resonance frequency 280-400 Hz, force constant 40-80 N/m) have been used. Images were obtained with a constant speed of 1 Hz with a resolution of 512 lines and 512 pixels each. The raw data were processed using the imaging processing software Nanoscope Analysis v.1.30. from Bruker (Nano Surfaces Corp. Santa Barbara, Ca, USA) and used mainly for slope corrections.

Scanning Electron Microscopy with Field Emission Gun (SEM-FEG)

SEM images were recorded with a Hitachi SU-70 Field Emission Gun Scanning Electron Microscope.

The samples were fixed on an alumina SEM support with a carbon adhesive tape and were observed without metallization. In-Lens Secondary electron detector (SE-Upper) was used to obtain characterize our samples. The accelerating voltage was 1 kV and the working distance was around 5 mm. At least five different locations were analyzed on each surface, arising to the observation of a minimum of 100 single bacteria observed.

Water contact angle measurements

Static water contact angles were measured under ambient conditions (at 20°C and 40% relative humidity) analyzing the drop profile of sessile drops. 10 µL droplet of millQ water was deposited on the sample surface using a Krüss DSA100 apparatus (Germany) equipped with a CCD camera and an image analysis processor. 4 droplets were analyzed on different locations on each sample and the test was performed in triplicate on 3 different samples. The reported values are the averages of these 12 measurements for each kind of surface.

Molecular drawing of SL and SL0 on a flat surface

Sophorolipid SL and SL0 molecules covalently bonded on cysteamine via an amide bond had been drawn respecting bond lengths, dihedral angles and sense of chirality for each atom. We referred to the IUPAC name for sophorolipids:

Antimicrobial activity

Strains and culture conditions

Non-human pathogenic bacteria Listeria ivanovii Li4pVS2 were used to investigate the modified surfaces. Unless otherwise indicated, bacterial suspensions were always prepared from stationary phase cultures incubated overnight in brain heart infusion (BHI) broth (BD Difco, France) at 37°C under agitation (250 rpm). The broth was then centrifuged at 10 000 g for 5 minutes, the supernatant was eliminated and the bacteria were re-dispersed in an isotonic sterile solution (NaCl 0.9%). The optical density of the broth was controlled at 620 nm and adjusted at 0.05, which corresponds to 5x10 6 colony forming unity (CFU) per mL.

Deposition of bacteria on samples

A 100 µL drop of bacteria solution freshly prepared was deposited on each surface sample, which were previously washed in a 70% ethanol aqueous solution and dried in sterile environment. In all experiments, unless otherwise mentioned, the inoculated surfaces were incubated for 3 hours at room temperature under a wet atmosphere.

Evaluation of bacteria adhesion by infrared spectroscopy

After bacterial deposition and incubation during 3 hours, surfaces were washed five times with 100 µL drops of isotonic sterile solution (NaCl 0.9%) and dried under a laminar air flow. Ten infrared spectra were acquired by PM-RAIRS on each sample in order to scan their entire surface and collect the signal from all the adhered bacteria. All measurements (on bare gold and modified surfaces) had been done during the same experimental session, thus reducing at minimum intensity variations of the background, beam intensity and alignment. The relative amounts of bacteria adsorbed were evaluated by considering the amide bands area, the bacteria IR fingerprints. The areas were integrated from 1700 cm -1 to 1500 cm -1 to include the whole signal due to amides I and II contributions, respectively at 1660 cm -1 and 1550 cm -1 . The attachment of bacteria onto the different surfaces is expressed as a percentage compared to gold substrates: Ad(%) = 100 *(area of amide bands on sample) / (area of amide bands on gold). The uncertainty attached to this percentage comes from the propagation of the uncertainties attached to the measurement of amide bands area on both the considered surface and the gold substrate. These results were confirmed by repeating the same procedure above on 3 different set of samples.

Observation of bacterial morphology by microscopy

Qualitative analysis of the bacterial morphology was done by mean of AFM and SEM-FEG microscopies, which enables to visualize the effect of antimicrobial products on bacteria and could help to identify general target sites [START_REF] Brogden | Antimicrobial Peptides: Pore Formers or Metabolic Inhibitors in Bacteria?[END_REF] . Since SEM-FEG observations are done under vacuum, samples were previously treated as follows: after the incubation period, 12 µL of formaldehyde at 37% were added in order to fix the bacteria and avoid collapsing of bacteria upon drying. After 15 minutes, samples were washed six times with 100 µL drops of filtered ultrapure water to remove non-adhered bacteria and dried under a laminar air flow.

Evaluation of bacteria viability by fluorescent staining

Damaged bacteria on the surface were counted using the Live/Dead Bacterial Viability Kit (BacLight©). 1.5 µL of each fluorescent stains, Syto9 and propidium iodide (PI), were added to 1 mL of ultrapure water. After incubation, surfaces were rinsed five times using 100 µL drops of an isotonic sterile solution and 10 µL of the fluorochrome solution were deposited on the surfaces. Samples were then left during 10 minutes in dark and at room temperature prior to microscopy analysis.

Surfaces were always kept under a humid environment during the whole time of the experiments to avoid water evaporation. Samples were then examined with an epifluorescence microscope (AXIO 100 Zeiss). Images were acquired with a 10 or 40 objective lens and recorded with a CCD camera (AxioCam MRm Zeiss). Fluorochromes were respectively excited and detected at 455-495 nm and 505-555 nm (for Syto9) and at 533-558 nm and 570-640 nm (for PI). About ten different locations of each surface were analyzed in order to have statistically relevant data; this experiment was conducted on 3 sets of samples and at least a thousand of bacteria were then enumerated. Bacterial counting (red: damaged membrane; green: intact membrane) was done using the software Image J and the viability is calculated as follows: Viability(%) = 100 * (number of green bacteria) / (number of green bacteria + number of red bacteria).

Bacteria growth capacity

The evaluation of bacterial growth capacity after being in contact with the samples completes the characterization data set and was done as follows: after bacterial deposition and incubation during 3 hours, the surfaces were washed five times with 100 µL drops of isotonic sterile solution. Samples were then transferred into a sterile tube containing 2 mL of isotonic sterile solution (NaCl at 0.9 %) and sonicated 5 minutes. The gold sample was removed and bacteria suspensions were diluted 100 and 1000 times. 50 µL of each dilution was deposited in duplicate on petri dishes filled with BHI agar (15 g/L). The plates were incubated at 37°C overnight before enumeration. Results are expressed in percentages of the number of attached and cultivable bacterial cells onto the different surfaces as compared to gold substrates: Growth(%) = 100* (number of colonies forming unity on sample) / (number of colonies forming unity on bare gold substrate). These tests were done in triplicate on each type of samples and the percentage of growth was averaged over the three samples. The uncertainty attached to these results follows from the statistical analysis of these repeated experiments.

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) determination

The MIC values of sophorolipids and derivatives used for surface grafting were determined using a liquid growth inhibition assay. An exponential-phase bacterial culture of Listeria ivanovii was diluted in broth to an optical density of 0.01 (10 6 CFU/mL). 50 µL of this bacterial suspension were mixed with 50 μl of two-fold serial aqueous dilutions of each product (final concentrations of sophorolipids and derivatives from 2 to 32 mg/mL). As OA, SA and SL0 are not soluble in water, these products were first solubilized into 10% of DMSO. The bacterial growth was monitored by measuring the change in optical density. This test was performed in triplicate with positive (0.7% formaldehyde) and negative (isotonic solution) inhibition controls. MIC is expressed as the lowest concentration that completely inhibits bacterial growth after overnight incubation.

The bacterial suspension with a concentration of sophorolipid superior or equal to the MIC are diluted and deposited on agar plate in order to determine the Minimum Bactericidal Concentration (MBC). The MBC is expressed as the lowest concentration for which no colony forming unit are observed on agar plates after overnight incubation.

Results

SL grafting on Au(111)

Figure 2 shows the PM-RAIRS spectra of gold surfaces after each functionalization step. After treatment in the cysteamine solution and rinsing, a typical IR spectrum of aminothiol is observed, with the expected absorption bands of NH and CH2 groups as described in literature [START_REF] Yala | Elaboration of Antibiofilm Materials by Chemical Grafting of an Antimicrobial Peptide[END_REF] The grafting of cysteamine is also confirmed by XPS analyses (bottom spectra in Figure S1 in the Supporting Information section). Then, the PM-RAIRS spectrum of the SL-modified sample (Au-cys-SL) shown in

Antimicrobial activity

Surface antimicrobial properties

In order to evaluate the antimicrobial activity of the SL-functionalized gold surfaces, we have combined AFM, SEM-FEG with epifluorescence microscopy and microbial viability tests, on both SLmodified (Au-cys-SL) and SL-free gold surfaces (Au and Au-cys). SEM-FEG is a fast screening technique to evaluate eventual morphological changes of the microorganisms. To avoid possible artifacts due to vacuum conditions, bacteria have been fixed on the wet surfaces with formaldehyde, known to cross-link the membrane proteins in their current state. Moreover, we always deposit bacteria on bare gold substrate as a control sample. On the contrary, imaging by AFM, a non-invasive microscopy technique commonly used to explore mechanical stress on biointerfaces, was performed on as deposited, non-cross linked, bacteria under ambient temperature and pressure conditions.

AFM and SEM-FEG images in Figure 3 show L. ivanovii deposited on SL-free samples (Au and Au-cys).

Bacteria are of about 1 µm in length and exhibit the expected elongated rod shape [START_REF] Robichon | Atomic Force Microscopy Imaging of Dried or Living Bacteria[END_REF] . On the SEM-FEG image, the plasma membrane of the Gram+ bacteria appears as a white line surrounding the ovoidal shape of the microorganism indicating that contact with these surfaces does not alter the cell envelope. On the contrary, on the SL-modified surfaces (Au-cys-SL), bacteria do not have their native oval shape and the cell envelopes are damaged and appear pierced, causing the leakage of the cytoplasmic content and the collapse of the bacteria, as indicated by the arrows in Figure 3 (Au-cys-SL). These observations show direct evidence that the gold surface-grafted with SL damages the plasma membrane of L. ivanovii in agreement with the antibacterial effect reported for sophorolipids in solution [START_REF] Lang | Antimicrobial Effects of Biosurfactants[END_REF][START_REF] Kim | Characteristics of Sophorolipid as an Antimicrobial Agent[END_REF][START_REF] Azim | Amino Acid Conjugated Sophorolipids: A New Family of Biologically Active Functionalized Glycolipids[END_REF] To confirm these findings and quantify the number of damaged bacteria, bacterial staining with fluorescent markers, Syto9 and PI, was conducted in such way that green-stained bacteria indicate an intact plasma membrane, while red-stained bacteria show damaged cell membrane. Our results

show that less than 5% of bacteria are damaged on both Au and Au-cys surfaces while almost half of the bacteria population has a damaged membrane on the Au-cys-SL sample (Figure 3).

However, epifluorescence microscopy does not constitute a direct proof of bacteria viability or death [START_REF] Roszak | Survival Strategies of Bacteria in the Natural Environment[END_REF] . Conversely, bacteria were also cultivated on agar plate after having been in contact with the modified surfaces in order to count the actual number of bacterial colonies. A drastic decrease of ca 45% of bacterial growth was systematically observed after contact with SL-containing surfaces compared to the Au and Au-cys samples (Growth (%): Au= 100%, Au-cys= 95%, Au-cys-SL= 54%).

These numbers are in very good agreement with the fluorescent staining (Intact bacteria (%):

Au= 98%, Au-cys= 96%, Au-cys-SL= 58%) and confirm the antibacterial effect of SL-modified surfaces via membrane lysis. Note in addition that the rate of damaged bacteria stays constant over time from 1 hour up to at least 6 hours in contact with the surface. Similarly, we have found the same biocidal efficiency at different growth stages (exponential phase or stationary phase) of L. ivanovii.

Understanding the origin of biocidal effect of SL

The data presented so far put in evidence several striking facts: 1) surface-grafted SL keep their biocidal action against Gram+ Listeria ivanovii; 2) the killing efficiency is in the 40 % -45 % range;

3) the apparent mechanism of action occurs through membrane lysis, as described for the same compounds (and related glycolipids) in aqueous solutions [START_REF] Chapman | Biocide Susceptibility and Intracellular Glutathione in Escherichia Coli[END_REF][START_REF] Glover | An Epr Investigation of Surfactant Action on Bacterial Membranes[END_REF][START_REF] Lang | Antimicrobial Effects of Biosurfactants[END_REF][START_REF] Kim | Characteristics of Sophorolipid as an Antimicrobial Agent[END_REF] . The persistence of the antimicrobial properties of SL after their grafting onto a surface demonstrates that the carboxylic acid group may not play a key role in their antibacterial property, since the COOH is neutralized by the anchoring strategy. This is in line with the comparisons of lactone-type and acid-type sophorolipids activity in solution, which show that the former has a higher antimicrobial activity [START_REF] Kim | Characteristics of Sophorolipid as an Antimicrobial Agent[END_REF] .

To put in evidence the role of the sophorose unit, we have controlled the possible biocidal action of OA-grafted gold surface. In addition, to understand the potential role of the conformation of sophorose at the outer surface we have tested the biocidal action of the fully saturated form of acidic sophorolipids (SL0) and the corresponding fatty acid (SA), as shown in Figure 1. It is, in fact, known that the anomer conformations of a synthetic glycolipids strongly influence their antibacterial activity in solution; for instance, the alpha anomer of the lauric ether derivative of methyl glucopyranoside has a MIC of 0.04 M, which is roughly 100 times smaller (hence, more efficient) than the corresponding beta anomer [START_REF] Nobmann | The Antimicrobial Efficacy and Structure Activity Relationship of Novel Carbohydrate Fatty Acid Derivatives against Listeria Spp. And Food Spoilage Microorganisms[END_REF] . For these reasons, even if OA and SA are not supposed to have antibacterial effects in solution [START_REF] Kabara | Fatty Acids and Derivatives as Antimicrobial Agents[END_REF] , and since we are not aware of such properties for SL0, we have tested the MIC in solutions of these compounds. Using DMSO to improve solubility, and then diluting the solution in water, SL0, OA and SA do not exhibit any inhibitory effect, even at high concentrations (32 mg/mL, i.e. eight-fold above the MIC value found in this work for SL, 4 mg/mL) and bacteria show their oval native shape and intact plasma membranes when observed by SEM-FEG (images not shown). Nevertheless, since SL0 forms supramolecular water-insoluble fibers [START_REF] Cuvier | Ph-Triggered Formation of Nanoribbons from Yeast-Derived Glycolipid Biosurfactants[END_REF] , it is difficult to obtain reliable antibacterial data in solution and therefore the corresponding grafted surfaces appear as an interesting tool to explore the interactions between these molecules and bacteria.

OA, SA and SL0-grafted surfaces have been elaborated and characterized following the same procedure as the one used for SL grafting (see description above): the corresponding infrared and XPS data (shown in Figure S3 and Figure S4 in the Supporting Information) confirm the grafting of SL0 on gold in equivalent amounts that SL (both layers have equivalent thickness, 30.0±2.0 Å for SL and 31±5 Å for SL0, according to estimation based on XPS analyses -see Supporting information), as well as OA and SA, making the comparison of antibacterial properties of these surfaces possible. Bacteria exhibit their native oval shape on SEM images and look intact on all samples. The harmless features of these surfaces is confirmed by fluorescent staining: more than 97% of the adhered bacteria appear green after fluorescent staining, meaning that their membrane was not damaged by any of the tested compounds. The corresponding colony counting data after exposure are reported in Figure 5 (green bars); however, these data must be weighted by the adhesion (% with respect to bare gold) of the bacteria onto the surfaces (grey bars in Figure 5). In fact, the adhesion capacity of L.

ivanovii is not constant on all modified surfaces. When in contact with SL-modified surfaces, the bacterial adhesion is very similar (95 %-100 %, within the experimental error bars) to what it is observed onto gold, but their growth is reduced by approximately 50 %. This difference nicely confirms, from a quantitative point of view, the biocidal action of grafted SL, previously highlighted by fluorescent staining. Interestingly, on OA-modified surfaces, both adhesion and growth are reduced by ~40 % with respect to the gold surface, thus meaning that this surface has a slight antiadhesive effect, but it is not biocidal, as also highlighted by the fluorescence staining experiments, cf.

Figure 4. Finally, the adhesive nature of SL0 and, at a lower extent, the one of SA are higher than bare Au or SL surfaces (the adhesion on those surface being respectively estimated to 160 and 130 % as compared to gold); the growth counting, which is found to be almost 60 % and 30 % higher with respect to gold for SL0 and SA, respectively, is then solely attributed to the adhesive properties of these surfaces. In order to better understand the interactions between bacteria and surfaces, water contact angles on the modified surfaces have been determined (Figure 6) as it may influence the contact between bacteria and surfaces, and hence the biocidal effect. The gold surface becomes more hydrophobic after functionalization with OA and SA, for which the water contact angles are respectively 55° ± 2° and 62° ± 2°. The sophorose units, hydroxyl-rich watersoluble disaccharides, of SL lead to an expected decrease of the contact angle with respect to OA from 55° ± 2° to 48° ± 2°, whereas the contact angle increases from 62° ± 2° to 71° ± 2° when passing from SA to SL0. The antinomic effect in terms of surface wetability between SL (48° ± 2°) and SL0 (71° ± 2°) is unexpected, if one looks at the chemical nature of both quasi identical molecules.

Nevertheless, this is not surprising; examples of conformational changes strongly affecting the wetability of coatings exist for peptides, where the chirality sense of the peptide backbone governs the hydrophilic/hydrophobic character of the coated surface [START_REF] Qing | Chirality-Driven Wettability Switching and Mass Transfer[END_REF] . It is also known that glucose-based surfaces/compounds can display different wetability properties. Cyclodextrins, for instance, are water-soluble compounds with a hydrophobic cavity [START_REF] Martinez | Cyclodextrin-Based Multivalent Glycodisplays: Covalent and Supramolecular Conjugates to Assess Carbohydrate-Protein Interactions[END_REF] , as the orientation of the C-OH groups in glucose identifies regions of different water-loving properties [START_REF] Hoja | Adsorption of Glucose, Cellobiose, and Cellotetraose onto Cellulose Model Surfaces[END_REF] . A striking example is constituted by two allomorphs of cellulose, Iα and Iβ, the (100) surfaces of which are, respectively, hydrophilic and hydrophobic [START_REF] Hoja | Adsorption of Glucose, Cellobiose, and Cellotetraose onto Cellulose Model Surfaces[END_REF] . The stronger wetability in Iα was demonstrated to come from both the larger exposure of the C-OH groups of glucose and more prominent surface roughness while the glucose moieties of the Iβ are parallel to the surface (low roughness) and more C-OH groups are involved in hydrogen bonding with their close neighbors. In the case of SL-modified surfaces, we then believe that the double bond present in the SL molecule favors the preferential exposure of C-OH groups compared to the SL0 coatings. Interestingly, if one lays SL and SL0 molecules on a flat surface respecting the theorethical bond lengths, dihedral angles and chirality for both the sugar and C17 atom in the aliphatic chain, it is then possible to see that the actual orientation of the C-OH groups is orthogonal to the surface for SL, while it is parallel to it for SL0, as shown in Figure 7, and thus explaining the hydrophilicity of the SL-containing substrate if compared to the SL0 substrate. In addition, the bending of the SL molecule may increase local disorder and a loss of molecular packing, similarly to differences of molecular packing and water-solubility encountered in solution: at room temperature and under acidic conditions, SL forms micelles [START_REF] Baccile | Practical Methods to Reduce Impurities for Gram-Scale Amounts of Acidic Sophorolipid Biosurfactants[END_REF] (water-soluble) while SL0 forms waterinsoluble crystalline fibers with nanoscale chirality [START_REF] Cuvier | Ph-Triggered Formation of Nanoribbons from Yeast-Derived Glycolipid Biosurfactants[END_REF] . Discussion.

By comparing the bacteria morphology and viability on OA, SA, SL0 and SL grafted samples, one can safely assert that only surfaces with the unsaturated form of sophorolipids have a biocidal activity (to this regard, please also see the comment in the PM-RAIRS section, page 4, of the SI). The simultaneous presence of the hydrophilic sophorose group and the double bonds appears to be necessary to induce bacterial killing. These two structural features have also a crucial influence on several physico-chemical properties. Sophorose is a bulky disaccharide and its steric hindrance influences the conformation of SL: the surface exhibits a loose packing as observed for antimicrobial peptides chemically grafted on thiolated self assembled monolayers, for instance [START_REF] Yala | Elaboration of Antibiofilm Materials by Chemical Grafting of an Antimicrobial Peptide[END_REF] . The presence of the double bond induces curvature in the alkyl chain layer, thus contributing to the loose, probably defect-rich, packing of the SL compound. The orientation of sophorose is also directly related to the constraints imposed by the C=C double bond (120° angle), and so is influencing the intermolecular hydrogen bonding network. The use of a fully saturated (stearic) alkyl chain in SL0 has several important consequences; it is in fact known that molecular packing is tighter for stearic acid based compound [START_REF] Small | Lateral Chain Packing in Lipids and Membranes[END_REF] . The so different values of the water contact angles of Au-cys-SL and Au-cys-SL0 suggest that the orientation of the sophorose is different on gold, maybe correlated to the so different biocidal properties (40% to 50% growth inhibition on SL modified-surfaces compared to almost none on SL0 modified-surfaces). Consequently, a variability of the killing-by-contact efficiency could be due to two combined reasons: the disaccharide orientation and local disorder.

To understand the importance of these findings, one must recall the role of carbohydrates in biocidal compounds, often mentioned but never clarified. In the first place, the biocidal properties of glycolipids have always been found for free molecules in solution, where it is impossible to dissociate the action of the sugar from its covalently bonded backbone. Nevertheless, some works show that the efficiency against a given organism depends on the nature of the sugar. For instance, sucrose monolaurate is less effective towards Enterococcus faecalis than lactose monolaurate, but their activities are comparable towards Listeria monocytogenes [START_REF] Devulapalle | Effect of Carbohydrate Fatty Acid Esters on Streptococcus Sobrinus and Glucosyltransferase Activity[END_REF][START_REF] Wagh | Effect of Lactose Monolaurate on Pathogenic and Nonpathogenic Bacteria[END_REF] . Besides, a stronger piece of evidence that the nature of the carbohydrate has an important role was reported by Nobmann et al. on the effect of alpha or beta anomer on the MIC, as discussed previously [START_REF] Nobmann | The Antimicrobial Efficacy and Structure Activity Relationship of Novel Carbohydrate Fatty Acid Derivatives against Listeria Spp. And Food Spoilage Microorganisms[END_REF] .

When polysaccharides or sugars are grafted on a surface, the main reported effect concerns the enhancement, or reduction, of microorganisms adhesion, but no biocidal effect [START_REF] Kesel | Carbohydrate Coating Reduces Adhesion of Biofilm-Forming Bacillus Subtilis to Gold Surfaces[END_REF] . The decrease of bacterial viability observed on gold samples modified with monounsaturated sophorolipids suggests a killing-by-contact action related to the presence of covalently grafted molecules, whereas the hypothetical role of micelles is excluded, since the initial sophorolipid concentration is of 50 µg.mL -1 , a value which is well below the critical micelle concentration (~110 µg.mL -1 ) [START_REF] Imura | Enzymatic Conversion of Diacetylated Sophoroselipid into Acetylated Glucoselipid: Surface-Active Properties of Novel Bolaform Biosurfactants[END_REF] . In addition, as shown by Quartz Crystal Microbalance experiments, the grafted layer remains stable after the rinsing procedure performed at the end of the grafting (cf. Figure S2 in Supporting Information). In any case, even if all sophorolipids present at the gold surface after rinsing (~180 ng according to QCM experiments, as exposed in the Supporting Information section) are released into the 100 µL drop of bacterial suspension used in the antibacterial tests, the concentration would be less than 2 µg.mL -1 , thus assuring to be below the typical MIC and the MBC for free sophorolipids (respectively 4 and 32 mg.mL -1 , as measured in this study). Excluding the hypothesis according to which bacteria may be killed by residual free sophorolipid molecules, it also automatically excludes the fact that grafted sophorolipids act via the classical "surfactant effect", as described in solution for many other glycolipids, and more in general surfactant molecules, or even peptides 21-23, 28, 46 . Here SL can only interfere with the bacterial cell envelope through the sophorose moiety, which is not able to diffuse neither through the cell wall nor the plasma membrane. We also make the assumption that sophorolipids, characterized by an ether covalent bond between sophorose and oleic acid (etherbased glycolipids are known to be stable during their antibacterial activity [START_REF] Nobmann | The Antimicrobial Efficacy and Structure Activity Relationship of Novel Carbohydrate Fatty Acid Derivatives against Listeria Spp. And Food Spoilage Microorganisms[END_REF] ), are stable at the gold surface towards potential hydrolysis by bacterial esterase. Another favorable argument consists in the fact that if sophorolipids would be hydrolyzed by L. ivanovii, we would not expect an antibacterial activity at all, as the residual oleic acid moiety has none. The killing process is then directly related to the contact between the cell envelope and the sophorose headgroup. Moreover, there is a general consensus on the fact that the most effective biocidal chemical groups (e.g. antimicrobial peptides, alkylammonium derivatives) are able to approach and pierce the cell wall thanks to their rigid and electrically-charged nature. The action of cationic polymers for instance is usually described as disrupting the lipidic membranes of bacteria through electrostactic and hydrophobic interactions [START_REF] Siedenbiedel | Antimicrobial Polymers in Solution and on Surfaces: Overview and Functional Principles[END_REF] .

Similar mechanisms are also suggested for antimicrobial peptides [START_REF] Brogden | Antimicrobial Peptides: Pore Formers or Metabolic Inhibitors in Bacteria?[END_REF] . In comparison, because of their electro-neutrality, monounsaturated sophorolipids have little chance to cause membrane lysis after grafting. We believe that the origin of their biocidal effect is rather related to specific sophorose/cell envelope interactions. However, these have never been described in the literature.

To understand the origin of cell envelope damage in our system, we thus make two hypotheses, biochemical and/or biophysical (or both simultaneously). The biochemical pathway refers to a possible interaction between sophorose and the microorganism which triggers a (series of) biochemical response(s) responsible for the cell envelope rupture. This is the classical way penicillin antibiotics work by inhibiting the transpeptidase, enzymes responsible for the cross-linking of the peptidoglycan layer. [START_REF] Yocum | The Mechanism of Action of Penicillin. Penicillin Acylates the Active Site of Bacillus Stearothermophilus D-Alanine Carboxypeptidase[END_REF] Similar arguments are also used to explain the mechanism of action of the moenomycin antibiotic, a well-known glycolipid, and its derivatives. They inhibit the in vitro transglycosylation reaction in E. coli, thus blocking the synthesis of the cell wall and its eventual death, with or without lysis [START_REF] Baizman | Antibacterial Activity of Synthetic Analogues Based on the Disaccharide Structure of Moenomycin, an Inhibitor of Bacterial Transglycosylase[END_REF][START_REF] Van Heijenoort | Polymerization by Transglycosylation in the Biosynthesis of the Peptidoglycan of Escherichia Coli K 12 and Its Inhibition by Antibiotics[END_REF][START_REF] Van Heijenoort | Biosynthesis of the Peptidoglycan of Escherichia Coli K-12: Properties of the in Vitro Polymerization by Transglycosylation[END_REF] . In addition, a potential role as signaling molecules that regulate gene expression has been discussed to explain the antiadhesive properties of some bacterial exopolysaccharides [START_REF] Bernal | Promising Biotechnological Applications of Antibiofilm Exopolysaccharides[END_REF] . If these specific mechanisms do not seem to be applicable to the case of surface-grafted SL against Listeria (since it seems difficult for anchoring SL to interfere with the inner metabolism of the bacteria), similar sophorose-induced biochemical pathways may not be completely excluded, even though unknown at the moment. The biophysical pathway refers to a mechanical, destabilizing, interaction between the carbohydrate and the Listeria cell envelope, which is mainly constituted by a peptidoglycan and phospholipid layers [START_REF] Ghosh | Isolation, Composition, and Structure of Membrane of Listeria Monocytogenes[END_REF][START_REF] Vazquez-Boland | Listeria Pathogenesis and Molecular Virulence Determinants[END_REF] . If the action of sophorose on the cell envelope is out of doubt, the way such interaction occurs is still unknown. Specific literature data neither exist on this specific system nor, to the best of our knowledge, on the more general destabilization of the peptidoglycan layer by mono-and disaccharides. As far as an action on the plasma membrane, the lysis of which is put in evidence here by the epifluorescence observations (if PI reaches the nucleic acid of red bacteria, it means their plasma membrane is damaged), one could formulate two different mechanistic hypotheses, admitting the previous interpenetration of the cell wall: 1) intra-membrane sugar intercalation [START_REF] Crowe | Interactions of Phospholipid Monolayers with Carbohydrates[END_REF][START_REF] Lenné | How Much Solute Is Needed to Inhibit the Fluid to Gel Membrane Phase Transition at Low Hydration?[END_REF][START_REF] Andersen | Reconciliation of Opposing Views on Membrane-Sugar Interactions[END_REF] , perturbing the local liquid crystalline order, as observed in the case of other non-reducing disaccharides (e.g., trehalose and sucrose) [START_REF] Moiset | Disaccharides Impact the Lateral Organization of Lipid Membranes[END_REF] .

2) Membrane destabilization mediated by recent fundamental studies by Abbott et al. go in this sense, showing the destabilizing effects the macromolecular adsorbents of biological relevance (e.g., proteins) may have on model phospholipid liquid crystal membranes [START_REF] Brake | Biomolecular Interactions at Phospholipid-Decorated Surfaces of Liquid Crystals[END_REF] . They have shown that not only weak binding of proteins drive the reorganization of the phospholipids and trigger orientational transitions in the liquid crystals, but also that similar events can also occur by ligand (biotin)-receptor (streptavidin) recognition phenomena, where streptavidin is contained in the liquid crystal layer and biotin in an external phospholipid vesicles approaching it [START_REF] Tan | Ordering Transitions Triggered by Specific Binding of Vesicles to Protein-Decorated Interfaces of Thermotropic Liquid Crystals[END_REF] . It is not known at the moment if any of these hypotheses, generally formulated in the case of interactions between free molecules and a cell envelope, can be applied to the present biocidal effect of surfaces-grafted sophorolipids and it deserves more fundamental studies. Besides, whatever the mechanism, the main reason for which the saturated SL0 sophorolipid does not seem to have any biocidal property could be multiple: poor accessibility of individual surface sophorose groups, enhanced bacterial adhesion, both limiting the accessibility of sophorose and consequent interaction with membrane penetration.

Conclusion

This paper reports the conception of an innovative biocidal coatings using carbohydrates immobilized on a surface. Sophorolipids, yeast-derived bolaform biosurfactants, were successfully attached to a model flat gold surface modified by a thiolamine primer through amidation of their COOH group. The resulting glycosylated surface, where sophorose (glucose β-1,2), a non-reducing disaccharide, lays on top of the surface, shows a biocidal activity towards Listeria ivanovii bacteria. So far, glycosylated surfaces have been known for their anti-adhesive properties. A combination of microscopy tools shows that bacteria are killed through membrane lysis. Bacterial growth and adhesive tests show that between 40 % and 50 % of exposed bacteria are damaged; these values are coherent with the quantitative data obtained from fluorescent staining. To better understand the mechanism of action, the effect of sophorolipids was compared to rational molecular variations of this compound: oleic acid, constituting the aliphatic backbone of sophorolipids, fully saturated sophorolipids and stearic acid, the aliphatic component of the latter, have been grafted on gold and no biocidal activity could be detected. This comparative study then underlines the crucial role played by sophorose but it also highlights the importance of the aliphatic chain configuration, influencing the substrate wettability, bacterial adhesion and biocidal effects. If this first example of a biocidal effect from a glycan array demonstrates the membrane disrupting properties of a non-reducing disaccharide, the actual mechanism of interaction between sophorose and the bacterial membrane is still unknown, and it may not still be confined to this particular sugar alone.

Financial Support

Figure 1 .

 1 Figure 1. Functionalization of gold surfaces with oleic acid (OA), acidic monounsaturated sophorolipid (SL), acidic fully saturated sophorolipid (SL0) and stearic acid (SA). Cysteamine (cys) constitutes the primer for all compounds.

  17-L-([2-O-β-D-glucopyranosyl-β-D-glucopyranosyl]-oxy)octadecenoic acid. Drawing and structure relaxation was done with the Gaussview software, version 2.1 while the images have been done using the PyMOL molecular graphic system, version 1.3.

Figure 2 Figure 2 .

 22 Figure 2 exhibits the typical vibrations expected for SL, as shown by the comparison with the corresponding IR-ATR powder spectrum, as well as the signature of the amide group at 1648 cm -1(νC=O) and 1538 cm -1 (νCN & δNH). These results confirm that the SL molecules are covalently grafted on the outer NH2 group of cysteamine via an amidation reaction with the COOH moiety of SL. They are corroborated by XPS spectra recorded on the Au-cys-SL sample (top spectra in FigureS1in the Supporting Information section). The additional weak band at 1738 cm -1 on the PM-RAIRS spectrum

Figure 3 .

 3 Figure 3. Bacteria (L. ivanovii) deposited on Au (left column), Au-cys (middle column) and Au-cys-SL (right column) surfaces observed by AFM (top range), SEM-FEG (middle range) and epifluorescence microscopy (bottom range). The charts below each image represent the percentage of adhering intact and damaged bacteria according to fluorescent staining. The scale bare represents 1 µm on AFM and SEM-FEG images and 50 µm on images from epifluorescence microscopy.

Figure 4

 4 Figure 4 displays the observations of bacteria deposit on these modified surfaces.

Figure 4 .

 4 Figure 4. Bacteria (L. ivanovii) deposited on Au-cys-OA (left column), Au-cys-SL0 (middle column) and Au-cys-AS (right column) surfaces observed by SEM-FEG (top range) and epifluorescence microscopy (bottom range). The charts below each image represent the percentage of adhering intact and damaged bacteria according to fluorescent staining. The scale bares represent 1µm on AFM images and 50 µm on images from epifluorescence microscopy.

Figure 5 .

 5 Figure 5. Adhesion (grey bars) and bacterial growth (green bars) of Listeria ivanovii after contact with Au-cys-OA, Au-cys-SL, Au-cys-SL0 and Au-cys-SA surfaces as compared to Au substrates.

Figure 6 .

 6 Figure 6. Water contact angle on bare gold surface and on functionalized surfaces.

Figure 7 .

 7 Figure 7. SL (left) and SL0 (right) lying on top of a model surface shown with highlighted C-OH group of sophorose using the sphere representation (Oxygen: red; Hydrogen: white; Sulfur: yellow; Nitrogen: green).

  This work was supported by French state funds managed by the ANR within the Investissements d'Avenir programme under reference ANR-11-IDEX-0004-02, and more specifically within the framework of the Cluster of Excellence MATISSE.

  .

		Au	Au-cys	Au-cys-SL
	AFM
	SEM FEG
	Fluorescent staining	damaged 2%	damaged 4%	damaged 42%
		intact 98%	intact 96%	intact 58%

Acknowledgements

The authors acknowledge IMPC (Institut des Matériaux de Paris Centre, FR2482) and the C'Nano projects of the Region Ile-de-France, for SEM-FEG and Omicron XPS apparatus funding. Christophe Méthivier is deeply acknowledged for his important help in XPS calculations. Frederik Tielens (LCMCP, Université Pierre et Marie Curie, Paris, France) is kindly acknowledged for his help in molecular drawing in the SI. Bastien Wild (LHYGES, Université de Strasbourg, France) is kindly acknowledged for his initial help in the experimental part.

Associated Contents

Supporting Information showing detailed XPS, QCM and PM-RAIRS additional data and analyses of SL, SL0, OA and SA on Au-cys substrates. The material is available free of charge via Internet at http://pubs.acs.org.