Hocquet
email: dhocquet@chu-besancon.fr

Charlotte Couchoud

Hygiène Hospitalière

Genome-based typing reveals rare events of patient contamination with Pseudomonas aeruginosa from other patients and sink traps in a medical intensive care unit

Keywords: Pseudomonas aeruginosa, whole-genome sequencing, phylogeny, cross-transmission, medical intensive care unit

Genome-based typing reveals rare events of patient contamination with Pseudomonas aeruginosa from other patients and sink traps in a medical intensive care unit

Introduction

P. aeruginosa (PA) is an opportunistic Gram-negative bacillus that can thrive in a wide variety of niches. Hence, PA is widespread in soil and water and frequently found in the environment, including in the wastewater evacuation network of hospitals (1). PA is also one of the most frequent species responsible for nosocomial infection in Europe and the USA (2,3). In intensive care units (ICUs), 10 to 15% of healthcare-associated infections are attributed to this pathogen (1). Such infections consist mostly of ventilatorassociated pneumonia or bacteremia, associated with high mortality [START_REF] Lynch | Emergence of antimicrobial resistance among Pseudomonas aeruginosa: Implications for therapy. Seminars in respiratory and critical care medicine[END_REF]. A high number of such infections are nosocomial, especially among mechanically ventilated patients [START_REF] Bukholm | An outbreak of multidrug-resistant Pseudomonas aeruginosa associated with increased risk of patient death in an intensive care unit[END_REF]. The PA genome can also readily acquire genetic material and thus gain new antibiotic resistance (6).

The rate of colonization by PA is low (2.3%) among healthy humans, more frequent among patients admitted to ICUs (4.1 to 11.6%), and can reach higher rates (57.8%) during hospitalization [START_REF] Botzenhart | Ecology and epidemiology of Pseudomonas aeruginosa[END_REF](8)[START_REF] Harris | Pseudomonas aeruginosa colonization in the intensive care unit: prevalence, risk factors, and clinical outcomes[END_REF][START_REF] Thuong | Epidemiology of Pseudomonas aeruginosa and risk factors for carriage acquisition in an intensive care unit[END_REF]. ICU patients can acquire PA from their environment and other patients, directly or via the hands of healthcare workers (8,[START_REF] Jefferies | Pseudomonas aeruginosa outbreaks in the neonatal intensive care unit-a systematic review of risk factors and environmental sources[END_REF]. Hence, PA can contaminate respiratory equipment, endoscopes, and sections of the hospital water network, such as faucets, shower drains, and sink traps (also known as U-bends or P-traps) [START_REF] Walker | Investigation of healthcare-acquired infections associated with Pseudomonas aeruginosa bio lms in taps in neonatal units in Northern Ireland[END_REF][START_REF] Aumeran | Pseudomonas aeruginosa and Pseudomonas putida outbreak associated with contaminated water outlets in an oncohaematology paediatric unit[END_REF][START_REF] Yapicioglu | Pseudomonas aeruginosa infections due to electronic faucets in a neonatal intensive care unit[END_REF][START_REF] Ambrogi | Transmission of metallo-b-lactamase-producing Pseudomonas aeruginosa in a nephrology-transplant intensive care unit with potential link to the environment[END_REF](16). The proportion of sink traps contaminated with PA varies from 15 to 50% in European ICUs and studies have reported that 7 to 50% of patients acquire PA from water points of use [START_REF] Cholley | The role of water ttings in intensive care rooms as reservoirs for the colonization of patients with Pseudomonas aeruginosa[END_REF]18). In addition, investigations of hospital outbreaks have identi ed the water supply system as the source of the PA outbreaks [START_REF] Kinsey | Pseudomonas aeruginosa outbreak in a neonatal intensive care unit attributed to hospital tap water[END_REF]. This has led to infection control departments to recommend sink trap disinfection or sink removal or redesign in high-risk wards, such as ICUs and hematology units [START_REF] Hota | Outbreak of multidrug-resistant Pseudomonas aeruginosa colonization and infection secondary to imperfect intensive care unit room design[END_REF][START_REF] Catho | Outbreak of Pseudomonas aeruginosa producing VIM carbapenemase in an intensive care unit and its termination by implementation of waterless patient care[END_REF].

The distribution of the sources of PA (endogenous, environmental, other patients) varies greatly between studies because of differing infection control procedures (contact precautions, sink trap disinfection). In addition, discrepancies in study conclusions may also result from differences in sampling protocols and bacterial typing methods. Hence, genome-based typing identi es transmission routes of pathogens with a higher accuracy than older typing methods, which probably overestimate the number of transmission events [START_REF] Salipante | Application of whole-genome sequencing for bacterial strain typing in molecular epidemiology[END_REF][START_REF] Lytsy | Time to review the gold standard for genotyping vancomycin-resistant enterococci in epidemiology: Comparing whole-genome sequencing with PFGE and MLST in three suspected outbreaks in Sweden during 2013-2015. Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases[END_REF].

We aimed to identify the acquisition pathways of PA by patients in a medical ICU (MICU) in which patients positive for PA were managed according to the recommendations of the French Society of Infection Control and, additionally, in which sink traps were disinfected with bleach twice a week. We sampled patients and sink traps for 10 months to collect 519 PA isolates, for which the genomes were entirely sequenced and compared at the nucleotide level. Such analysis elucidated the precise network of PA transmission in this MICU and identi ed the routes of PA acquisition by the patients.

Methods

Study characteristics. This prospective monocentric study took place in a 15-bed MICU between January and November 2019 in a university hospital in France. This MICU has 11 individual rooms distributed within three subunits (A, B, and C) with one water point of use and a four-bed room with two water points of use (Fig. 1). The MICU admits ~ 700 patients/year and all 549 patients admitted during the time of the study were included. Each patient received care from ~ 10 members of the healthcare staff per day.

Healthcare workers were dedicated to a subunit but could help in another subunit when needed. We conducted this study in the absence of an identi ed outbreak of PA.

Infection control procedures. All 13 water points of use were equipped with a lockable sink trap (Geberit, France) that was bleachdisinfected twice a week and at patient discharge. Brie y, locked sink traps were treated 15 min with 20 ml 2.6% liquid bleach and then rinsed with tap water. This unit systematically applies standard precautions of hygiene according to the recommendations of the French Society of Infection Control [START_REF] Sfhh | Recommandations nationales -prévention de la transmission croisée: Précautions complémentaires contact[END_REF]. Bacteriological methods. Patients admitted to the MICU were screened for PA carriage upon admission and twice a week thereafter using nasal swabs, rectal swabs, and tracheal aspirates when intubated. In parallel, we collected all PA isolates retrieved from diagnostic samples. All sink traps were sampled every week for PA detection by collecting 50 ml sink-trap content with a suction catheter and a syringe. The sample was centrifuged 5 min at 5000 x g at room temperature and the supernatant discarded. Swabs and pellets were streaked on PA-selective cetrimide agar plates (Bio-Rad) and incubated 48 h at 35°C. All colony phenotypes were identi ed using a MALDI-TOF mass spectrometer (Maldi Biotyper, Bruker). We assessed the activity of 13 antipseudomonal agents (listed in Supplemental Table 1) from three classes (β-lactams, aminoglycosides, uoroquinolones) by the agar diffusion method as recommended by the EUCAST (www.eucast.org). Morphologically different colonies of PA recovered from the same sink trap sample were retained for further analysis when they showed distinct resistance pro les. The resistance pro le of isolates susceptible to all tested antibiotics was considered to be wildtype. Acquired genes encoding β-lactamases (including carbapenemases) were sought within the genomic data against the ResFinder database (25).

Data. Each isolate collected was associated with its date of isolation, its patient or sink trap of origin, and its antibiotic resistance pro le. See Supplemental Material and Methods for details on bacteriological and sequence analysis. We sequenced the full genome of all PA isolates (Supplemental Table 1). Sequencing data are available in the NCBI BioProject PRJNA788732. We rst identi ed the sequence type (ST) of each isolate by MultiLocus Sequence Typing (MLST) (26). Then, isolates for which the genome contained ≤ 30 different genes were clustered into groups with Core Genome MLST (cgMLST) using 3,867 core genes [START_REF] Tonnies | Establishment and evaluation of a core genome multilocus sequence typing scheme for whole-genome sequence-based typing of Pseudomonas aeruginosa[END_REF]. We further measured the genetic relatedness of isolates within each group by the number of single nucleotide polymorphisms (SNPs) between genomes. See Supplemental Fig. 1 for the justi cation of the threshold.

De nitions. (i) Isolates from a given group were de ned as clonal when their genomes clustered with a threshold of seven SNPs. (ii) Cross-contamination was de ned by the identi cation of clonal isolates in two sampling points (patient or sink trap). (iii) In cases of cross-contamination between two sampling points ≥ 7 days apart, we de ned the older one as the source. (iv) Infections were de ned according to Sepsis-3.

Statistical analysis. The data were analyzed with R Studio (v 1.4) using the circlize and vegan libraries. Differences between the distributions of resistance pro les of the two PA reservoirs (patients and sink traps) were tested using Fisher's exact test at a threshold of 0.01. a In cases of transmission between two sampling points ≥ 7 days apart, we de ned the older one as the source.

b Isolated low-level resistance to uoroquinolones. Most of the links occurred within a given sink trap, showing that such niches were contaminated with a signature ora that was stable over time. However, we identi ed 22 cross-transmission events between sink traps of different rooms, with 10 between sink traps of different subunits (Fig. 2A).

We identi ed six events of PA cross-transmission between patients, four involving one clonal isolate and one involving two (Table 1, Fig. 2B). These events involved ve non-high-risk clones (ST198, ST274, ST1197, ST1238, ST3218) and nine patients.

Isolate ST198 group34_1 was shared by two patients hospitalized in room A1 during the same week, but whose hospitalization period did not overlap. Isolates ST1197 group25_1 and ST1238 group27_1 were transmitted between patients hospitalized during the same week in different rooms (C1 and C2 for ST1197, A1 and B5 for ST1238). Two patients from room C2 shared two isolates (ST274 group30_1 and ST3218 group15_1). The temporal proximity of the nding of these isolates prevented the identi cation of the source of contamination. In one case, we could document the direction of contamination of a patient of room C2 with the isolate ST3218 group15_1 from a patient hospitalized in room A3 (Table 1, Fig. 2B). Overall, four patients shared the isolate ST3218 group15_1: two patients were hospitalized in April 2019 in room C2 and two others six months later in rooms C2 or A3

(Table 1). We never retrieved this isolate from any trap (Supplemental Table 1).

In terms of environment-to-patient contamination, only one transmission of a PA isolate occurred from a sink trap to a patient. A high-risk ST253 clone, repeatedly found in the sink trap of rooms B2 and B3 from January to September 2019, was isolated from a patient hospitalized in May in room A3 (Table 1, Fig. 2B). In addition, we identi ed six transmission events of PA from patients to the sink traps of their rooms (Fig. 2B). The six STs involved (ST27, ST234, ST253, ST308, ST309, ST671) were transmitted in rooms A1, A2, A5, B2, and B3 (Table 1). Overall, among the 65 patients infected or colonized with PA, one patient (1.5%) acquired a PA isolate from a sink trap and ve other patients (7.7%) were contaminated with a PA isolate from another patient.

Resistance pro les of P. aeruginosa. The proportion of isolates susceptible to all antibiotics tested was higher for the PA of human origin (74.8%; 86/115) than the PA found in the sink traps (48.0%, 194/404) (Fisher's exact test, p = 2.8x10 -7 ; Supplemental Table 1). The only clone that produced extended-spectrum β-lactamase (VEB-1) belonged to ST357 and was represented by eight isolates found in the sink trap of room B1. Isolates non-susceptible to carbapenems were more frequently found in sink traps (152/404, 37.6%) than in patients (19/115, 16.5%) (Fisher's exact test, p = 1.6x10 -5 ). The 13 isolates resistant to all antibiotics tested (Supplemental Table 1) were exclusively retrieved from sink traps and clustered within two clones belonging to ST111 (room C2) and ST357 (room B1).

Four of the ve PA isolates transmitted between patients (ST274 group30_1, ST1197 group25_1, ST1238 group27_1, ST3218 group15_1) displayed wildtype resistance pro les. Of note, isolate ST3218 group15_1 was transmitted on two occasions involving four patients. The fth isolate (ST198 group34_1) displayed an isolated low level of resistance to cipro oxacin. Finally, the isolate ST253 group0_6 transmitted from a sink drain to a patient had a wildtype resistance pro le to antibiotics (Table 1, Supplemental Table 1, Supplemental Fig. 3).

Discussion

We investigated the cross-transmission of PA between patients and sink traps over 10 months in the MICU of a university hospital in France in the absence of a recognized outbreak. Among the 65 patients infected or colonized with PA, one (1.5%) was contaminated with a clone originating from a sink trap and ve (7.7%) from one originating from another patient.

The proportion of patients contaminated with a PA isolate previously found in a sink trap (1.5%) was lower than that previously reported (7-50%) [START_REF] Cholley | The role of water ttings in intensive care rooms as reservoirs for the colonization of patients with Pseudomonas aeruginosa[END_REF]18). Such a discrepancy could be due to the more accurate typing method used here relative to older typing techniques based on DNA restriction, such as pulsed-eld gel electrophoresis (PFGE). Although PFGE can detect local outbreaks caused by PA, comparison at the nucleotide level is required to identify contamination routes of the pathogens [START_REF] Salipante | Application of whole-genome sequencing for bacterial strain typing in molecular epidemiology[END_REF][START_REF] Lytsy | Time to review the gold standard for genotyping vancomycin-resistant enterococci in epidemiology: Comparing whole-genome sequencing with PFGE and MLST in three suspected outbreaks in Sweden during 2013-2015. Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases[END_REF]28). The implementation of infection control procedures, with improved hand hygiene, presumably accounted for the low transmission rate from the environment to patients. In addition, we identi ed ve patients from among the 65 (7.7%) who acquired PA from another patient (Table 1, Fig. 2B). This is the rst quanti cation of the rate of patient cross-contamination in a non-epidemic context. For each cross-contamination event, the genomes of the PA isolates retrieved from the two patients were completely identical (Supplemental Fig. 3). Three transmission events involved patients hospitalized in different rooms, indicating transmission by healthcare workers (Fig. 2B). Of note, the six cross-transmission events between patients were concentrated in the geographically close rooms A1 (two events) and C2 (four events), which frequently shared healthcare workers (Table 1, Fig. 1). Hence, the proximity of the beds and the sharing of sinks in the four-bed room C2 could enhance the risk of cross-contamination with PA. All clinical PA transmitted to the sink traps originated from patients occupying the room (Table 1), probably during their bathing.

Among the 65 patients positive for PA, 29 (44.6%) tested negative at admission. Only two (2/29; 6.9%) acquired a PA isolate from another patient. In other words, the vast majority of patients who became positive with PA during their hospitalization acquired an isolate not previously found in the other patients or sink traps. Other studies have reported higher proportions (50.0-93.6%) of patients contaminated with an exogenous isolate in ICUs with no detectable PA outbreak and no bleach-disinfection of sink traps [START_REF] Salipante | Application of whole-genome sequencing for bacterial strain typing in molecular epidemiology[END_REF][START_REF] Bortolaia | ResFinder 4.0 for predictions of phenotypes from genotypes[END_REF]. Although we cannot rule out contamination with PA isolates originating from unexplored sources (e.g. healthcare workers, invasive devices), our data show that endogenous sources (i.e. patient ora) predominate over exogenous sources in a nonepidemic context [START_REF] Salipante | Application of whole-genome sequencing for bacterial strain typing in molecular epidemiology[END_REF].

Overall, we can assume that the cleaning and disinfection procedures, together with infection control procedures, applied in this MICU limit the risk of PA transmission. Sinks were cleaned daily and sink traps were disinfected twice a week with 2.6% bleach. Disinfection procedures using bleach, acetic acid, electrochemically-activated solutions, or self-disinfecting sink drains fail to sterilize sink traps because bacterial bio lms in wastewater plumbing systems resist disinfectants and are not easy to access. However, such procedures likely limit the inoculum size, which, in turn, reduces the risk of contamination of the surrounding sink area and further transmission [START_REF] Valentin | A prospective multicentre surveillance study to investigate the risk associated with contaminated sinks in the intensive care unit[END_REF]. Additionally, other mechanisms may limit the transmission of PA from sink traps to patients.

Hence, sub-inhibitory concentrations of bleach can promote horizontal gene transfer [START_REF] Zhang | Subinhibitory concentrations of disinfectants promote the horizontal transfer of multidrug resistance genes within and across genera[END_REF], thus favoring the adaptation of pathogens to a harsh environment. Such adaptation, illustrated here by the stability and low diversity of PA populations in sink traps (Fig. 2A, Supplemental Fig. 2), could impair the ability of PA to colonize patients [START_REF] Rohmer | Are pathogenic bacteria just looking for food? Metabolism and microbial pathogenesis[END_REF].

Our study focused on PA, but other pathogens (Klebsiella pneumoniae, Acinetobacter baumannii, Elisabethkinga meningoseptica) have already been retrieved from sink traps [START_REF] Snitkin | Tracking a hospital outbreak of carbapenemresistant Klebsiella pneumoniae with whole-genome sequencing[END_REF][START_REF] Moore | Waterborne Elizabethkingia meningoseptica in adult critical care[END_REF][START_REF] Forgia | Management of a multidrug-resistant Acinetobacter baumannii outbreak in an intensive care unit using novel environmental disinfection: a 38-month report[END_REF]. One can assume that disinfection also limits the risk of outbreaks with these pathogens. However, the implementation of sink bleaching alone has not been shown to be systematically associated with the cessation of outbreaks and the implementation of a bundle of measures is recommended for infection control [START_REF] Gordon | The hospital water environment as a reservoir for carbapenem-resistant organisms causing hospital-acquired infections -A systematic review of the literature. Clinical infectious diseases: an o cial publication of the Infectious[END_REF]36).

Outbreaks of multidrug-resistant bacteria speci cally linked to drains or sinks are overrepresented in the literature [START_REF] Gordon | The hospital water environment as a reservoir for carbapenem-resistant organisms causing hospital-acquired infections -A systematic review of the literature. Clinical infectious diseases: an o cial publication of the Infectious[END_REF]. We found all PA isolates transmitted from and to patients to be fully susceptible to β-lactams and aminoglycosides. Clinicians and microbiologists should not neglect the potential spread of strains with unspectacular resistance pro les [START_REF] Cremers | Surveillanceembedded genomic outbreak resolution of methicillin-susceptible Staphylococcus aureus in a neonatal intensive care unit[END_REF].

This monocentric study was not designed to assess the effect of infection control procedures on the transmission of PA. Hence, the absence of a comparable ICU using different hygiene practices prevented assessment of the e cacy of sink trap disinfection in preventing the transmission of PA to patients. Instead, our design focused on breadth and depth sampling to identify the contamination routes of PA to patients. We isolated PA from both sink traps and patients over 10 months, collecting the largest dataset yet used for an epidemiological study (38). As we were aware of the complex ora of the sink traps [START_REF] Hota | Outbreak of multidrug-resistant Pseudomonas aeruginosa colonization and infection secondary to imperfect intensive care unit room design[END_REF], we sampled all sink traps weekly and analyzed all colonies with various colony phenotypes and resistance pro les in each sample to obtain a complete picture of the PA population in this environmental niche. The use of isolates collected over 10 months increased the chance of nding clonal isolates that overlapped among patients and sink traps. The genomes of all isolates were fully sequenced and compared at the nucleotide level. This contrasts with typing methods previously used in epidemiological studies [START_REF] Bortolaia | ResFinder 4.0 for predictions of phenotypes from genotypes[END_REF][START_REF] Trautmann | Common RAPD pattern of Pseudomonas aeruginosa from patients and tap water in a medical intensive care unit[END_REF]. We circumvented the absence of a consensus threshold for clonal isolate identi cation from genome-based data by performing a twostep analysis of the genomes. First, we clustered the isolates with cgMLST and grouped all isolates using a threshold (30 alleles of difference) higher than that found in the literature (15 alleles of difference) to avoid missing any clonal isolates [START_REF] Tonnies | Establishment and evaluation of a core genome multilocus sequence typing scheme for whole-genome sequence-based typing of Pseudomonas aeruginosa[END_REF]. Second, we called variants between isolates within a group and performed a second clustering to access clonal strains with a threshold of seven SNPs of difference (Supplemental Fig. 3). This threshold was experimentally optimized (see Supplemental Fig. 2), consistent with previous studies (6-10 SNPs), and is fully compatible with the evolution rate of the bacterial pathogen [START_REF] Duchene | Genome-scale rates of evolutionary change in bacteria[END_REF].

Conclusions

Genome-based typing revealed the contamination of patients by PA isolates originating from sink traps to be rare (1.5%) in a MICU with sink trap bleaching measures and that 7.7% of the patients acquired PA from cross-contamination.

List Of Abbreviations

cgMLST, core genome multilocus sequence typing ICU, intensive care unit Figures

c

  Patient32 and Patient49 shared both isolates ST274, group30_1 and ST3218, group15_1. d ST3218, group15_1 was shared by Patient32 and Patient49 and transmitted from Patient8 to Patient39.

Table 1

 1 Details of the transmission of P. aeruginosa isolates involving patients in the medical intensive care unit at Besançon University Hospital (France) between January and November 2019.

	Type of	Isolate (ST,	Resistance	Reservoir	Room Date	Direction of	Reservoir	Room Date
	transmission	group)	phenotype	1		(2019)	transmission a	2		(2019)
	Patient-to-patient
		group34_1	FQs b	Patient43	A1	Apr. 14	Unknown	Patient49	A1	Apr. 18
		ST274, group30_1	Wild-type	Patient49 c C2	Apr. 12	Unknown	Patient32 c C2	Apr. 18
		ST1197,	Wild-type	Patient1	C1	Aug. 7	Unknown	Patient13	C2	Aug.
		group25_1								12
		ST1238,	Wild-type	Patient9	B5	Jul.	Unknown	Patient48	A1	Jul.
		group27_1				22				22
		ST3218, group15_1 d	Wild-type	Patient32 c C2	Apr. 15	Unknown	Patient49 c C2	Apr. 18
		ST3218,	Wild-type	Patient8	A3	Oct.	→	Patient39	C2	Oct.
		group15_1 d				14				22
	Sink trap to
		ST253,	Wild-type	Sink trap	B2	May	→	Patient14	A3	May
		group0_6				13				24
	Patient sink trap
		ST27,	Wild-type	Patient36	B3	Feb.	→	Sink trap	B3	Mar. 4
		group21_1				23
		ST234,	Wild-type	Patient5	A5	May 2	→	Sink trap	A5	Jul.
		group33_1								29
		ST253,	Wild-type	Patient17	A1	Oct.	→	Sink trap	A1	Nov. 4
		group0_3				23
		ST308,	Wild-type	Patient54	A2	Jul. 8	→	Sink trap	A2	Jul.
		group1_6								15
		ST309,	Wild-type	Patient10	B2	Sep. 2	→	Sink trap	B2	Sep.
		group2_3								23
		ST671,	Wild-type	Patient19	A2	Mar.	→	Sink trap	A2	Apr. 8
		group32_1				30

Acknowledgements. We warmly thank Dr. Catherine Llanes for helpful discussions. Computations were performed at the Mésocentre de Calculs de Franche-Comté (Besançon, France).

Funding. This work was supported by a grant from the University Hospital of Besançon, France (API 3A -Risques 2019 to X.B.). The funders had no role in the study design, data collection or analysis, decision to publish, or preparation of the manuscript.

Availability of data and materials. The datasets generated during the current study are available in the NCBI repository as the BioProject PRJNA788732.

Results

Contamination of sink traps and patients by P. aeruginosa. During the 10 months of the study, the 13 sink traps of the MICU were sampled 42 times each, for a total of 546 samples, of which 282 (51.6%) were positive for one or more isolates of PA. This led to the collection of 404 environmental isolates. All sink traps were positive for PA at some point of the study and could be contaminated with multiple STs (mean: 5, min: 2, max: 10), with clones persisting in each sink trap for long periods of time (mean: 242 days, max: 286 days). Among the 549 patients included, 65 (11.8%) were positive for PA. We collected 115 clinical isolates from these patients during their hospitalization. Three patients (3/65, 4.6%) were infected with PA.

Population structure of P. aeruginosa. The 519 isolates were distributed within 62 different STs, with ve STs accounting for 54.9% of the entire collection. Hence, the high-risk clones ST253, ST308, ST298, and ST244 were represented by 90 (17.3%), 69 (13.3%), 39 (7.5%), and 32 (6.2%) isolates, respectively, and ST309 was represented by 55 isolates (10.6%) (Supplemental Table 1). Isolates from sink traps and patients were distributed within 27 and 48 different STs, respectively, with 13 STs in common. We then compared the clonal diversity of the population of isolates retrieved from the sink traps with that of the clinical isolates and found that the community of clinical PA was 4.1-times richer and 2.6-times more diverse than that retrieved from the sink traps (Supplemental Fig. 2).

Transmission routes of P. aeruginosa. The routes of transmission of PA within the MICU were accurately identi ed by comparing the genomes of all isolates with a pipeline that allowed variant calling. This method clustered the isolates into 36 groups (Supplemental Fig. 3). We combined these genomic data with spatiotemporal data to identify intra-and inter-reservoir transmission events (Table 1, Fig. 2).

MALDI-TOF

Supplementary Files

This is a list of supplementary les associated with this preprint. Click to download.

CouchoudetalSupplementary le.pdf


Gabriel Dulaquais
email: gabriel.dulaquais@univ-brest.fr.

Pierre Fourrier

Jean François Maguer

Corentin Denis

Matthieu Waeles

Ricardo Riso

Size exclusion chromatography and stable carbon isotopes reveal the limitations of solid phase extraction with PPL to capture autochthonous DOM production

Introduction

Marine dissolved organic matter (DOM) is a large reservoir of reduced carbon (662 Pg, [START_REF] Hansell | Dissolved organic matter in the ocean: A controversy stimulates new insights[END_REF] involved in key aquatic processes. The main constituent of DOM is carbon (~50%) that supports the metabolism of heterotrophic bacterioplankton production [START_REF] Azam | Size distribution and activity of marine microheterotrophs 1[END_REF][START_REF] Ducklow | The bacterial component of the oceanic euphotic zone[END_REF]. DOM also consists of various heteroatoms making DOM an alternative bioavailable reservoir of nitrogen and phosphorus for marine micro-organisms [START_REF] Lønborg | Bioavailability and bacterial degradation rates of dissolved organic matter in a temperate coastal area during an annual cycle[END_REF][START_REF] Stepanauskas | Summer inputs of riverine nutrients to the Baltic Sea: bioavailability and eutrophication relevance[END_REF]. Thereby, DOM plays an integral role in the biogeochemistry of aquatic systems as well as in the biological carbon pump.

Historically DOM has been studied through the prism of dissolved organic carbon (DOC) since the early 1980. With major analytical advances (high-temperature combustion method), intercomparison efforts [START_REF] Hedges | Measurement of dissolved organic carbon and nitrogen in natural waters[END_REF] and international oceanographic programs (e.g. JGOF, CLIVAR) the global distribution of oceanic DOC was assessed in the mid 2000 (Hansell andCarlson, 2002, Hansell et al., 2009). DOC mapping demonstrated that its concentration was not uniform along the water column but exhibited concentration gradients across biogeochemical domains and a distribution strongly influenced by overturning circulation and deep sea mineralization [START_REF] Hansell | Dissolved organic matter in the ocean: A controversy stimulates new insights[END_REF]. However, despite the growing community interest in DOM, its study was still limited by analytical issues. Low ambient concentrations (down to 30 µM-C), salts, and wide diversity in the molecular composition [START_REF] Zark | Molecular properties of deep-sea dissolved organic matter are predictable by the central limit theorem: Evidence from tandem FT-ICR-MS[END_REF] have been a barrier for decades to a molecular characterization of marine DOM.

For DOM isolation, solid-phase extraction (SPE) using historical amberlite sorbents (e.g. DAX 8 and 4) was developed early to define hydrophobic and transphilic fractions of DOM. However, their implementation and evaporation steps were often time consuming and required large sample volumes. In the late 2000, [START_REF] Dittmar | A simple and efficient method for the solid-phase extraction of dissolved organic matter (SPE-DOM) from seawater[END_REF] published a rapid and simple method to isolate DOM from any aquatic matrix, which was a new step for the scientific community. Among the 6 sorbents studied by [START_REF] Dittmar | A simple and efficient method for the solid-phase extraction of dissolved organic matter (SPE-DOM) from seawater[END_REF] the styrene divinyl benzene polymer (PPL) was the most promising with extraction efficiency of 43% for DOC for deep sea water and up to 65% for DOC in freshwater. This study permitted the emergence of the first analyses of oceanic DOM composition along the entire water column and recently in different biogeochemical domains [START_REF] Broek | Low molecular weight dissolved organic carbon: Aging, compositional changes, and selective utilization during global ocean circulation[END_REF][START_REF] Hertkorn | High-field NMR spectroscopy and FTICR mass spectrometry: powerful discovery tools for the molecular level characterization of marine dissolved organic matter[END_REF]Martinez-perez et al., 2017 ;[START_REF] Medeiros | Dissolved organic matter composition and photochemical transformations in the northern North Pacific Ocean[END_REF][START_REF] Osterholz | Accumulation of DOC in the South Pacific subtropical gyre from a molecular perspective[END_REF][START_REF] Seidel | Molecular-level changes of dissolved organic matter along the Amazon River-to-ocean continuum[END_REF]. PPL is also widely used for the isolation of soil organic matter [START_REF] Patel | Soil texture and environmental conditions influence the biogeochemical responses of soils to drought and flooding[END_REF] and for the development of molecular formula-based indices (notably the aromaticity index of DOM) used to study DOM reactivity [START_REF] Zherebker | Aromaticity Index with Improved Estimation of Carboxyl Group Contribution for Biogeochemical Studies[END_REF]. SPE-DOC can be processed for stable isotope (δ 13 C), a useful tool to trace the sources of DOC.

δ 13 C of SPE-DOC helps to trace the estuarine mixing [START_REF] Zhou | Spatial changes in molecular composition of dissolved organic matter in the Yangtze River Estuary: Implications for the seaward transport of estuarine DOM[END_REF], to distinguish the main source of DOC (C3 vs C4 photosynthetic pathway) in estuary [START_REF] Marques | Dissolved black carbon in the headwaters-to-ocean continuum of Paraíba do Sul River, Brazil[END_REF] and to trace terrigenous inputs in coastal systems [START_REF] Takasu | Contribution of riverine dissolved organic carbon to organic carbon decomposition in the Ariake Sea, Japan, a coastal area suffering from summer hypoxia[END_REF] and in the ocean (Zigah et al., 2017). δ 13 C of SPE has usually the same or lower δ 13 C than bulk seawater (Broek et al., 2017;Zigah et al., 2017), suggesting that PPL resin selectively isolates compound groups and may have difficulty in capturing the DOM derived from the degradation of marine phytoplankton.

Extraction with PPL resin has also been applied to culture experiments with the aim of defining the molecular composition of DOM of phytoplankton or bacterial origin (Landa et al., 2013 ;[START_REF] Liu | Stable isotope probing identifies bacterioplankton lineages capable of utilizing dissolved organic matter across a range of bioavailability[END_REF] or to study the lability of oceanic DOM [START_REF] Shen | Mixing it up in the ocean carbon cycle and the removal of refractory dissolved organic carbon[END_REF]2020). These works have opened up a new field of research related to DOM ecology that focuses on the complex interactions between organic compounds and microbial cells [START_REF] Dittmar | Enigmatic persistence of dissolved organic matter in the ocean[END_REF].

Because PPL resin has become the most popular sorbent, understanding the representativeness of the DOM isolated using PPL resin is crucial. The few studies that address the selectivity of this sorbent have shown contrasting results. While DOM of terrigenous origin does not appear to be significantly fractionated during extraction [START_REF] Raeke | Selectivity of solid phase extraction of freshwater dissolved organic matter and its effect on ultrahigh resolution mass spectra[END_REF], the sample matrix composition has been shown to affect the extraction efficiency of a given compound [START_REF] Johnson | Extraction efficiency and quantification of dissolved metabolites in targeted marine metabolomics[END_REF]. Furthermore, incomplete elution of the PPL resin retentate can induce a bias in the determination of stable and radiocarbon isotopic composition [START_REF] Lewis | Isotopic and optical heterogeneity of solid phase extracted marine dissolved organic carbon[END_REF]. It has also been shown that for seawater samples, the optical signature of the retentate from a PPL resin is substantially different from that of the corresponding bulk samples (Wünch et al., 2018). Another key issue is the high selectivity (chemically based) of the PPL sorbent. [START_REF] Li | How representative are dissolved organic matter (DOM) extracts? A comprehensive study of sorbent selectivity for DOM isolation[END_REF] analyzed retentates after SPE extractions using PPL resin for Suwannee River and North Sea water samples. Their results showed high compositional similarity in these two samples of different origin raising questions about the use of this sorbent to study DOM modifications. Considering the results of [START_REF] Li | How representative are dissolved organic matter (DOM) extracts? A comprehensive study of sorbent selectivity for DOM isolation[END_REF] and the growing number of studies reporting high similarities between DOM isolated across different oceanic basins and depths [START_REF] Broek | Low molecular weight dissolved organic carbon: Aging, compositional changes, and selective utilization during global ocean circulation[END_REF], it is important to investigate the selectivity/fractionation of DOM by the PPL sorbent during the extraction process using PPL sorbent.

The objectives of our study are to define the representativeness of DOM isolated using a PPL resin and to determine whether this type of extraction is adequate to capture internal processes in estuarine systems and to trace the origin of DOM inputs (allochthones vs autochthones). We first tested the accuracy of carbon stable isotope measurements of three different types of organic compounds after their isolation on a PPL resin and measured the stable carbon isotope composition in the PPL resin retentates. These experiments were done to determine if SPE using PPL can be used to trace the origin of DOM in estuarine and marine waters. Secondly, we studied the DOC size class distributions and DOM composition using size exclusion chromatography (SEC) of estuarine samples before and after their passage through the PPL resin in order to define the representativeness of the PPL retentate. SEC has the advantage of not requiring a pre-concentration step and allows access to the DOM composition without fractionation effects. This analytical tool has been successfully used for the analysis of freshwater [START_REF] Marie | Dynamics and sources of reduced sulfur, humic substances and dissolved organic carbon in a temperate river system affected by agricultural practices[END_REF], estuarine [START_REF] Dulaquais | Measuring dissolved organic matter in estuarine and marine waters: size-exclusion chromatography with various detection methods[END_REF] and marine waters [START_REF] Fourrier | Characterization of the vertical size distribution, composition and chemical properties of dissolved organic matter in the (ultra) oligotrophic Pacific Ocean through a multi-detection approach[END_REF] and allows in this study to determine precisely the fractionation of DOM by the PPL resin in terms of size class and composition (including aromaticity and nitrogen content).

Material and Methods

Sampling

11 samples were collected along the salinity gradient of the Aulne estuary-Bay of Brest estuarine system (Britanny, France) on May 31 2021. For salinities below 20 of practical salinity unit (PSU), sampling was operated from the R/V Hésione (INSU-CNRS-UBO) and was carried out 0.5 m below the surface with the arm fully covered by a plastic glove (92 cm, Polysem®) using acid cleaned high density polyethylene (HDPE) bottles. For salinities above 20, sampling was operated onboard of the R/V Albert Lucas (INSU-CNRS-UBO) and was done at 1 m depth using a Niskin bottle. All samples collected were filtered onboard the R/V Albert Lucas within two hours after sampling using precombusted 0.7-µm GF/F filters (Whatman®). The filtrates were then collected in acid-cleaned HDPE bottles, double bagged and stored in the dark at 4°C. It is worth noting that the cruise occurred during a low discharge period (9.1 m 3 s -1 ). This water flow was preceded by a month of low water regime. This choice of water regime was made in order to have a residence time of the waters in the estuary (~15 days) significantly longer than the timescale of sampling (10 hours) allowing a better visualization of the biogeochemical processes occurring in the estuary. Salinity (S) and pH (NBS scale) were measured in-situ using a multi-parameter probe (Hanna Instruments© 9829) calibrated on the day of sampling. The accuracy of the measurements is ± 0.1 g kg -1 and ± 0.01 pH units respectively. Salinity and pH of the samples were remeasured in the lab at 25 °C. The marine end-member sample was collected in the Iroise Sea on October 22, 2018 during the FeLINE cruise [START_REF] Riso | Distribution, speciation and composition of humic substances in a macro-tidal temperate estuary[END_REF]) and stored at -20°C from collection until analysis. For all cleaning procedures and the preparation of all aqueous solutions we used ultrapure water (resistivity > 18.2 MΩ.cm, MilliQ Element, Millipore®) and HCl (Suprapur®, Merck).

Experimental and solid phase extraction procedures

The chart of the experimental procedure used in this work is shown in figure 1 and the details of the different steps are provided in the following sections.

For estuarine samples, solid phase extraction was operated once per sample. After acidification at pH 2 (Suprapur®, Merck), 0.5 L (S < 20) or 1 L of estuarine samples (S > 20) were passed through a 100 mg PPL cartridge (Agilent, Santa Clara, CA, USA) following the procedure described in [START_REF] Dittmar | A simple and efficient method for the solid-phase extraction of dissolved organic matter (SPE-DOM) from seawater[END_REF]. The cartridges were first washed with 1 column volume of methanol. Methanol (HPLC grade, Merck©) was rinsed from the cartridge with 5 volumes of ultrapure water acidified at pH 2 (HCl, Suprapur®, Merck) before the extraction started.

Extractions were performed at a flow rate of 4 mL/min using a peristaltic pump (Watson Marlow © 205S) with TYGON® tubing. Before air-drying, the cartridges were rinsed with 5 volumes of ultrapure water acidified at pH 2 (HCl, Suprapur®, Merck) in order to remove salts.

Then, after 5 min of air drying we started the elution of the cartridges. We used 1.5mL of HPLC grade methanol, which is within the range of the recommended volume (12 mL/g of resin) to avoid bias resulting from incomplete elution [START_REF] Lewis | Isotopic and optical heterogeneity of solid phase extracted marine dissolved organic carbon[END_REF]. After elution, the methanol extracts were evaporated directly into smooth wall tin capsules (Elemental microanalysis ©) on a hot plate at 50°C. All of these steps were performed under a laminar flow bench (ISO-5).

The tin capsules were then stored in precombusted glass vials in a desiccator until determination of elemental C and δ 13 C (by Elemental Analyzer coupled to Isotope ratio mass spectrometer, EA-IRMS) within a week after extraction. It is worth noting that volatile organic compounds may be lost during the drying step inducing bias for elution recovery assessment. For the evaluation of stable carbon isotopic accuracy, solid phase extractions were performed for three certified referenced materials: Suwannee River Fulvic Acid (INTERNATIONAL HUMIC SUBSTANCES SOCIETY (IHSS), SRFA 1S101F), Leonardite Humic Acids (International humic substances society , LHA 1S104H) and Atropine (Elemental microanalysis , OAS 279202). These compounds were selected by considering their isotopic signature and elemental composition (Table 1). These materials were first dissolved in artificial seawater (final concentrations 2 mg / L) prepared as described in Fourrier et al., 2022 (see supplementatry informations, SI).

Artificial seawater was then acidified to pH 2 (HCl) and 250 mL of these solutions were then passed through the PPL resin, eluted and evaporated as for the estuarine samples. theoretical plates, Toso, Japan), permits the separation of DOM into six fractions of organic compounds with an optimal resolution. These fractions were described in order of retention as biopolymers (BP, high molecular weight compounds (HMW) > 10 kDa), humic substances (HS, 0.5 -10 kDa), building blocks (BB, 0.3 -0.5 kDa), low molecular weight acids (LMW acids, < 0.3kDa, charged), low molecular weight neutrals (LMW neutrals < 0.3 kDa) and the nonchromatographable DOC (retained by the column at a pH of 7) called hydrophobic DOC (HOC).

The respective compositions of these operationally defined fractions are described in details in [START_REF] Huber | Characterisation of aquatic humic and non-humic matter with size-exclusion chromatography-organic carbon detection-organic nitrogen detection (LC-OCD-OND)[END_REF] and Dulaquais et al. (2018b). We provide additional information considering the recent work of [START_REF] Fourrier | Characterization of the vertical size distribution, composition and chemical properties of dissolved organic matter in the (ultra) oligotrophic Pacific Ocean through a multi-detection approach[END_REF]. The BB fraction, initially thought as degradation by-products of HS, may actually correspond to degradation by-products of BP;

LMW monoprotonic acids and LMW neutral compounds correspond to small-degradated humic substances and small hydrophilic compounds, respectively.

Stable carbon isotopic measurements

Stable carbon isotope ratios and C content analyses were performed using an Elemental Analyzer (Thermo Scientific EA Flash 2000) coupled to an Isotope Ratio Mass Spectrometer (IRMS Thermo Scientific Delta Plus). Isotopic ratios were expressed in conventional δ notation and were reported in parts per thousands (‰) as :

δ 13 C = [(( 13 C/ 12 C) Sample / ( 13 C/ 12 C) VPDB) -1] *1000
where ( 13 C/ 12 C) VPDB = 0.0112372, the ratio of 13 C to 12 C in the international reference Vienna-Pee Dee Belemnite (V-PDB) standard. Replicate analysis, using atropine as working standard (calibrated to the VPDB) indicated an analytical precision of ± 0.1‰ and ±0.6% for δ 13 C and for C content, respectively.

The determination of bulk δ 13 C composition (before solubilization) of the SRFA (-27.6 ‰ ± 0.1 n = 3) and LHA (-23.7 ‰ ± 0.1 n = 3) reference material were in keeping with the values (-27.6

‰ and -23.8 ‰ respectively) provided by IHSS for these compounds (https://humicsubstances.org/elemental-compositions-and-stable-isotopic-ratios-of-ihss-samples/), ensuring accurate of δ 13 C measurements for natural organic matter.

Statistical analysis

T-tests were operated to determine if the PPL extraction procedure significantly affects the initial δ 13 C signature of the 3 organic compounds tested in this study (SRFA, LHA and atropine).

If p-value < 0.05 then the dataset are considered significantly different. The possibility of an effect of the PPL extraction procedure onto DOM size fractionation was tested using a two factor Wilcoxon test (two groups, permate and retentate) for each of the six size class fractions.

Results and discussion

Accuracy of δ 13 C measurement after extraction

Table 1 shows the δ 13 C results (blank corrected) for the three referenced materials after their solubilization in artificial seawater and extraction using the PPL sorbent. We found that the PPL resin (≥ 88%) efficiently extracted SRFA and Leornadite Humic Acids. Their δ 13 C isotopic signatures were in agreement with those certified by the IHSS (table 1; p-value > 0.05). In contrast, atropine was not extracted efficiently (33%) and we measured a δ 13 C isotopic signature (-19.9 ± 0.3; p-value < 0.05) significantly lower than the certified value (-18.1 ± 0.1).

On the one hand, our results demonstrate that humic substances of both freshwater and terrigenous origin can be efficiently extracted from seawater matrix using a PPL sorbent. This process does not induce isotopic fractionation of stable carbon isotopes. On the other hand, our results suggest that hydrophilic molecules with low C/N ratio are not efficiently retained by the PPL sorbent. This low extraction yield seems to induce an important bias for the δ 13 C isotopic signature. Here we suggest that the δ 13 Cmeasurement of a sample after solid phase extraction using the PPL sorbent is strongly influenced by the type of compounds in solution but not sensitive to the salinity of the sample. The extraction yield (EY) of the PPL resin was determined for DOC and for the different size classes operationally defined by SEC analysis. The elution recoveries were all > 95% ensuring no elution bias for EA-IRMS analysis of the retentate. Along the salinity gradient, DOC EY (Table 2) decreased from 52% in the Aulne freshwater (S = 0) to 35% in the marine sample (S = 35.15).

A similar decrease in EY from fresh to marine waters was initially observed by [START_REF] Dittmar | A simple and efficient method for the solid-phase extraction of dissolved organic matter (SPE-DOM) from seawater[END_REF].

The different operational fractions exhibited varying EYs along the salinity gradient. On the one hand, the EY% of the HS fraction decreased from ~60% to 45%. Those of the biopolymers and LMW neutrals also decreased from freshwater to marine waters, from ~40% to ~13% and ~30 % to ~1%, respectively. These results demonstrate the weak ability of the PPL sorbent to capture these different hydrophilic compounds in marine samples. On the other hand, the EY of the most hydrophobic organic compounds was enhanced for higher salinities (EY% range 90-100% for S>30) compared to the low salinity samples (EY% range 35-65%). Because riverine and soil humic substances were accurately recovered in artificial seawater (S = 35, see section 3.1, table 1), salinity can not be considered as the controlling parameter for DOM adsorption onto the PPL sorbent. Thereby, the changes of EY we observed along the salinity gradient indicate changes in the DOM molecular composition. This selectivity of the PPL sorbent implies that analyses of PPL retentate will only be representative of a fraction of DOM. at S > 20 (Figure S1). DOM partitioning was similar to those previously reported in the same estuary [START_REF] Dulaquais | Measuring dissolved organic matter in estuarine and marine waters: size-exclusion chromatography with various detection methods[END_REF] with ( 1) the predominance of HS across the system (from 60% of DOC at S < 10 to 45% at S > 32 ); (2) a decrease in HOC contribution of HOC from 15 % in freshwater to 4 % in the most marine waters (36 µM at S = 0.17 to 4 µM at S> 32.5); (3) an increase in the contribution of LMW neutrals with salinity (from 10% in freshwater to 25% in marine waters), and (4) a low and near constant concentration of biopolymers (HWM DOC, 10.5 ± 2.3 µM, n = 11). With the exception of HOC, the mixing diagrams show non-conservative behavior for all fractions studied (Figure S1), indicating that internal process were affecting the distribution of DOM during the sampling period behind the quasi-conservative distribution of DOC along the salinity gradient.

Selectivity of the PPL sorbent

In order to study the representativeness of a PPL resin retentate we studied the size class partitioning of DOC along the salinity between the PPL retentate (referred to as SPE-DOC hereafter) and its permeate (referred to as non-SPE-DOC hereafter) .

The size class fractionations shown in Figure 2 clearly demonstrate a high selectivity of the PPL sorbent for some fractions. The size class composition of the SPE-DOC samples was completely different from that of the corresponding bulk samples (Figure 2) with an over-representation of HOC and humic fractions in the SPE-DOC, at the expense of biopolymers and LMW neutrals fractions. To further identify which size fractions are selectively retained by the PPL sorbent, we determined the discrete (Figure S1) and average (with associated standard deviation) contribution of each size fraction to the SPE-DOC pool and to the unextractable DOC (using PPL) along the salinity gradient (Figure 3). Among the six fractions studied, five were retained with a selectivity that induced a significant difference (Wilcoxon test) between the retentate and the permeate (Figure 2, 3 and S1). Furthermore the high selectivity of the PPL sorbent induced a similar distribution of SPE-DOC size classes between freshwater and the marine waters, whereas before the extraction procedure, the samples had different compositions (Figure 2 & 3).

The previous results demonstrate that during the extraction procedure, the size fractionation of DOC changes. Thereby the DOC retained by the PPL resin is not representative of the bulk sample. Such fractionation may lead to biased interpretations in the absence of additional data (e.g. SEC measurements). Our results also showed that the selectivity of the resin was however not total for a given fraction, suggesting that in a fraction operationally defined by the SEC, the PPL resin is able to retain specific compounds according to their chemical properties. To investigate this hypothesis, we compared the elemental C/N ratios and aromaticity of compounds retained by the PPL sorbent to those that escape the extraction procedure. The C/N ratios measured in the PPL permeate were consistently lower than those measured in the samples before the extraction procedure indicating that the PPL resin has a low affinity for N-containing molecules. Since high nitrogen content is accepted as a proxy of DOM lability [START_REF] Fourrier | Characterization of the vertical size distribution, composition and chemical properties of dissolved organic matter in the (ultra) oligotrophic Pacific Ocean through a multi-detection approach[END_REF] and C/N ratio increases with DOM ageing [START_REF] Walker | Pacific carbon cycling constrained by organic matter size, age and composition relationships[END_REF]; the ability of the PPL resin to capture labile and freshly produced DOM seems limited. This result is of particular importance because the decrease of C/N after PPL processing was observed in both humic substances (Figure 4a) and biopolymer (data not shown) fractions, which have different production pathways and labilities. Biopolymers ( > 10kDa) can be considered as young and labile DOM derived from phytoplankton degradation (cell lysis). Humic substances are compounds that result from more intense processing (condensation, bacterial degradation), they can be considered as a more refractory fraction of DOM. Our results also show that the aromaticity of the biopolymers and humic substances fractions was also affected by the extraction procedure. We observed a much higher aromaticity of DOM in the PPL permeate at salinities higher than 20 compared to the untreated samples (Figure 4b). This result indicates that the PPL resin has a lower affinity for marine aromatic DOM than for riverine/terrigenous aromatic compounds. Considering the low affinity of PPL for chemical nitrogen groups (Figure 4a) our observation can be related to a higher proportion of aromatic moities containing heteroatoms (e.g. O, N) in the marine DOM compared to the freshwater DOM. Overall, such high selectivity for hydrophobic and aliphatic compounds at the expense of aromatic and N-rich compounds strongly limits the use of the PPL resin to monitor the production or to study the composition of labile DOM in marine waters.

Isotopic composition of SPE-DOC along the estuary

Along the Aulne estuary, δ 13 C values measured inPPL eluates ranged from -28.6 ± 0.1 ‰ in the Aulne river freshwater to -24.2‰ ± 0.1 at S = 35.15. The depletion of δ 13 C in the riverine endmember is consistent with the terrigenous origin of DOM in the river [START_REF] Opsahl | Major flux of terrigenous dissolved organic matter through the Arctic Ocean[END_REF].

Since δ 13 C was not determined in the marine sample we must assume an isotopic signature of -22.7 ‰ +-0.1 in North Atlantic waters identical to the one reported in the work of [START_REF] Medeiros | A novel molecular approach for tracing terrigenous dissolved organic matter into the deep ocean[END_REF] for North Atlantic SPE-DOC (using PPL). The increasing δ 13 C isotopic signature we observed along the estuary (Figure 5) is consistent with the increasing contribution of marine waters. From these two end-members (e.g. Aulne River and North Atlantic), a mixing line can be generated using a mass balance of the freshwater fraction (f) at each point according to Equation 3. Two distinct processes can explain this discrepancy. First, autochthonous DOM production in the salinity range 25-31 resulted in an input of N-rich humic substances. Considering their aquatic origin, these humic substances produced in the estuary are probably enriched in 13 C.

These N-rich humic substances are weakly retained by the PPL resin (Figure 4a) and thus not available for the δ 13 C measurement. Second, the extraction efficiency of HOC compounds increasing along the salinity gradient (Figure 6) may have affected the δ 13 C values. Indeed, the PPL retained HOC very efficiently (> 95%) at salinities above 25, the extraction yield was much lower (35-65%) at salinities below 20. These HOC are primarily river-borne (Figure 6) are likely characterized by a light δ 13 C consistent with their terrigenous origin. Therefore, any changes in the recovery of these compounds by the PPL sorbent will also affect the SPE-DOC δ 13 C measurements.

Implication for oceanic studies

Extraction with PPL as sorbent is commonly used to isolate DOM in order to trace molecular changes in DOM through biogeochemical processes [START_REF] Osterholz | Accumulation of DOC in the South Pacific subtropical gyre from a molecular perspective[END_REF]. Here, we observed that extraction with a PPL sorbent alters the size class distribution of the initial DOM and that this resin is not optimal for capturing N-rich or aromatic marine DOM (Figure 4). In addition, we observed a high selectivity of the PPL resin for specific DOM fractions. This selectivity, already reported in other studies [START_REF] Li | How representative are dissolved organic matter (DOM) extracts? A comprehensive study of sorbent selectivity for DOM isolation[END_REF]Wünch et al., 2018), generates a quasi-constant composition of the DOM isolated by the PPL resin whether in freshwater or marine waters (Figure 2 and4). The PPL sorbent is often reffered as having a high affinity for refractory DOM compounds, which would make this sorbent effictive for studying this DOM fraction. It is clear that an PPL extraction of DOM with PPL in the deep ocean will trap a substantial part of the refractory DOC (up to 60%), however any analysis at the molecular level of the retentate from a PPL resin cannot be assigned to the intrinsect signature of refractory DOC. This is supported in particular by the work of [START_REF] Broek | Low molecular weight dissolved organic carbon: Aging, compositional changes, and selective utilization during global ocean circulation[END_REF]. These authors observed significant compositional differences between bulk DOM and PPL DOM retentates in term of C/N ratio, δ 13 C and Δ 14 C in the deep Pacific waters. At these Pacific depths, DOM there can be considered fully refractory [START_REF] Fourrier | Characterization of the vertical size distribution, composition and chemical properties of dissolved organic matter in the (ultra) oligotrophic Pacific Ocean through a multi-detection approach[END_REF], so the existence of compositional differences between retentate from a PPL resin and untreated sample demonstrates that the PPL resin captures only a fraction of the refractory DOM. Furthermore in a controlled laboratory experiment conducted by [START_REF] Wienhausen | The exometabolome of two model strains of the Roseobacter group: a marketplace of microbial metabolites[END_REF], the PPL resin retained a portion of the freshly produced (labile) DOM, again supporting that this sorbent fractionates the DOM not in terms of lability but in terms of chemical properties.

These important results provide evidence that PPL resin fractionates DOM through chemical properties and that DOM refractability can not only be defined as the chemical properties of compounds retained by a PPL resin.

Due to the high selectivity of the PPL resin, we suggest that both labile and refractory DOM, can be isolated using a PPL resin. Consequently, a DOM isolated using the PPL resin will have the same chemical properties and molecular composition (in broad terms) in any aquatic system. As a result, the refractory nature of DOM can not be assigned solely in terms of chemical composition. This hypothesis is supported by systematic fractionation of DOM that we observed in PPL retentated from marine, estuarine and freshwater (Figure 2) while the main origin of DOM is still discernable using carbon isotopes (Figure 5). This is also supported by the accumulation of recent studies showing extraordinary homogeneity in DOM composition from surface to the deep ocean , between oceanic basins and in different environments [START_REF] Broek | Low molecular weight dissolved organic carbon: Aging, compositional changes, and selective utilization during global ocean circulation[END_REF][START_REF] Li | How representative are dissolved organic matter (DOM) extracts? A comprehensive study of sorbent selectivity for DOM isolation[END_REF][START_REF] Osterholz | Accumulation of DOC in the South Pacific subtropical gyre from a molecular perspective[END_REF]. Analysis of PPL retentates by any powerful technique (e.g. Fourier Transform Ion Cyclotron Resonance Mass Spectrometry ; Nuclear Magnetic Resonance) provides useful information to study this defined fraction. However, one should not lose sight of the fact that the PPL retentate is not representative the refractory DOM alone but fractionate DOM in both in the refractory and non-refractory pool.

Conclusions

We studied the fractionation of DOM by solid-phase extraction with a PPL sorbent along an estuarine system. Our study confirms that the composition of the material is the main parameter controlling the extraction efficiency of DOM by the PPL resin. PPL sorbent has a high affinity for aliphatic hydrophobic compounds and retains poorly high molecular weight compounds as well as those enriched in nitrogen. Salinity does not seem to affect the extraction significantly. In this work we have demonstrated that at low extraction yields (< 33%) of a compound, the measurement of δ 13 C in the retentate of a PPL resin is in error. For yields > 88% the δ 13 C measurement is valid.

The study of the size class fractionation of estuarine samples before and after their treatment on a PPL resin allowed us to observe that (i) the composition of a PPL resin retentate was not representative of the original sample, (ii) the distribution of DOC by size class was constant in the different retentates without taking into account the origin of the sample (freshwater and marine water). We observed a high selectivity of the PPL resin based on chemical properties (C/N ratio, aromaticity). This selectivity was observed for both refractory (humiques substances) and semi-labile (biopolymers) organic matter.

The carbon isotope study showed that the extraction efficiency of the compounds strongly influenced the δ 13 C data obtained from the retentate analysis of a PPL resin. In this work, a high retention of the most hydrophobic compounds and a low extraction of humic substances enriched in nitrogen and produced in the estuary induced an underestimation of the δ 13 C at the highest salinities (S > 20).

This work shows that without control of the extraction process, biased interpretations can be made based on the analysis of a PPL resin rententate alone. We call for a cautious interpretation of the DOM data from PPL extraction because this sorbent strongly fractionates the DOM pool on chemical and molecular properties and not on the lability.

ForWith

  each sample, the DOC size class distribution was measured by size exclusion chromatography (SEC) before (bulk sample) and after (permeate, Non SPE-DOM) the sample passed through the PPL resin. The permeate is constituted of all the water samples that passed through the PPL resin during the extraction procedure. The retentate and eluate refer to the DOM retained by the PPL resin and recovered after a methanol elution. The extraction yield (EY%) for each class of compounds was determined according to equation 1. Since the retentate could not be injected in the SEC, the size class distribution of the retentate (SPE-DOC) was calculated by the difference between bulk and permeate DOC concentrations in each size class fraction. Elution recoveries (ER %) were estimated according to equation 2 by comparing the amount of C in the eluate (measured by an Elemental Analyze, EA , (Thermo Scientific EA Flash 2000) to the amount of DOC in the retentate (SPE-DOC multiplied by sample volume). We observed a contamination (with methanol) of the permeate during the extraction procedure for the sample at salinity 15.5. For this sample, the DOC recovery (50.7%) was estimated from the amount of C measured by an EA and considering 95% of ER. Recovery of the different fractions can not be assessed for this latter sample. EY the Extraction yield; ER the Elution recovery; [DOC]eluate and [DOC]bulk the dissolved organic carbon concentration measured by size exclusion chromatography before and after the sample passes through the PPL resin respectively. OCEA is the amount of organic carbon measured by the elemental analyser in the tin capsule after elution and evaporation of the PPL resin; Vsample the volume of sample that passes through the PPL resin.

Figure 1 :

 1 Figure 1: Chart of the experimental procedure followed in this study 2.3 DOM size class distribution Measurements of the organic compound class distribution were performed by SEC coupled with C, N and UV detectors (DOC-Labor®, Karlsruhe, Germany) as previously described by Huber et al. (2011) for freshwater, and adapted for estuarine and marine waters by Dulaquais et al. (2018b). All the chemicals used for SEC analyses were those described in Fourrier et al. (2022). Repeated analysis of Deep Sea Reference samples (DSR, Hansell lab, Miami, USA [DOC]DSR = 43.2 ± 1.7 µmol L -1 ; n = 5; consensus value of lot #10-18: 43 -45 µmol L -1 ) ensures an accurate determination of DOC. The determination of C:N ratios and of the percentage of aromatic carbon (%Carom) were conducted similarly as described in Riso et al. (2021) and Fourrier et al. (2022) (see SI file for additional information regarding the procedures). The device, equipped with two chromatographic columns (250 mm × 20 mm, TSK HW-50S, 3000

  distribution and partitioning along the salinity gradient The distributions of DOC and the six operationally defined size fractions along salinity gradient are shown in figure 2a. DOC ranged from 234 µM-C in the freshwater sample (Aulne River) to 96.3 µM-C at S=32.5 (Bay of Brest). The North Atlantic surface water used in this work is a sample collected from the Iroise Sea in 2018 (Riso et al., 2021) with a DOC concentration of 89.8 µM-C. At the time of sampling, DOC showed a nearly conservative distribution along the salinity gradient (DOC = 235µM-C -3.9*Salinity ; n = 11; R² = 0.96) with a slight positive anomaly

Figure 2 :

 2 Figure 2: Dissolved organic carbon (DOC in µM-C) concentrations (left panels) and relative contribution (right panels) in the six fractions operationally defined by size exclusion chromatography for (a) filtered sample, (b) the PPL-retentate (SPE-DOM), (c) the PPL-permeate (Non SPE-DOM). It is worth noting that a contamination with methanol prevents the analysis of the S=15.5 sample by SEC after the extraction procedure (PPL permeate and retentate). ** Sample collected in 2018 and kept frozen until analysis.

Figure 3 :

 3 Figure 3: Mean DOC distribution in the bulk sample (grey bars), PPL permeate (non-SPE-DOC, white bars) and in the PPL-retentate (SPE-DOC, dark bars) for the 11 samples collected along the Aulne-Iroise Sea land-sea continuum. * indicate a significant difference between white and dark bars based on a two-factor Wilcoxon test (two groups, permate and retentate). Insert represents the DOM fractionation of PPL permeate and of PPL retentae for the freshwater (S = 0.17) and marine (S = 35.15) endmembers.

Figure 4 :

 4 Figure 4 : Elemental C/N ratios (a) and percentage of aromatic carbon (b) measured in the bulk samples (dark dots), in the permeate of a PPL resin (white dots) and calculated for the retentate of a PPL resin (grey squares) for the humic substances fraction along the land-sea continuum of the Aulne-Iroise Sea.

Equation 3

 3 δ 13 C SPE-DOCtheo = {(δ 13 CAulne* [SPE-DOC]Aulne * f ) + (δ 13 CAtlantic* [SPE-DOC]Atlantic * (1-f))} / {([SPE-DOC]Aulne * f) + ([SPE-DOC]Atlantic * (1-f))} With δ 13 C SPE-DOCtheo the theoretical stable isotopic carbon signature of the PPL retentate; [SPE-DOC]Aulne and [SPE-DOC]Atlantic the DOC concentrations of the PPL retentate in the Aulne river and in the Iroise Sea samples respectively; f the fraction of freshwater, f = 1 if S = 0.17 ; f = 0 if S = 35.15 Along the estuary, values of δ 13 C (Figure 5, white dots) were close to the mixing line. Closer examination, however, indicates some deviations from this line, with heavier δ 13 C values in the upstream part of the estuary (S < 21) and lighter δ 13 C when S > 21. Given the uncertainties of both mixing line and samples, five data points drift significantly from the mixing line. Based on the SPE-DOC excess estimate (Equations 4 & 5), we used a mass balance (Equation6) to determine the δ 13 C signature of the apparent SPE-DOC excess (δ 13 C SPE-DOCexcess, Figure5, yellow dots). The calculated δ 13 C SPE-DOCexcess was -26.3 ± 0.2 for the two points in the salinity range 10-16 and was-27.8 ± 0.2 for the three points in the 25 -31 salinity range.Equation 4 [SPE-DOC]excess = [SPE-DOC]measured -[SPE-DOC]theoEquation 5 [SPE-DOC]theo = {([SPE-DOC]Aulne -[SPE-DOC]Atlantic) / (SAulne -S Atlantic)} * SmeasuredEquation 6 δ 13 C SPE-DOCexcess = {(δ 13 CSPE-DOC measured * [SPE-DOC]obs ) -(δ 13 C SPE-DOC theo * [SPE-DOC]theo)} / [SPE-DOC]excessInterpretation based strictly on δ 13 C data, may lead to a false conclusion and/or biased interpretations. The δ 13 C SPE-DOCexcess values in the salinity range 25-31 indicate a lighter organic δ 13 C source that could be erroneously associated with terrigenous DOM inputs. Our study of the distribution of DOM size class by SEC clearly indicates an internal input of humic substances in this part of the estuary (FigureS1). At these salinities, the humic substances have aromaticities and C/N ratios much lower than the values measured for the Aulne endmember (Figure4). Based on these properties, we relate this input of humic substances to an internal autochthonous production that may result from bacterial degradation of phytoplankton cells rather than to an external terrigenous input. If correct, these autochthonous humic substances should have a high δ 13 C (phytoplankton origin) and increase the δ 13 C signature of the DOM. Thereby there is a discrepancy between the SPE-DOC δ 13 C values that suggest a terrigenous input and the SEC measurements that indicate autochthonous input.

Figure 5 :

 5 Figure 5: Stable isotopic organic carbon composition (δ 13 C) measured in the PPL resin eluate (white dots) along the land-sea continuum of the the Aulne-Iroise land-sea continuum. Solid and dashed lines predict the conservative mixing and associated uncertainties calculated using the δ 13 C value measured in the Aulne river (this study) and the δ 13 C of the surface Atlantic ocean (red dot) determined by Medeiros et al. (2016). The δ 13 C associated with excess SPE-DOC (yellow dots) were calculated by mass balance between δ 13 C measurements and the excess SPE-DOC. See text for details of calculations.

Figure 6 :

 6 Figure 6: Hydrophobic DOC concentrations (left axis) in the bulk samples (dark dots) and in the PPLpermeates (white dots) measured along the Aulne-Iroise Sea land-sea continuum. The associated percentages of hydrophobic DOC extraction yields (red diamonds, right axis) increase from fresh to marine waters.

Table 1 :

 1 Certified elemental and stable carbon isotopic composition (δ 13 C) of the three certified referenced materials used in this study. Recovery and δ 13 C isotopic signature measured after their dissolution in artificial seawater and extraction using a PPL sorbent. Retention yields for the samples collected in the salinity gradient

					SRFA	LHA	Atropine
	Elemental O/C			0.60	0.37	0.18
	Elemental H/C			0.99	0.7	1.37
	Elemental C/N			85.0	60.5	17.0
	δ 13 C certified			-27.6	-23.8	-18.1
	δ 13 C measured in solid (n = 3)	-27.6 ± 0.1	-23.7± 0.1	-18.1 ± 0.1
	Number	of	independent	10	3	3
	extraction
	% recovery in artificial	88	92	33
	seawater
	δ 13 C	measured	after	-27.8 ± 0.2	-23.9 ± 0.1	-19.9 ± 0.3
	solubilization and	PPL
	extraction
	3.2

Table 2 :

 2 Dissolved organic carbon concentrations (µM-C) measured in estuarine samples before and after

	(permeate) the solid phase extraction for the 6 operationally defined fractions. Associated extraction yields (EY
	%) for each fraction and total load of carbon in the PPL cartridge are provided. ND means not determined due to
	a methanol contamination during the extraction procedure.

* Calculated using EA-IRMS carbon measurement and an elution recovery of 95% (ER = 95%). See text for details

Acknowledgement

We thank Jerôme Lepioufle, professeur agrégé d'anglais, for his help in proofreading and editing. We thank F. Quéré, E. Armice and the crew of the R/V Albert Lucas for help during the cruise and for collecting samples along the Aulne-Iroise Sea Land Sea continuum. This work is a contribution to the FeLINE project (Fer Ligands In the aulNe Estuary, Ifremer, Politique de site DS, 2019-2020) and was funded by Ifremer, grant number R204-12-MS-02.