Sébastien Gorecki

Fabrice Nesslany

Daniel Hube

Jean-Ulrich Mullot

Paule Vasseur

Eric Marchioni

Valérie Camel

Laurent Noel

Bruno Le Bizec

Thierry Guérin

Daniel Hubé

Laurent Noël

Cyril Feidt

Xavier Archer

Aurélie Mahe

Gilles Rivière

Human health risks related to the consumption of foodstuffs of plant and animal origin produced on a site polluted by chemical munitions of the First World War

Keywords: Chemical munitions, explosives, polluted site, food contamination, health risk assessment

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

Introduction __________________________________________________________________

During the First World War (WWI), it is estimated that over 1 billion shells and projectiles were fired in Europe by the belligerents on the battlefield. The projectiles contained different high explosives and some of them were loaded with a wide variety of chemicals (toxic warfare agents, incendiary agents). As far as explosives are concerned, >400,000 tons of nitroaromatic compounds were manufactured by Germany, the most important being 2,4,6-trinitrotoluene (TNT), along with dinitrobenzene (DNB) and dinitrotoluene (DNT) isomers [START_REF] Bausinger | Exposure assessment of a burning ground for chemical ammunition on the Great War battlefields of Verdun[END_REF]. Different chemical warfare agents were also massively produced and used on a large-scale on the battlefield for their lethal or incapacitating properties. Among the most commonly used chemicals in ammunitions were arsenical compounds (emetic agents), sulfur mustard (also called yperite, a blister agent), phosgene (a choking agent) and hydrogen cyanide (a blood agent) [START_REF] Pitschmann | Overall view of chemical and biochemical weapons[END_REF]. Additionally, about 11,000 tons of arsenical warfare agents were produced in Germany and France during WWI. Due to their vomiting properties, diphenylchloroarsine ("Clark 1"), diphenyloarsinous cyanide ("Clark 2"), phenylarsine dichloride ("Pfiffikus") and dichloroethylarsine ("Dick") were some of the most commonly used arsenicals, only used by the German armies for the loading of "Blue Cross" or "Green Cross 2,3" shells which appeared late in Great War [START_REF] Bausinger | Environmental remnants of the first World war: soil contamination of a burning ground for arsenical ammunition[END_REF]. Half of the poison-gas shells fired on the battlefield by Germans during the last year of WWI were Blue Cross shells. The French did not use organo-arsenical compounds to load chemical shells. However, arsenic trichloride (AsCl3, "Marsite" for the French) just like tin tetrachloride (SnCl4) ("Opacite" for the French) were mixed with suffocating agent as smoke-producing compounds in French chemical projectiles of the Great War (Belot, 1997unpublished).

At the end of the war, the large quantity of fired and unfired munitions remaining on the former front and innumerous ammunition dump sites in the former backlines needed to be broken down. Hundreds of thousand tons of these weapons were dumped in the Northern European seas after WWI, in shallow waters [START_REF] Missiaen | Evaluation of a chemical munition dumpsite in the Baltic Sea based on geophysical and chemical investigations[END_REF]. Recent research shows that >1.7 billion tons of ammunitions remained at the Armistice in dumps as surpluses and coming from salvaging conducted by the allies [START_REF] Hubé | Sur les traces d'un secret enfoui[END_REF]. In 1920,1.1 million tons had to be broken down for safety purposes and to recover scrap, metals, and some chemical constituting the shells. A large amount of these remaining shells was destroyed in specific sites what led to the contamination of the surroundings in Belgium and in France [START_REF] Bausinger | Exposure assessment of a burning ground for chemical ammunition on the Great War battlefields of Verdun[END_REF][START_REF] Bausinger | Environmental remnants of the first World war: soil contamination of a burning ground for arsenical ammunition[END_REF]. [START_REF] Bausinger | Environmental remnants of the first World war: soil contamination of a burning ground for arsenical ammunition[END_REF] showed that the soil nearby a former ammunition destruction facility in Belgium was polluted by arsenic, copper, lead, nitroaromatic explosive compounds and their derivatives (ni-trobenzenes, nitrotoluenes), as well as triphenylarsine (a pyrolysis byproduct of organoarsenical agents).

These studies enabled to shed a light on the impact of ammunition destruction activities on soil contamination in Europe. In 2015, the French Bureau of Geological and Mining Research (BRGM) undertook an environmental assessment of a well-known former site used for the destruction of WWI chemical shells, called "La place à gaz" [START_REF] Brgm | Site de la « Place à gaz[END_REF] and previously studied by [START_REF] Bausinger | Environmental remnants of the first World war: soil contamination of a burning ground for arsenical ammunition[END_REF]. This site is a small zone located in the North East of France, close to the city of Verdun, in the Spincourt forest. "La place à gaz" was operated during 1926-1928 and over 200,000 German arsenical containing weapons were burnt there. However, historical research recently conducted by the BRGM revealed that this zone was part of a much larger-scale site, called the "Muzeray-Spincourt-Vaudoncourt" complex (also referred to as the "Clere & Schwander Meuse" complex). This site was operated from 1919 to 1925 by the historic French ministry of war ammunitions. One and a half million chemical shells and 300,000 explosive shells have been gathered there by the armies for destruction, making it the largest chemical ammunitions breaking down facility known on the former western front.

The "Clere & Schwander Meuse" site now covers >100 ha of farmlands and grasslands. The agricultural productions implanted in this zone are cereals for human consumption (soft winter wheat, winter and spring barley) and maize silage for the feeding of the dairy cow herds of two local farms. The analysis undertaken by the BRGM in the "Clere & Schwander Meuse" area revealed the presence in agricultural soils of nitrates, zinc, arsenic, organo-arsenical agents, tetrabromoethane (a solvent used for the manufacturing of arsenic warfare agents) and locally high levels of nitroaromatic compounds [START_REF] Brgm | Site de la « Place à gaz[END_REF].

Consequently, the French Agency for food, environmental and occupational health & safety (Anses) was asked to determine whether the consumption of plant and animal products originating from this area was safe for consumers. In this paper a specific methodology to assess the health risks related to the consumption of foodstuffs produced on areas with a battlefield history is proposed, and the results obtained in the particular case of the "Clere & Schwander Meuse" site are presented. To this end, food products of plant and animal origin were sampled in 2015-2016 and concentrations of organic and inorganic chemical contaminants were measured using developed specific analytical methods. Human exposure was assessed based on local individual consumption or/and national diet study. Risk was assessed for adults and children using health-based guidance values (HBGVs), margins of exposure or the toxicological concern concept in the case limited toxicological information were available.

Material and Methods __________________________________________________________

Selected contaminants

Based on the preliminary soil analysis undertaken by the BRGM the following substances were measured as a priority in different foodstuffs (see Section 2.3 for details):

Trace elements (TEs); primarily Zn, As, Pb and Cd; from shells, fuses and loading, Nitroaromatic explosives: primarily trinitrotoluene (TNT, 2,4,6trinitrotoluene), TNT impurities (2,4-dinitrotoluene (2,4-DNT) and 2,6-dinitrotoluene (2,6-DNT)), as well as 2-amino-4,6dinitroluene (2-ADNT) and 4-amino-2,6-dinitrotoluene (4-ADNT), which are the primary metabolites of TNT in plants;

Primarily phenylarsenic compounds including diphenylarsinic acid (DPAA -oxidation compound of phenylarsine) and triphenylarsine (TPA -pyrolysis transformation compound of phenylarsine); Perchlorate ion (high explosive used in some French artillery mines); Tetrabromoethane (TBE), a solvent that was used to produce arsines as warfare agents Vinyl bromide: given that this substance is a metabolite of tetrabromoethane in soil and that this substance has been classified as "probably carcinogenic to humans" (group 2A) according to the International Agency for Research on Cancer (IARC), it was deemed necessary to include this compound in the assessment.

Hazard characterization

For TEs, health-based guidance values (HBGVs) selected in the context of the second French total diet study (TDS2) were used (Anses, 2011a). For the other contaminants, an in-depth literature search was carried out. For certain contaminants (e.g. nitroaromatic explosives), HBGVs were available, and the most robust were selected. For the other substances, since no HBGVs have been established to date, toxicological benchmarks have been proposed based on the available data.

It should however be noted that these toxicological benchmarks were specifically determined for this study and are not intended to be used for another health risk assessment without an exhaustive analysis of the literature.

For diphenylarsinic acid and triphenylarsine, in order to allow the Threshold of Toxicity Concern (TTC) approach, the Cramer classification was determined with the Organization for Economic Co-operation and Development (OECD) Quantitative Structure-Activity Relationship (QSAR) Toolbox and Toxtree (version 2.5.0) software programs.

Contamination data

Sampling protocol

Samples of maize silage, barley and wheat were collected for analysis from seven growing plots located on the "Clere & Schwander Meuse" site. Regarding straw cereals (barley and wheat), in order to ensure representative samples for each plot, ten sampling points were randomly selected in the field using a "W" design (Belp, 1986). These ten sub-samples were pooled into equal shares to make composite samples before the analysis. Additional samples were taken in highly contaminated areas (referred to as "hot spots") determined by the BRGM based on the preliminary soil analyses.

As for foodstuffs of animal origin, two dairy cattle farms were considered in this study because their animals were fed with maize silage grown on the "Clere & Schwander Meuse" site. Therefore, 32 animals including 16 dairy cows, one heifer, three young bulls and 12 calves from these two farms were slaughtered. For each of these animals, muscle, liver and kidney tissues were sampled for analyses. Composite samples of raw milk were constituted for analysis. All the samples were shipped in frozen state to the laboratories in charge of the analyses.

Analytical methods

Arsenic and zinc in foodstuffs of plant origin were analyzed by inductively coupled plasma mass spectrometry (ICP-MS) after mineralization undertaken according to the NF EN 14084 standard (AFNOR, 2003a). Lead and cadmium in cereals were analyzed by atomic absorption spectroscopy according to the NF EN 14082 standard (AFNOR, 2003b). For TEs in foodstuffs of animal origin (milk, muscle, liver and kidneys) were analyzed by ICP-MS according to the method validated by [START_REF] Millour | Simultaneous analysis of 21 elements in foodstuffs by ICP-MS after closed-vessel microwave digestion: method validation[END_REF]. Details of the method are described in the supplementary material. The perchlorate analyses were carried out by liquid chromatography coupled to tandem mass spectrometry based on a previously reported method [START_REF] Fda | Rapid Determination of Perchlorate Anion in Foods by Ion Chromatography-Tandem Mass Spectrometry[END_REF].

For the other compounds, specific analytical methods were developed and validated by the participating laboratories as summarized in Table 1 along with the reported analytical performances. Details of these methods are described in the supplementary material.

Contamination data

For each studied foodstuff/contaminant pair, as part of a "worst-case" scenario, the maximum contamination value obtained in this study was retained for risk assessment (Table 2).

Results below the limit of detection (LoD) or quantification (LoQ) were processed using the "substitution method" recommended by the World Health Organization (WHO) [START_REF] Who | Reliable evaluation of low-level contamination of food -Addendum of the report on GEMS/Food-EURO second workshop of the 26-27th[END_REF]. It consists of defining the lower bound (LB) and upper bound (UB) for a measured value. The LB was calculated by considering that all values below the LoD were equal to zero and those between the LoD and LoQ were equal to the LoD. The UB was calculated by considering that all values below the LoD were equal to the LoD and those between the LoD and LoQ were equal to the LoQ.

Consumption data

The consumption data used for these two populations were taken from the second French consumption study (INCA2) (Anses, 2009). Exposure levels were estimated for children and adolescents between 3 and 17 years old and adults over the age of 17 years. In order to take into account local consumption habits, INCA2 respondents surveyed in the vicinity of Meuse, i.e. 136 individuals (77 adults and 59 children), were selected.

Exposure calculations

Based on the individual consumption data and contamination data, exposure was calculated using the following equation: where:

Ei is the total daily exposure of an individual i (μg kg body weight -1 day -1), Ci,k is the daily consumption of the food k by an individual i (g day -1),

Lk is the estimated level for the studied contaminant in the food k (mg kg -1 fresh food), BWi is the body weight of the individual i (kg), n is the total number of foods consumed by the individual i.

Mean exposure levels for the population were calculated in addition to exposure levels for the most exposed individuals (95th percentile).

Scenarios considered for exposure calculations

Given that the foodstuffs produced on the "Clere & Schwander Meuse" site are intended to be distributed on a national scale, the exclusive consumption of locally produced both foodstuffs of animal origin and cereals is highly unlikely. Consequently, two protective and more realistic scenarios were considered for exposure calculations. On one hand, the exposure through the consumption of foodstuffs of animal origin only was considered (scenario A) and, on the other hand, the exposure through the consumption of wheat only was considered (scenario B). The Health Risk Assessment (HRA) was undertaken according to the procedure presented in Fig. 1.

Kidneys were not taken into account for the calculation of exposure since the 136 local individuals considered in this study declared not eating kidneys. Similarly, for maize silage and barley, several data were lacking to quantitatively assess the risk related to the consumption of these foodstuffs (spatial variability of contaminant levels in soil, soil-plant, plant-animal and soil-animal transfer rates). Consequently, these foodstuffs were not taken into account.

Scenario A: Exposure via the consumption of foodstuffs of animal origin produced on the "Clere & Schwander Meuse" site For milk, meat, liver and wheat, individual consumption data from INCA2 were taken into account.

Trace elements (TEs)

It was assumed that the contaminants contained in the raw foodstuffs are fully transferred to finished products (with no dilution or concentration effect).

Total dietary exposure was calculated only for substances for which mean contamination levels measured in this study were higher than those previously reported in the framework of the TDS2 (it was indeed considered that for substances for which the contamination levels were below those of the TDS2, consumption of the foodstuffs produced on the "Clere & Schwander Meuse" site will not lead to overexposure compared to the general population studied in the TDS2). In this case, total dietary exposure was calculated by combining the contamination data for the total diet (excluding meat, liver and milk) from TDS2 with those measured in the present study (meat, liver and milk). When the calculated exposure levels (mean and 95th percentile) were higher than those of TDS2, a specific HRA was undertaken.

Nitroaromatic explosives, perchlorate ions, brominated compounds and arsines

For substances not considered in the TDS2 study (nitroaromatic explosives, perchlorate ions, brominated compounds and arsines), in order to take into account exposure related to the consumption of dairy products that could be produced from raw milk harvested on the "Clere & Schwander" site, contamination levels for dairy products like ultra-fresh dairy, butter and cheese were estimated using a table of dairy equivalents available on the website of the French National Federation of Dairy Cooperatives (Table 3).

Scenario B: Assessment of exposure via the consumption of wheat produced on the "Clere & Schwander Meuse" site For wheat, which is integrated in the form of flour in many foodstuffs, the total daily consumption of this foodstuff was estimated for the French population using available information in the INCA2 database (flour content of foodstuffs and daily consumption of these foodstuffs). The total consumption of wheat was 125 g day -1 on average and 220 g day -1 at the 95th percentile for adults and 80 g day -1 on average and 190 g day -1 at the 95th percentile for children. Exposure levels were estimated by considering only the consumption of foodstuffs including wheat flour, taking into account mean wheat consumption of the INCA2 French population.

Health risk assessment (HRA)

For substances with threshold dose effects, HRA was performed either by calculating a margin of exposure (MOE) (US-EPA, 2012) or a hazard quotient (HQ). HQ was calculated for substances with an available health-based guidance value (HGBV). For 2,6-DNT and tetrabromoethane, MOEs were calculated based on their no observed adverse effect level (NOAEL). For carcinogenic substances with no-threshold dose effects (TNT, 2,4-DNT, 2,6-DNT and vinyl bromide), HRA was performed by calculating an individual excess cancer risk (IECR) (US-EPA, 2005) based on their respective slope factors and considering a lifetime of exposure (70 years).

Results

______________________________________________________________________

Hazard characterization

Trace elements

For TEs, health-based guidance values selected in the context of the TDS2 were used (Anses, 2011a).

.2,4,6-trinitrotoluene

TNT is currently classified by IARC as belonging to Group 3, "not classifiable as toits carcinogenicity to humans" (IARC, 1996). Available toxicity studies indicate that chronic oral exposure to TNT can lead to effects on the liver, kidneys and blood (ATSDR, 1995).

The United States Environmental Protection Agency (US EPA) set a Reference Dose at 0.5 Eıg kg bw -1 day -1 based on a study on dogs exposed for 26 weeks, considering hepatotoxic effects as the critical effect (US-EPA, 1988). Regarding nonthreshold dose effects, the US EPA also established a slope factor of 0.03 (mg kg bw -1 day -1 ) -1 based on a two-year carcinogenicity study in rats, using combined urinary tract tumors as the critical effect (US-EPA, 1988). The risk related to dietary exposure to TNT was assessed using these two guidelines to take into account threshold dose effects on the one hand and non-threshold dose effects on the other hand.

2,4-Dinitrotoluene

Toxicity studies indicate that chronic oral exposure to 2,4-DNT can lead to adverse effects on the liver, kidneys and blood. Carcinogenicity studies in rats revealed that 2,4-DNT induces tumors in the renal tubules, hepatocellular carcinomas, adenomas of the mammary gland, and fibromas and fibrosarcomas of the skin. Consequently, IARC classified 2,4-DNT as belonging to group 2B, "possibly carcinogenic" to humans (IARC, 1996).

The US EPA set a minimum risk level of 2 Eıg kg bw -1 day -1 based on a two-year study on oral exposure in dogs, considering hematological effects as the critical effect (US-EPA, 1992). Regarding the non-threshold dose effects of 2,4-DNT, a slope factor of 0.31 (mg kg bw -1 day -1) -1 was established by the Office of Environmental Health Hazard Assessment (OEHHA) based on the incidence of tumors of the liver and mammary gland [START_REF] Oehha | Air Toxics Hot Spots Program Risk Assessment Guidelines (Part II. Technical Support Document for Describing Available Cancer Potency Factors[END_REF]. The risk related to dietary exposure to 2,4-DNT was assessed based on these two values to take into account non-threshold dose effects on the one hand and threshold dose effects on the other hand.

2,6-Dinitrotoluene

2,6-DNT is classified by the IARC as belonging to group 2B (IARC, 1996). Only one carcinogenesis study is reported by ATSDR for 2,6-DNT that indicates that 2,6-DNT can cause hepatocellular carcinomas in rats exposed to oral intake for 52 weeks (ATSDR, 2013).

As part of the Superfund Program, the US EPA set a provisional Toxicological Reference Value (TRV) of 0.3 Eıg kg bw -1 day -1 for 2,6-DNT, based on a lowest observed adverse effect level (LOAEL) of 4 mg kg bw -1 day -1 (US-EPA, 2013). Regarding non-threshold dose effects, the US EPA set a provisional slope factor of 1.5 (mg kg bw -1 day -1) -1 based on a one-year study on oral exposure in rats, using the onset of hepatocellular carcinomas as the critical effect (US-EPA, 2013).

Given the provisional nature of the TRV of 0.3 Eıg kg bw -1 day - 1and the uncertainties regarding the toxicity of 2,6-DNT, as reflected in the choice of a safety factor of 10,000 to derive this TRV (lack of toxicity studies on reproduction and development, few chronic studies), it was deemed preferable to assess the risk related to dietary exposure to 2,6-DNT by calculating a margin of exposure (MOE) based on the LOAEL of 4 mg kg bw -1 day -1 . The risk related to dietary exposure to 2,6-DNT was also estimated based on the provisional slope factor to take into account possible non-threshold effects.

2-amino-4,6-dinitrotoluene and 4amino-2,6-dinitrotoluene

Since there were no available specific toxicological data for 2-ADNT or 4-ADNT, the reference value set for 2,4-DNT (2 Eıg kg bw -1 day -1) (US-EPA, 1992) was found suitable to assess the risk related to dietary exposure to 2-ADNT and 4-ADNT due to molecular similarity. Given that 2-ADNT and 4-ADNT have the same toxicological benchmark and are the most commonly measured TNT metabolites in plants [START_REF] Burken | Phytoremediation and plant metabolism of explosives and nitroaromatic compounds[END_REF][START_REF] Vanek | Phytoremediation of explosives in toxic wastes[END_REF], the risk was assessed for the sum of these two compounds.

Perchlorate ions

Perchlorate ions inhibit iodine uptake [START_REF] Greer | Health effects assessment for environmental perchlorate contamination: the dose response for inhibition of thyroidal radioiodine uptake in humans[END_REF]. In its Opinion of July 2011, Anses proposed a TRV for perchlorate ions by ingestion of 0.7 Eıg kg bw -1 day -1 that was used in the present study to assess the risks related to dietary exposure to this substance. This TRV was based on the study by [START_REF] Greer | Health effects assessment for environmental perchlorate contamination: the dose response for inhibition of thyroidal radioiodine uptake in humans[END_REF] undertaken in healthy subjects exposed to perchlorate in drinking water for 14 days, in which a small decrease in thyroidal radioiodine uptake was measured (Anses, 2011b).

Diphenylarsinic acid

Neurotoxic effects were observed in people chronically exposed to diphenylarsinic acid through the ingestion of water from a contaminated well [START_REF] Ishii | Detection of diphenylarsinic acid and its derivatives in human serum and cerebrospinal fluid[END_REF]. In these studies, this substance was found to have toxic effects on the cerebellum, brainstem and brain. Diphenylarsinic acid tends to persist in the brain over a long period of time, having long-term repercussions. Mental retardation associated with brain atrophy has been observed in some poisoned children. These neurotoxic effects have also been observed in animals [START_REF] Negishi | Developmental subchronic exposure to diphenylarsinic acid induced increased exploratory behavior, impaired learning behavior, and decreased cerebellar glutathione concentration in rats[END_REF][START_REF] Ozone | Toxicity and oxidative stress induced by organic arsenical diphenylarsinic acid and inorganic arsenicals and their effects on spatial learning ability in mice[END_REF]. However, these studies cannot be used to determine a toxicological point of departure (POD) since the experimental conditions and/or exposure levels associated with the observed neurological symptoms are not known. In the absence of chronic or subchronic oral toxicity studies for establishing a POD, the "Threshold of Toxicological Concern" (TTC) approach, which offers a "minimum threshold value", was deemed the only approach that could be used for this study. In this case, the specific value that could be used was that determined by the compound's classification in Cramer Class III (according to the OECD Toolbox software), with no alert for genotoxicity, corresponding to 90 Eig person -1 day -1 (i.e. 1.5 Eig kg bw -1 day - 1 considering a person with a body weight of 60 kg) (EFSA, 2012).

Triphenylarsine

Toxicological data for triphenylarsine are limited. Consequently, an assessment was undertaken with the QSAR Toolbox and Toxtree software programs. No alerts for in vitro mutagenicity, in vivo genotoxicity or carcinogenicity were found with these softwares, which classified triphenylarsine in Cramer Class III. Consequently, for triphenylarsine, the risk assessment was undertaken based on the TTC of 1.5 Eig kg bw -1 day -1 as in the case of diphenylarsinic acid.

Tetrabromoethane

1,1,2,2-Tetrabromoethane was shown to cause neurotoxic, pulmonary and hepatotoxic effects to occupationally exposed workers by the inhalation route [START_REF] Morrow | PET and neurobehavioral evidence of tetrabromoethane encephalopathy[END_REF][START_REF] Van Haaften | Acute tetrabromoethane (acetylene tetrabromide) intoxication in man[END_REF]. In animals, the susceptibility of four-day-old newborn rats treated for 17 days with 1,1,2,2-TBE administered orally was studied and compared with that of young rats between the ages of five and six weeks at the beginning of a 28-day study. The young rats appeared to be more susceptible than the newborns to the toxic effects of TBE in the range of the tested concentrations. A NOAEL at 6 mg kg bw -1 day -1, based on hepatotoxic effects was selected [START_REF] Hirata-Koizumi | Susceptibility of newborn rats to hepatotoxicity of 1,3dibromopropane and 1,1,2,2-tetrabromoethane, compared with young rats[END_REF]. The risks related to dietary exposure to TBE were assessed by calculating a MOE based on this NOAEL.

Vinyl bromide

Like vinyl chloride, a chemical structure analogue, vinyl bromide is mutagenic both in vitro and in vivo. Vinyl bromide also induces DNA fragmentation in vivo in several organs (stomach, liver, kidneys, bladder, lungs and brain) in mice (IARC, 1986;[START_REF] Ntp | Report on Carcinogens (Thirteenth Edition[END_REF]. Furthermore, vinyl bromide is suspected to be carcinogenic to humans based on the induction of tumors in multiple organs in rats (NTP, 2014). To date, there is no HBGV available for vinyl bromide. However, the available data described above demonstrate similarities with vinyl chloride in terms of metabolism, genotoxicity and carcinogenicity. Consequently, in the absence of a specific HBGV, risks related to dietary exposure to vinyl bromide were assessed using the slope factor for vinyl chloride (oral route) of 1.5 (mg kg bw -1 day -1) -1 set by the US-EPA (US-EPA, 2000).

Exposure and risk assessment

Scenario A: Exposure via the consumption of foodstuffs of animal origin

Trace elements

The mean contamination levels measured in foodstuffs from animal origin produced on the "Clere 8r Schwander Meuse" site (meat, liver and milk) were compared with those previously reported in the context of the TDS2 (Anses, 2011a). These results were in the same order of magnitude. It was therefore considered that the consumption of milk, meat and offal produced on the "Clere 8r Schwander Meuse" site was not likely to result in overexposure to TEs in relation to the general population studied in TDS2.

Nitroaromatic explosives, arsines, brominated compounds and per-chlorate ions

With the exception of perchlorate ions for which quantifiable levels were measured in milk, meat, liver and kidneys, and triphenylarsine for which quantifiable levels were measured in offal (liver and kidneys), none of the other substances were detected (Table 2). Exposure levels were calculated according to UB hypothesis (Table 4). Results of the risk assessment are presented in Table 5 (for threshold effects) and Table 6 (for no-threshold effects).

For TNT, 2,4-DNT, 2-ADNT, 4-ADNT, diphenylarsinic acid, triphenylarsine, and perchlorate ions, the calculated exposure levels were lower than the toxicological values, regardless of the population.

For 2,6-DNT and tetrabromoethane, the lowest calculated MOEs were 24,000 and 288,000 respectively (for childrenexposure at the 95th percentile as the UB). In light of these MOEs, exposure to 2,6-DNT and tetrabromoethane through milk, liver and meat produced on the "Clere & Schwander Meuse" site was unlikely to be of health concern.

For TNT, an individual excess cancer risk (IECR) slightly above 10 -6 was calculated (for children, considering exposure at the 95th percentile as the UB). For other populations, the IECRs were below 10 -6 . For 2,4-DNT, 2,6-DNT and vinyl bromide, IECRs above 10 -5 were calculated for all populations; the highest IECR was calculated for 2,6-DNT in children at the 95th percentile (as the UB) with a value of 2.5 × 10 -4 .

It should be emphasized that the risk was assessed using a worst-case scenario since it was assumed that individuals consume, for their entire lifetime (70 years), only foodstuffs of animal origin produced on the "Clere & Schwander Meuse" site. Consequently, in light of the above results, it was considered that the consumption of foodstuffs of animal origin produced on the "Clere & Schwander Meuse" site was unlikely to be of health concern.

Scenario B: Exposure through the consumption of wheat

Trace elements

Pb and Cd levels measured in wheat were compliant with their respective regulatory limits [START_REF] Ec | Scientific Opinion on Exploring Options for Providing Advice about Possible Human Health Risks Based on the Concept of Threshold of Toxicological Concern (TTC)[END_REF]. For other substances for which regulatory limits in wheat have not been set out, the health risk was assessed.

Exposure levels were calculated according to UB hypothesis, considering mean wheat consumption (125 g day -1for adults and 80 g day -1 for children). The results obtained are presented in Table 7.

The corresponding health based guidance values were not exceeded for Zn. Therefore, exposure to this element through the consumption of wheat products is unlikely to be of health concern.

With regard to inorganic arsenic, depending on both the scenario and the population, the MOEs calculated with regard to the benchmark dose lower bound (BMDL01 0.3 to 8 pg kg bw -1 day -1) ranged from 8 to 665 (Table 8). The average exposure levels calculated for adults and children in this study accounted for 5 and 7% of the mean exposure levels calculated in TDS2 for adults and children respectively.

Although not quantified in the three composite samples taken randomly, it should be noted that arsenic was quantified in one "hot spot" sample (0.01 mg kg -1, Table 2).

However, the scenario considered to calculate exposure levels via the consumption of wheat and wheat products appears conservative. Indeed, it was assumed that all wheat products consumed by the considered populations (bread, pastries, biscuits, etc.) were prepared using only wheat produced on the "Clere & Schwander Meuse" site. Therefore, it was considered that the batch of wheat produced on this site is unlikely to pose a health risk. However, given the presence of hot spots detected on certain wheat plots, it appears likely that on certain plots, wheat (and probably any other crops) should not be grown.

Nitroaromatic explosives, arsines, brominated compounds, perchlorate ions

None of these substances were detected in wheat (see Table 2). Exposure levels were therefore calculated according to the UB hypothesis, considering mean daily wheat consumption (125 g d -1 for adults and 80 g d -1 for children). Results of the theoretical exposure calculations are shown in Table 9. Results of the HRA are given in Table 10 (for dose-threshold effects) and Table 11 (for no-dose-threshold effects).

Considering dose-threshold effects, for TNT, 2,4-DNT, 2-ADNT and 4-ADNT, diphenylarsinic acid, triphenylarsine, and perchlorate ions, the calculated exposure levels were lower than the corresponding health based guidance values used for the HRA, regardless of the population. For 2,6-DNT and tetrabromoethane, the lowest calculated MOEs were 150,000 and 1,000,000 respectively (for childrenexposure at the 95th percentile as the UB). In light of these MOEs, exposure to 2,6-DNT and tetrabromoethane through the consumption of wheat produced on the "Clere & Schwander Meuse" site was unlikely to be of h Considering no-dose-threshold effects, for TNT, IECRs of approximately 10 -8 to 10 -7 were calculated. As for 2,4-DNT, 2,6-DNT and vinyl bromide, IECRs slightly higher than 10 -5 were calculated for overall populations. So, it can be concluded for these effects and pollutants that the consumption of wheat produced on the "Clere & Schwander Meuse" site was unlikely to be of health concern.

Discussion ___________________________________________________________________

The Meuse site illustrates the potential long-term impact of the destruction and disposal of weapons on the contamination of both soil and the food chain. Other similar destruction sites after WWI, WWII and Cold war have already been identified elsewhere in France and Europe by the BRGM where similar health concern may be encountered. Itis also well established that chemical weapons were disposed in the sea after WWI and WWII [START_REF] Bełdowski | Arsenic concentrations in Baltic Sea sediments close to chemical munitions dumpsites[END_REF] what raises a more general question on the impact of the disposal and/or destruction of weapons on marine ecosystems. Given the nature of the chemical substances involved (arsenical compounds, explosives and derivatives), their persistence and toxicity, further consideration related to this issue is needed.

A specific methodology was developed for the health risk assessment related to the consumption of foodstuffs produced on the "Clere & Schwander Meuse" site, enabling the proposal of risk management measures in that particular case. For wellknown contaminants (such as TEs), for which the dietary exposure of French population has already been assessed in the framework of a total diet study, it was determined if the consumption of locally grown foodstuffs could lead to overexposure compared to the general population. For unknown contaminants, such as explosives and chemical warfare agents, specific analytical methods were determined. Concomitantly, a review of the available toxicity data was performed in order to determine toxicological benchmarks robust enough to conduct a health risk assessment. The same logic could easily be applied to other sites polluted by both well-known and unknown contaminants.

It should however be highlighted that several uncertainties can be listed. First of all, for some chemical substances considered, in the absence of health based guidance values, toxicological benchmarks were selected by default (e.g. the TTC was used for triphenylarsine and the slope factor for vinyl chloride was extrapolated to vinyl bromide). Specific toxicological data would therefore be required to fully characterize the hazards related to these compounds. Moreover, other compounds such as thiodyglycol (a metabolite of sulfur mustard and cyanide) also could have been taken into account and this would require the development of ad hoc analytical methods. In the framework of this study, some analytical methods were specifically developed, but it was non-feasible to develop additional methods to investigate other possible pollutants. Lastly, given the persistence of these contaminants and their presence in soil leachate as well as groundwater samples, the contamination of surface water and groundwater would need further consideration. For this particular matter, detailed environmental investigations conducted by the BRGM are foreseen in 2017 on the "Clere & Schwander Meuse" complex to implement the risk assessment by the characterization of groundwater, surface water, flesh of fish and sediments of the river Othain of the ponds close to the site. Deep soil will also be sampled.

Lastly, this work also highlighted the fact that additional data are required on the transfer of these contaminants from soil to plant and from animal feed to animals, to fully assess the risks related to the consumption of foodstuffs grown in contaminated areas. Further research is needed to improve knowledge on the toxicity, space and time fate of compounds present in high explosives, propellants and chemical warfareagents to assess the risk for health, water resource and environment in relation to these sites, in the specific historical, regulatory and environmental frame of France.

a During analysis, since tetrabromoethane broke down to tribromoethene, tribromoethene was systematically analyzed. b During analysis, triphenylarsine was oxidized into triphenylarsine oxide. Consequently, both compounds were systematically analyzed. a Values compliant with the regulatory limits set for foodstuffs: 0.020 mg kg for raw milk, 0.010 mg kg -1 for meat, 0.50 mg kg -1 for offal and 0.20 mg kg -1 for cereals (Regulation (EC) No 1881/2006). b Values compliant with the regulatory limits set for foodstuffs: 0.050 mg kg -1 for meat, 0.50 mg kg -1 for liver, 1 mg kg -1 for kidneys and 0.10 mg kg -1 for cereals (Regulation (EC) No 1881/2006). c Arsenic was quantified in one "hot spot" wheat sample and was not quantified in the three composite samples (LoQ = 0.01 mg kg -1). d The analysis showed that triphenylarsine is easily oxidized. Therefore, the two compounds were systematically tested for.

e During analysis, tetrabromoethane broke down to tribromoethene. Tribromoethene was systematically tested for with an LoQ of 0.5 μg kg-1. Indication: Numbers correspond to the PCB congeners. Framed congeners are dioxin-like PCBs. Bold numbers were congeners transferred at a high level ranking from 38 to 78% and from 30 to 80% respectively for milk and eggs.

Figure 1 .

 1 Figure 1. Procedure followed for the Health Risk Assessment.

Table 2 . Maximum contamination levels observed in foodstuffs produced on the "Clere & Schwander Meuse" site (Value ± Uncertainty).

 2 When the results are <LoD or <LoQ, the respective values of the LoDs and LoQs are given in parentheses.

	TEs

Table 3 . Table of dairy equivalents.

 3

		Equivalent volume
	Dairy products	of whole milk (L)
	1 kg butter	22
	1 kg Emmental	12
	1 1 whole-milk yoghurt
	cheeses	1
	Four 250 g Camembert	8

Table 4 . Exposure levels calculated according to the UB hypothesis for nitroaromatic explosives, brominated compounds, perchlorate and arsines, taking into accoun t milk, meat and liverproduced on the "Clere & SchwanderMeuse" site aswell as other dairy products that couldbe made from this milk (butter, ultra-fresh dairy, cheese).

 4

				HRA -threshold	effects -HQ or ΜΟΕ calculation
			Selected toxicological value	Children		Adults
	Substances	Calculation type	in ng kg bw -1 day -1	Mean exposure	Exposure at the 95th percentile	Mean exposure	Exposure at the 95th percentile
	TNT	ΗQ	500	0.006	0.011	0.004	0.011
	2,4-DIT	HQ	2000	0.014	0.027	0.009	0.027
	2,6-DIT	MOE	4,000,000	>350,000	>150,000	>550,000	>400,000
	2-ADNT + 4-ADNT	HQ	2000	0.003	0.005	0.002	0.005
	Diphenylarsinic acid	HQ	1500	0.009	0.018	0.006	0.018
	Triphenylarsine	HQ	1500	185 x 10 -4	3.58 x 10 -4	1.18 x 10 -4	3.58 x 10 -4
	Tetrabromoethane	MOE	6,000,000	>2000,000	>1,000,000	>3,000,000	>2,500,000
	Perchlorate ions	HQ	700	0.002	0.004	0.001	0.004

a Exposure levels for these two substances were added up since these compounds have the same TRV and are common metabolites of TNT during plant metabolism.

Table 5 . Risk assessment (tier 2A approach) according to the UB hypothesis: results of the Hazard Quotient and Marge Of Exposure calculations (substances with dose-threshold effects).

 5

	Risk assessment
	Children	Adults
	Selected toxicological value

a Exposure levels for these two substances were added up since these compounds have the same TRV and are common metabolites of TNT during plant metabolism.

Table 6 . Risk assessment (tier 2A approach) according to the UB hypothesis: results of the individual excess cancer risk calculation (substances with no-dose- threshold effects).

 6

				Risk assessment -IECR result
		Selected slope factor	Children			Adults
	Substances	(in ng kg • bw -1 day -1) -1	Mean exposure	95th percentile	Mean exposure	95th percentile
	TNT	3 x 10 -8	5.09 x 10 -7	125 x	10 -6	2.85 x 10 -7	5.48 x 10 -7
	2,4-DIT	3. 1	x 10 -7	5.26 x 10 -5	129 x	10 -4	2.95 x 10 -5	5.66 x 10 -5
	2,6-DIT	15	x 10 -6	102 x 10 -4	2.50 x	10 -4	5.70 x 10 -5	110 x 10 -4
	Vinyl bromide	15	x 10 -6	2.55 x 10 -5	6.25 x	10 -5	143 x 10 -5	2.74 x 10-5

Table 7 . Exposure to trace metal elements via wheat consumption estimated according to the UB hypothesis

 7 Speciation hypothesis used: 70% of total arsenic in wheat is considered to be in inorganic form(EFSA, 2009)

	Substances	Units	Children (n = 59)		Adults (n = 77)
			Mean	95th percentile	Mean	95th percentile
	Pb	μg kg • bw -1 day -1	0.11	0.22	0.07	0.10
	Zn	mg day -1	5.84	5.84	9.13	9.13
	Cd	μg kg • bw -1 week -1	1.13	2.18	0.72	0.96
	Total As	μg kg • bw -1 day -1	0.054	0.054	0.018	0.024
	Inorganic As a μg kg • bw -1 day -1	0.038	0.038	0.012	0.016

a

Table 8 . Inorganic arsenic exposure levels and risk assessment considering wheat consumption

 8 MOEs were calculated with regard to the BMDL01 of 0.3 μg kg bw -1 day -1 (EFSA, 2009).

	Population			MOE	MOE
		Mean exposure	95th percentile	for mean	for exposure at the
				exposure	95th percentile
		(µg•kg bw -1 day -1 )	(µgkg•bw -1 day-1
	Adults	0.012	0.016	25-665	19-505
	Children	0.020	0.038	15-400	8-215

Table 9 . Exposure estimated for nitroaromatic explosives, brominated compounds, perchlorateand arsines considering wheat consumption (tier 2B approach).

 9 Exposure levels for these two substances were added up since these compounds have the same TRV and are common metabolites of TNT during plant metabolism.

		Exposure level (ng kg bw -1 day -1 )
		Children (n = 59)	Adults	= 77)
	Substances	Mean	95th percentile	(n Mean	95th percentile
	TNT	2.8	5.4	177	2.36
	2,4-DIT	27.7	53.7	17.7	23.6
	2,6-DIT	11.1	215	7.1	9.4
	2-ADNT + 4-ADIT a	5.5	10.7	3.5	4.7
	Diphenylarsinic acid	13.9	26.9	8.9	11.8
	Triphenylarsine	0.277	0.537	0.177	0.236
	Vinyl bromide	8.3	16.1	5.3	7.1
	Tetrabromoethane	2.8	5.4	18	2.4
	Perchlorates	14	2.7	0.89	118

a

Table 10 . Risk assessment: Results of the Hazard Quotient and Marge Of Exposure calculations (substances with dose-threshold effects). Scenario taking into account the consumption of wheatproduced on the "Clere & Schwander Meuse" site.

 10 Exposure levels for these two substances were added up since these compounds have the same TRV and are common metabolites of TNT during plant metabolism.

				HRA -threshold effects -HQ or MOE calculation
				Children		Adults
			Selected toxicological value
	Substances	Calculation type	(ng kg bw -1 day -1)	Mean exposure Exposure at the 95th percentile	Mean exposure	Exposure at the 95th percentile
	TNT	HQ	500	0.034	0.083	0.019	0.083
	2,4-DIT	HQ	2000	0.085	0.208	0.048	0.208
	2,6-DIT	MOE	4,000,000	58,900	24,005	105,193	54,760
	2-ADNT + 4-ADNTa	HQ	2000	0.017	0.041	0.009	0.042
	Diphenylarsinic acid	HQ	1500	0.012	0.030	0.007	0.030
	Triphenylarsine	HQ	1500	3.40 x 10 -4	8.33 x 10 -4	190 x 10 -4	8.33 x 10 -4
	Tetrabromoethane	MOE	6,000,000	706,795	288,066	1,262,312	657,118
	Perchlorate ions	HQ	700	0.236	0.592	0.132	0.592

a

Table 11 . Results of the individual excess cancer risk calculations (substances with no-dose-threshold effects). Scenario taking into account the consumption of wheat produced on the "Clere &Schwander Meuse" site.

 11

	Risk assessment -IECR result


Tedj El

Moulouk Khaldi

Mounira Kebouchi

Claire Soligot

ORCID Mohamed Amine Gomri

Kharroub

ORCID Yves Le Roux

ORCID Emeline Roux
email: emeline.roux@univ-lorraine.fr

ORCID Karima Kharroub

Keywords: Streptococcus macedonicus strains isolated from traditional fermented milks: resistance Streptococcus macedonicus, Multi Locus Sequence Typing MLST, gastrointestinal tract, bile salts, 45 adhesion, epithelial cells. 46

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Streptococcus macedonicus strains isolated from traditional fermented milks: resistance to gastrointestinal environment and adhesion ability

Tedj El Moulouk Khaldi, Mounira Kebouchi, Claire Soligot, Mohamed Amine Gomri, Karima Kharroub, Yves Le Roux, Emeline Roux

To cite this version:

Tedj El Moulouk Khaldi, Mounira Kebouchi, Claire Soligot, Mohamed Amine Gomri, Karima Kharroub, et al.. Streptococcus macedonicus strains isolated from traditional fermented milks: resistance to gastrointestinal environment and adhesion ability. Applied Microbiology and Biotechnology, 2019, 103 (6), pp.2759-2771. 10.1007/s00253-019-09651-z. hal-02111693 al. 2009). Streptococcus thermophilus (S. thermophilus), widely used in dairy industry, is presently the sole 76 species among the Streptococcus genus to have the GRAS status obtained from the American Food and Drug 77 Administration (FDA 2002[START_REF] Fda | GRN No.378 Cultured [dairy sources, sugars, wheat, malt, and fruit-and vegetable based sources] 418 fermented by [Streptococcus thermophilus, Bacillus coagulans, Lactobacillus acidophilus, 419 Lactobacillus paracasei subsp. paracasei, Lactobacillus plantarum, Lactobacillus sakei, Lactobacillus 420 bulgaricus and Proprionibacterium freudenreichii subsp. shermanii or mixtures of these strains[END_REF] and the Qualified Presumption of Safety (QPS) status from the European Food 78 Safety Authority (EFSA). This bacterium is recognized for its beneficial effects such as the prevention of chronic 79 gastritis [START_REF] Rodríguez | Therapeutic effect of Streptococcus thermophilus CRL 1190-fermented milk on chronic 512 gastritis[END_REF] or the alleviation of lactose intolerance (EFSA Panel on Dietetic Products, Nutrition 80 and Allergies 2010; Savaiano 2014).

Introduction

81

The beneficial effects are not restricted to this bacterium since other species belonging to the Streptococcus 82 genus and which have been isolated from dairy products, could also exert positives actions on the host after 83 consumption as well as on the flavor of fermented milks. Among them, S. macedonicus was first isolated from 84 Greek Kasseri cheese [START_REF] Tsakalidou | The 538 Combined Use of Whole-cell Protein Extracts for the Identification (SDS-PAGE) and Enzyme Activity 539 Screening of Lactic Acid Bacteria Isolated from Traditional Greek Dairy Products[END_REF]) and later from Italian cheeses [START_REF] Lombardi | Characterization of Streptococcus 463 macedonicus strains isolated from artisanal Italian raw milk cheeses[END_REF]. It is a 85 thermophilic bacterium and homofermentative [START_REF] Tsakalidou | Identification of streptococci from Greek Kasseri cheese and description of Streptococcus 543 macedonicus sp[END_REF]) derived from the sub-group 86 Streptococcus gallolyticus [START_REF] Schlegel | Reappraisal of the taxonomy of the 518 Streptococcus bovis/Streptococcus equinus complex and related species: description of Streptococcus 519 gallolyticus subsp. gallolyticus subsp. nov., S. gallolyticus subsp. macedonicus subsp. nov. and S. 520 gallolyticus subsp. pasteurianus subsp. nov[END_REF]. However, it is generally considered non-pathogenic 87 [START_REF] Maragkoudakis | In vitro and in vivo safety evaluation of the bacteriocin producer 467 Streptococcus macedonicus ACA-DC 198[END_REF] and has even been reported to have a "food grade" status (De Vuyst and Tsakalidou 88 2008). Nevertheless, genomic analysis based on S. macedonicus genomes allow to establish its non-pathogenic 89 status [START_REF] Papadimitriou | Complete 486 genome sequence of the dairy isolate Streptococcus macedonicus ACA-DC 198[END_REF][START_REF] Papadimitriou | Whole-Genome Sequence of the 489 Cheese Isolate Streptococcus macedonicus 679[END_REF]. 90 Furthermore, the genomes of S. macedonicus published up to date all contain bacteriocin-related sequences. This 91 corroborates previous publications on the ability of S. macedonicus strains to produce antimicrobial peptides 92 directed against the alteration flora and the pathogenic species [START_REF] Anastasiou | Inhibition of 361 Clostridium tyrobutyricum by Streptococcus macedonicus ACA-DC 198 under conditions mimicking 362 Kasseri cheese production and ripening[END_REF][START_REF] Georgalaki | Macedocin, a Food-Grade Lantibiotic Produced by Streptococcus macedonicus 429 ACA-DC 198[END_REF] 93 [START_REF] Georgalaki | 425 Macedovicin, the second food-grade lantibiotic produced by Streptococcus macedonicus ACA-DC 198. 426[END_REF][START_REF] Grazia | Bacteriocin-like inhibitory substance 431 (BLIS) activity of Streptococcus macedonicus MBF10-2 and its synergistic action in combination with 432 antibiotics[END_REF][START_REF] Papadelli | Characterization of the gene cluster 479 involved in the biosynthesis of macedocin, the lantibiotic produced by Streptococcus macedonicus[END_REF][START_REF] Pieterse | Bacteriocin ST91KM, produced by Streptococcus gallolyticus 491 subsp. macedonicus ST91KM, is a narrow-spectrum peptide active against bacteria associated with 492 mastitis in dairy cattle[END_REF][START_REF] Zoumpopoulou | Incidence of Bacteriocins Produced by Food-Related Lactic Acid 562 Bacteria Active towards Oral Pathogens[END_REF]).

94

S. macedonicus strains exhibit up to 10 genes related to bacteriocins and ribosomally synthesized antimicrobial 95 peptides. In contrast, only a few sequenced strains of S. thermophilus possess those bacteriocins-related 96 sequences [START_REF] Treu | Whole-Genome 535 Sequences of Three Streptococcus macedonicus Strains Isolated from Italian Cheeses in the Veneto 536 Region[END_REF], making S. macedonicus of great technological interest. 97 Finally, S. macedonicus seems to have a good immunomodulatory activity [START_REF] Cho | Physiological Characteristics and Immunomodulating 383 Activity of Streptococcus macedonicus LC743 Isolated from Raw Milk[END_REF][START_REF] Fda | GRN No.378 Cultured [dairy sources, sugars, wheat, malt, and fruit-and vegetable based sources] 418 fermented by [Streptococcus thermophilus, Bacillus coagulans, Lactobacillus acidophilus, 419 Lactobacillus paracasei subsp. paracasei, Lactobacillus plantarum, Lactobacillus sakei, Lactobacillus 420 bulgaricus and Proprionibacterium freudenreichii subsp. shermanii or mixtures of these strains[END_REF] and produce 98 exopolysaccharides, which could not only be of technological interest but also display bioactivities (Tiwari and 99 Misra 2008;[START_REF] Vincent | Structure and properties of the 554 exopolysaccharide produced by Streptococcus macedonicus Sc136[END_REF]. Additionally, S. macedonicus, in co-culture with other LAB, leads to naturally 100 folate bio-enriched fermented milk [START_REF] Laiño | Milk fermented with selected 457 strains of lactic acid bacteria is able to improve folate status of deficient rodents and also prevent folate 458 deficiency[END_REF]. All these results indicate that S. macedonicus could 101 potentially be a good candidate as probiotic.

102

Nevertheless, little information is available on prerequisites of S. macedonicus probiotic interest such as the 103 tolerance to the hostile conditions of gastrointestinal tract and the adhesion ability to the intestinal mucosa. Up to 104 date, only one complete assembled genome is available in databases (strain Papadimitriou et al. 105 sequences were obtained using ITS16S (5'-TTGTACACACCGCCCGTCA-3') and ITS23S (5' -134 GGTACCTTAGATGTTTCAGTTC-3') primers (Dufour et al. 2008;[START_REF] Kostman | Molecular epidemiology of Pseudomonas cepacia 451 determined by polymerase chain reaction ribotyping[END_REF].

135

PCR reactions were carried out in a Mastercycler pro Thermocycler apparatus (Eppendorf, Hamburg, Germany) 136 with a final volume of 20 µL containing 100 ng of genomic DNA, 1X PCR buffer, 3 mM MgCl2, 0.2 mM dNTP, 137 0.5 µM of each primer and 0.5 U of Taq polymerase (Invitrogen, Thermo Fisher Scientific, Waltham, MA, 138 USA). Thirty cycles of denaturation (95°C, 30 s), annealing (55°C, 30 s), and extension (72°C, 1 min) steps were 139 repeated to amplify the 16S rRNA. For ITS, hybridization temperature was 54°C and extension time 45s. 152 macedonicus ACA-DC 198 genome [START_REF] Papadimitriou | Complete 486 genome sequence of the dairy isolate Streptococcus macedonicus ACA-DC 198[END_REF], and were synthesized by the Eurogentec 153 society. These primers sequences are listed in Table 1. PCR and gel electrophoresis conditions were those 154 previously described by Delorme et al. (2007). Genes dpr, gmk, rpoD, parC, pta, pyrC and recN were amplified 155 using Shibata's primers and PCR conditions [START_REF] Shibata | Development of a multilocus sequence typing scheme for 523 Streptococcus gallolyticus[END_REF]. The Sanger sequencing of each fragment on 156 both strands was realized by Beckman Coulter Genomics Society. A concatenated sequence of the 10 variable 157 housekeeping genes (pyrC, ilvC, pta, recN, ddlA, dpr, gmk, parC, rpoD

206

All cells were maintained in culture medium with antibiotics at 37°C in a humidified atmosphere with 10% CO2 207 for 21-25 days. Two days before the adhesion assays, cells were kept in culture medium without antibiotics.

208

Adhesion assay

209

The in vitro adhesion ability of S. macedonicus strains, LMD-9 and LGG strains to cultured cell lines was 210 studied. This study was performed according to Kebouchi et al. (2016). Succinctly, bacterial cells from an 211 overnight culture (14 h) were collected and the bacterial pellets were washed with HBSS and re-suspended at a 212 final concentration of 10 10 CFU/mL in culture medium free of antibiotics. In order to have the bacterial cell to 213 epithelial cell ratio of 1000 to 1, two milliliters of each bacterial suspension were added per well to cell 214 monolayers, which were previously washed with HBSS to remove dead cells and cellular debris. The six-well 215 plates containing the bacterial cells on the cell monolayers were incubated during 2 h at 37°C in a humidified 216 atmosphere with 10% CO2. Supernatants were then collected and epithelial cells were washed thoroughly to 217 recover non-adherent bacteria. Adherent bacteria were obtained by scraping cell wells with Triton ® X-100.

218

Adhesion was evaluated by enumeration of adherent and non-adherent bacteria after plating of serial dilutions on 219 LM17 and MRS agar. Two independent experiments were performed in duplicate.

220

Statistical analysis 221

Experimental data for acid, bile salts survival and bacterial adhesion were analyzed using analysis of variance 222 (ANOVA) followed by comparison of multiple averages with Tukey correction. Results were expressed as 223 means ±SEM. Differences were considered statistically significant when P value < 0.05.

224

Results

225

Identification of S. macedonicus strains 226

Isolation and phenotypic identification

227

First of all, the selection scheme adopted (Fig. 1) led to the recovery of 670 isolates -growing at 42°C on LM17 228 medium, being cocci Gram positive and catalase negative -from the 40 products (23 Rayeb and 17 Lben) 229 provided by 12 farmers and 13 retailers (Table 2). In a second time, the isolates were selected for their non-230 ability to grow on hostile medium (NaCl 6.5% (w/v) and pH 9.6) and to hydrolyze esculin. Only 6 fermented 231 products had strains responding to those criteria and were further investigated by molecular biology techniques 232 to confirm the presence of S. macedonicus strains.

233

Genotypic identification 234 Amplification and sequencing of 16S rRNA gene (partial sequence around 900 bp) (Table 3) indicated the 235 presence of four S. macedonicus strains having 100% identity with ACA-DC 198 and two S. thermophilus 236 strains (Lf9 having 100% identity with S. thermophilus JIM8232 or MN-ZLW-002; LA14, 100% identity with S.

237

thermophilus LMD-9). These results were comforted by amplification and sequencing of ITS 16S-23. The S.

238

thermophilus strain Lf9 has an ITS type I and the LA14 strain type II. 239 Phylogeny 240 Sixteen housekeeping genes were partially sequenced in four S. macedonicus strains. No differences were found 241 for 6 genes: glcK, pepO, thrS, tkt, pyrE and dnaE, with 100% sequence identity between the four strains isolated 242 and the Greek strain ACA-DC 198 [START_REF] Papadimitriou | Complete 486 genome sequence of the dairy isolate Streptococcus macedonicus ACA-DC 198[END_REF]). These last genes were remote in the MLST study.

243

The nucleotide sequences of 363 to 842 bp internal fragments were determined for the ten other loci (ddlA, ilvC, 244 serB, dpr, gmk, rpoD, parC, pta, pyrC and recN). The sequence diversity within the S. macedonius genes allows 245 distinguishing of two to three alleles per locus; the maximal percent nucleotide sequence divergence within S.

246

macedonicus ranges from 0.2% to 1.5% (Table 4).Among these genes, PyrC have the highest number (7 sites) 247 and the highest proportion (1.5%) of polymorphic sites, followed by ddlA (6 sites, 0.7%). Genes rpoD and serB 248 are the least polymorphic ones (2 sites, 0.2%).

253

Acid and bile salts tolerance 254

Survival to acid stress

255

The viability of strains S. macedonicus PA, LMD-9 and LGG was not affected after exposure to pH 4 during 2 h 256 (data not shown). At pH 3 (Fig. 3a), the PA strain was also more resistant than other S. macedonicus strains with 257 a loss of viability lower than 1 log and the same results were obtained for LMD-9 strain, whereas no 258 susceptibility of LGG strain was observed. The three other S. macedonicus strains P2, LK13 and LS5 were more 259 sensitive to an exposure at this pH with a survival decrease higher than 1.5 log. Moreover, no S. macedonicus 260 strain was detected after an exposure during 1 h at pH 2, contrariwise to LMD-9 and LGG strains which were 261 more resistant (data not shown).

262

Survival to bile salts stress

263

The four S. macedonicus strains were more resistant than LGG and LMD-9 strains. Furthermore, no viable 264 bacteria were detected at 10 mM and 4 mM of BSM for LGG and LMD-9 strain, respectively. 265 At the concentration of 6 mM BSM (Fig. 3b), the four S. macedonicus strains underwent a significant loss of 266 viability ranging from 1 log for the LS5 and LK13 strains, 2.5 log for the PA strain to 4.6 log for the most 267 sensitive strain P2. Furthermore, except the P2 strain, the three S. macedonicus conserved viability after 2 h 268 exposure to 10 mM BSM (Fig. 3b) and no significant effect was detected at 4 mM BSM (data not shown).

269

Bacterial adhesion to IEC 270

The ability of S. macedonicus strain (PA, P2, LS5 and LK13) to adhere to intestinal epithelial Caco2-TC7 and 271 HT29-MTX cells (IEC) in vitro is shown in Fig. 4. The LMD-9 and LGG strains were also included as 272 references.

273

Strains PA, P2 and LK13 of S. macedonicus studied have an adhesion capacity to Caco2-TC7 cells similar to 274 that LGG strain (Fig. 4a). LS5 strain has a lower adhesion capacity than the other S. macedonicus strains. These 275 values were even higher than those of LMD-9 strain. 276 However, among S. macedonicus strains, only PA strain have shown a significant high adhesion ability to

296

In the second part of this study, we examined in vitro some properties that probiotic strains should possess. In 297 addition, two strains, S. thermophilus LMD-9 and LGG were used as references. LGG strain is recognized as a 298 probiotic and it is known to have a good adhesion capacity and a high tolerance to the harsh conditions of the 299 digestive tract. Regarding LMD-9 strain, it was used as a reference because it is part of streptococci and also for 300 its best adhesion capacity and survival to acid and bile salt stresses among other S. thermophilus strains tested 301 [START_REF] Junjua | A large scale in vitro screening of Streptococcus 444 thermophilus strains revealed strains with a high anti-inflammatory potential[END_REF].

302

We chose pH values close to the physiological conditions of the GIT varying from 1 to 2 when the stomach is 303 empty and up to 4 -5 during meals [START_REF] Chen | The acid tolerance association with expression of H+ -ATPase in 381 Lactobacillus casei[END_REF]. Our results indicate that S. macedonicus strains tolerate 304 acidity up to pH value of 3, with a progressive reduction in viability between pH 4 and 3 depending on the 305 strains (data not shown). According to [START_REF] Ripamonti | Screening of species-specific lactic acid bacteria for veal calves 510 multi-strain probiotic adjuncts[END_REF], a strong reduction in the viability of fecal origin 306 strains S. macedonicus SB230 and SB330 was observed after exposure to pH 3 (Table 6). Furthermore, although 307 S. macedonicus ACA-DC 198 strain does not survive after 1 hour exposure to pH 2.5 [START_REF] Zoumpopoulou | Lactobacillus 557 fermentum ACA-DC 179 displays probiotic potential in vitro and protects against trinitrobenzene 558 sulfonic acid (TNBS)-induced colitis and Salmonella infection in murine models[END_REF], it has been isolated from mouse stools at a concentration close to that of the oral administration 309 [START_REF] Maragkoudakis | In vitro and in vivo safety evaluation of the bacteriocin producer 467 Streptococcus macedonicus ACA-DC 198[END_REF]. Therefore, the S. macedonicus strains should be resistant enough to cross the GIT 310 in a food matrix context.

311

The majority of survival studies to bile salts used the extract of bile whose exact composition is not well-known.

312

We preferred to use a bile salts mixture containing conjugate and free bile salts according to the study of 313 Kebouchi et al. (2016). The physiological bile salt concentrations vary from more than 40 mM to less than 1 mM 314 [START_REF] Ruiz | Bile resistance mechanisms in Lactobacillus and Bifidobacterium[END_REF]) with a concentration of 10 mM in small intestine [START_REF] Bajor | Bile acids: short and long term effects in the intestine[END_REF], which justifies the 315 concentrations adopted in this study. Our strains, except strain P2, seem to well tolerate high concentrations of 316 BSM, up to 10 mM. According to the literature, the behavior of bacteria towards bile salts is strain-dependent. 317 Indeed, S. macedonicus strains ACA-DC 198, LC743, SB230, SB330 and ACA-DC 208 resist quite well to bile 318 salts [START_REF] Cho | Physiological Characteristics and Immunomodulating 383 Activity of Streptococcus macedonicus LC743 Isolated from Raw Milk[END_REF][START_REF] Ripamonti | Screening of species-specific lactic acid bacteria for veal calves 510 multi-strain probiotic adjuncts[END_REF][START_REF] Zoumpopoulou | Lactobacillus 557 fermentum ACA-DC 179 displays probiotic potential in vitro and protects against trinitrobenzene 558 sulfonic acid (TNBS)-induced colitis and Salmonella infection in murine models[END_REF]Zoumpopoulou et al. ,2017)). However, the ten strains of S. 319 macedonicus tested by [START_REF] Blaiotta | Effect of chestnut extract and 369 chestnut fiber on viability of potential probiotic Lactobacillus strains under gastrointestinal tract 370 conditions[END_REF] display very different levels of resistance to bile salts (from 1% to 320 100% of survival). LAB can counteract bile salts by expressing a bile salt hydrolase (BSH). Although, the bsh 321 gene was shown in silico to be a pseudogene in the ACA-DC 198 genome [START_REF] Papadimitriou | Comparative genomics of the dairy isolate Streptococcus 483 macedonicus ACA-DC 198 against related members of the Streptococcus bovis/Streptococcus equinus 484 complex[END_REF], partial 322 enzyme activity of BSH type was observed in vitro for this strain [START_REF] Zoumpopoulou | Lactobacillus 557 fermentum ACA-DC 179 displays probiotic potential in vitro and protects against trinitrobenzene 558 sulfonic acid (TNBS)-induced colitis and Salmonella infection in murine models[END_REF]. In fact, bile salt 323 resistance is not always correlated with BSH activity, as reported for Lactobacillus salivarius (Fang et al. 2009).

324

Other mechanisms, unknown to date in S. macedonicus, are developed by bacteria to respond to bile stress, such 325 as cell wall composition, induction of stress protein or production of exopolymers. Concerning our four S.

326

macedonicus strains, they are more tolerant to BSM than LGG (used as a positive control in several studies) or S.

327

thermophilus LMD-9 and their integration in a food matrix could certainly allow them to cross the GIT.

328

The in vitro adhesion ability of the four S. macedonicus strains to human intestinal epithelial cell lines Caco-2 329 TC7 and HT29-MTX was investigated. Caco-2 cells are able to differentiate spontaneously in vitro culture 330 forming a cell monolayer expressing apical and basolateral proteins as enterocytes of the human small intestine 331 whereas HT29-MTX cells differentiate into polarized goblet cells and secrete massive amounts of mucus mainly 332 composed with MUC5AC mucins (stomach origin) (Kebouchi et al. 2016;[START_REF] Lesuffleur | Growth adaptation to methotrexate of HT-29 human 460 colon carcinoma cells is associated with their ability to differentiate into columnar absorptive and 461 mucus-secreting cells[END_REF]) and Pinto et al. 333 1983). To our knowledge, there are few studies on the adhesion capacity of S. macedonicus strain to intestinal 334 epithelial cells. Our results revealed that S. macedonicus strains, except one, adhere at the same level as LGG to 335 Caco-2 TC7 cells. Regarding HT29-MTX, PA strain was characterized by its moderate adhesion capacity 336 compared to LGG strain. [START_REF] Boleij | Novel clues on the specific association of Streptococcus gallolyticus subsp gallolyticus with 373 colorectal cancer[END_REF]) reported that S. macedonicus CIP105865T (SM) strain adheres 337 moderately to Caco-2 and HT29 cells, however a weak adhesion to the same cells was observed for S. 338 macedonicus strains tested by Zoumpopoulou et al. (2017). The adhesion of bacteria to epithelial cells are 339 generally related to the bacterial cell surface structures, among which pili and other surface proteins (Boleij et al. 340 2009;[START_REF] Jans | Genomics, evolution, and molecular epidemiology of the 440 Streptococcus bovis/Streptococcus equinus complex (SBSEC)[END_REF]. In S. gallolyticus (phylogenetically close to S. macedonicus), pil3 contributes to the 341 adhesion to HT29-MTX mucus since it binds specifically to mucin MUC5AC [START_REF] Martins | 470 Streptococcus gallolyticus Pil3 Pilus Is Required for Adhesion to Colonic Mucus and for Colonization 471 of Mouse Distal Colon[END_REF].The analysis

345

To conclude, we isolated four new S. macedonicus strains from traditional Algerian fermented milks, 346 differentiable on the genetic level from the Greek strain ACA-DC 198 (whole genome available on databases).

347

These strains have a sufficient survival capacity to gastric acidity and bile salts in the context of a food bowl.

348

Their in vitro adhesion ability to Caco-2 TC7 is significantly higher than the one of LMD-9 strain. A moderate 349 adhesion ability to HT29-MTX is obtained with one particular strain (PA). This first characterization encourages 350 the exploration of their probiotic effects in animal models. The tree was based on concatenated sequences of ten housekeeping genes (pyrC, ilvC, pta, recN, ddlA, dpr, gmk, 596 parC, rpoD and serB). The tree was constructed using the Maximum Likelihood method based on the Tamura-597

Nei model [START_REF] Tamura | Estimation of the number of nucleotide substitutions in the control region of 525 mitochondrial DNA in humans and chimpanzees[END_REF]. The percentage of trees in which the associated taxa clustered together is shown 602

The survival at pH 3 was evaluated for 1 and 2 h compared to the negative control (pH 7) and expressed as Log10

  Sequence Typing study was adapted from Delorme et al. (2007; 2010); El-Sharoud et al.149 (2012); Junjua et al. (2016) and Shibata et al. (2014). Primers used for amplification of internal fragments of 150 ddlA, pepO, glcK, ilvC, thrS, tkt, dnaE, pyrE and serB genes were specifically designed for S. macedonicus with 151 Primer3Plus program (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi), based on S.

  249 strains used in this study. Phylogenetic analysis of the concatenated sequences (total length of 5126 bp) yielded the tree shown in Fig. 2. It revealed a clear separation of the species S. macedonicus and S. gallolyticus, 251 supported by significant bootstrap values and a clear separation between Algerian and Greek strains of S. 252 macedonicus.

  277 mucus-secreting cells HT29-MTX (around 13 bacteria per cell) (Fig. 4b). Low adhesion values were obtained 278 with other S. macedonicus strains (P2, LS5 and LK13). 279 Discussion 280 The first objective of this work was to identify different strains, in particular of S. macedonicus, in raw milks 281 fermented spontaneously (Lben and Rayeb). The majority of the isolates from these raw milks was enterococci 282 and was consequently eliminated from the selection. In fact, enterococci were frequently isolated from 283 traditional dairy products (Cogan et al. 1997) and their predominance in Algerian traditional dairy products was 284 confirmed by Bensalah et al. (2009). S. thermophilus and S. macedonicus share the same characters targeted by 285 the phenotypical identification protocol. Only genetic analysis of 16S rRNA gene and ITS sequences allowed 286 separating the species. Thus, from the 40 traditional fermented milks subject of this work, we could only identify 287 four strains of S. macedonicus and two strains of S. thermophilus. These results show a presence of low 288 frequency of S. macedonicus in traditional fermented milks.289The MLST technique was often used to discriminate bacterial species: Streptococcus bovis/Streptococcus 290 equinus complex (SBSEC)[START_REF] Jans | Phylogenetic, epidemiological and functional analyses of the 437 Streptococcus bovis/Streptococcus equinus complex through an overarching MLST scheme[END_REF][START_REF] Shibata | Development of a multilocus sequence typing scheme for 523 Streptococcus gallolyticus[END_REF]. Our strains, sharing the same geographical 291 surface present a small degree of genetic variability. This low polymorphic character of S. macedonicus strains 292 was highlighted by[START_REF] Jans | Phylogenetic, epidemiological and functional analyses of the 437 Streptococcus bovis/Streptococcus equinus complex through an overarching MLST scheme[END_REF]: they detected by MLST a very low but visible genetic diversity on the 293 phylogenetic tree of S. macedonicus strains isolated from various origins (Greek, Somali, Ivorian and Malian). In 294 the same way, our four Algerian strains constitute a distinct genetic entity from the Greek strain S. macedonicus 295

  342of the genome of S. macedonicus ACA-DC 198 revealed the presence of the operon pil3(Papadimitriou et al.

  seems to have been preserved in S. macedonicus along evolution. An heterogeneity in the level of 344 pili encoding-gene expression in different strains may explain the affinity of strain PA for HT29-MTX cells.

  , Tsakalidou E (2008) Streptococcus macedonicus, a multi-functional and promising species for dairy 395 fermentations. Int Dairy J 18:476-485. doi: 10.1016/j.idairyj.2007.10.006 396 Delorme C, Bartholini C, Bolotine A, Ehrlich SD, Renault P (2010) Emergence of a cell wall protease in the 397 Streptococcus thermophilus population. Appl Environ Microbiol 76:451-460. doi: 398 10.1128/AEM.01018-09 399 Delorme C, Poyart C, Ehrlich SD, Renault P (2007) Extent of horizontal gene transfer in evolution of 400 Streptococci of the Salivarius group. J Bacteriol 189:1330-1341. doi: 10.1128/JB.01058-06 401 Dufour D, Nicodème M, Perrin C, Driou A, Brusseaux E, Humbert G, Gaillard J-L, Dary A (2008) Molecular 402 typing of industrial strains of Pseudomonas spp. isolated from milk and genetical and biochemical 403 characterization of an extracellular protease produced by one of them. Int J Food Microbiol 125:188-404 196. doi: 10.1016/j.ijfoodmicro.2008.04.004 405 EFSA Panel on Dietetic Products, Nutrition and Allergies (2010) Scientific Opinion on the substantiation of 406 health claims related to live yoghurt cultures and improved lactose digestion (ID 1143, 2976) pursuant 407 to Article 13(1) of Regulation (EC) No 1924/2006. EFSA J 8:1763 (p1-18) 408 El-Sharoud WM, Delorme C, Darwish MS, Renault P (2012) Genotyping of Streptococcus thermophilus strains 409 isolated from traditional Egyptian dairy products by sequence analysis of the phosphoserine 410 phosphatase (serB) gene with phenotypic characterizations of the strains. J Appl Microbiol 112:329-411 337. doi: 10.1111/j.1365-2672.2011.05212.x 412 Fang F, Li Y, Bumann M, Raftis EJ, Casey PG, Cooney JC, Walsh MA, O'Toole PW (2009) Allelic variation of 413 bile salt hydrolase genes in Lactobacillus salivarius does not determine bile resistance levels. J 414 Bacteriol 191:5743-5757. doi: 10.1128/JB.00506-09 415 FDA (2002) GRN No.49 Bifidobacterium lactis strain Bb12 and Streptococcus thermophilus strain Th4. GRAS 416 Not Inventory

Fig. 1 Fig. 2

 12 Figure legends

600 Fig. 3

 6003 Fig. 3 Survival of S. macedonicus, S. thermophilus LMD-9 and L. rhamnosus GG (LGG) strains after exposure

Fig. 4 Fig. 2 Fig. 3 Fig. 4

 4234 Fig. 4 Adhesion of S. macedonicus, S. thermophilus LMD-9 and L. rhamnosus GG (LGG) S. strains to Caco-2

161 Nucleotide sequence accession numbers and strain accession number 162 183 to pH 7 (control), pH 4, pH 3 and pH 2. Bacteria were then incubated at 37°C for 2 hours. The viable bacteria 184 were

  determined by plate count of CFU and results were expressed as Log10 CFU/mL.

	158	and serB) was created and the
	159	phylogenetic tree was built with Maximum Likelihood method based on the Tamura-Nei model (Tamura and Nei
	160	1993) (bootstrap: 1000 replicates) on the MEGA 7 software. Sequences of the genes from S. gallolyticus strains
		UCN34, ATCC43143 and ATCCBAA-2069 were used to root the tree.

185 Bile salts survival assay 186

	193	concentrations (0, 4, 6 and 10 mM). After plating serial dilutions on LM17 and MRS agar and incubating of plate
	194	dishes for 48 h at 42°C for streptococci strains and 37°C for LGG, the number of viable bacteria was determined
		by enumeration of CFU and results were expressed as Log10 CFU/mL.
	187	The survival of S. macedonicus strains to high concentrations of bile salts was carried out according to the
	188	method described previously by Iyer et al. (2010) and Kebouchi et al. (2016)with some modifications. Briefly,
	189	after an overnight culture (14 h), bacterial pellets were washed with HBSS and re-suspended in LM17 and MRS
	190	for Streptococcus genus and L. rhamnosus, respectively, at a final concentration of 10 10 CFU/mL. Bacterial
	191	suspensions were exposed for 2 h at 37°C to an equimolar bile salts mixture (BSM) containing sodium
		taurocholate, sodium cholate, and sodium deoxycholate (Sigma Aldrich®) (Kebouchi et al. 2016) at different

195 Bacterial adhesion to intestinal epithelial cells (IEC) 196 Cell culture conditions 197

	198	Two cells lines Caco-2 TC7 and HT29-MTX were used to study the in vitro adhesion ability of S. macedonicus
	199	strains. The cell culture conditions of Caco-2 TC7 (UMR 7274, Laboratoire Réactions et Génie des Procédés,
	200	Nancy, France) and mucus secreting cells HT29-MTX (INSERM UMR S 938, Paris, France) (Lesuffleur et al.
	201	1990) were performed using the method previously described by Kebouchi et al. (2016). For adhesion assay, the
	202	cell lines were seeded in six well culture plates (Dominique DUTSCHER) with an effective area of 9.7 cm 2 at a
	203	concentration of 11.10 6 cells per well. All kind of cells were cultured in Dulbecco's Modified Eagle's Minimal
	204	Essential Medium (DMEM, Glutamax, Fisher Scientific) supplemented with glucose, heat-inactivated fetal calf
	205	serum (FCS), antibiotics (penicillin/streptomycin) and eventually non-essential amino-acids (medium
		composition available in Kebouchi et al. (2016).

  Table 5 summarizes the allelic profiles of the five S. macedonicus

Tables 568 Table 1 .

 5681 Oligonucleotide primers for S. macedonicus(Multilocus Sequence Typing Method)

	569
	Locus	Primer	Sequence (5' -3')	Gene function
	glcK	glcK-for	GGGATTGACCTTGGTGGGAC	Glucose kinase
		glcK-rev	AAGACTTGCTGCACCGATGA
	ddlA	ddlA-for	CTGAGAGTGTCATGCGTGCG	D-alanine D-alanine ligase
		ddlA-rev	ATGGCTTTCGCGTTTTTCAA
	pepO	pepO-for	CCACATTGGGTCTCCTGCTT	Endopeptidase
		pepO-rev	GGCGCCAAATGCAAGAAGAA
	ilvC	ilvC-for	TGGTTATGGCTCACAAGGTCA	Ketol acid-reductoisomerase
		ilvC-rev	TGAATGCGTCGTCATCGTTT
	thrS	thrS-for	TGGACCGTAAAAGGCTGCTT	Threonyl-tRNA synthetase
		thrS-rev	CCTGACCACGAAGATGCACT
	tkt	tkt-for	CAGGACATGGTTCTGCCCTT	Transketolase
		tkt-rev	CGTGAGTTGGTCCGTCTTCA
	pyrE	pyrE-for	TCACAAATTGCATCAGATTTGCT	Orotate phosphoribosyltransferase
		pyrE-rev	TCACGCCAAGTTTCTTGATTTTCT
	dnaE	dnaE-for	GTCAGACCTCTATCCACGCG	DNA polymerase III
		dnaE-rev	CGCAAATAGACTCCCGAGCT
	serB	serB-for	TGGTAAGGCGCTTCTTTTCG	Phosphoserine phosphatase
		serB-rev	TGGATGTGGACTCTACCCTCA
	dpr	dpr-for	CCAAGCGGTTGCTGATTTGTC	Peroxide resistance protein
		dpr-rev	ATTTGATTCCTGGTGCTTGTCC
	gmk	gmk-for	CCGTTTCTATGACAACACGCC	Guanylate kinase
		gmk-rev	CGTCCGATAACACGATCAACA
	rpoD	rpoD-for	CGGACAAAGAAGGAAATCCATC	RNA polymerase sigma factor
		rpoD-rev	GTTTCAACCATGTGAACAGGA
	parC	parC-for	TGGCTTGAAGCCTGTTCAAC	DNA topoisomerase IV
		parC-rev	TCAATGAGGTAAACAACCGC	subunit A
	pta	pta-for	TGTTTGCTGACTGTGCGATT	Phosphate acetyltransferase
		pta-rev	CCATCAATAGCTTGAGCAGCT
	pyrC	pyrC-for	GGCTTTGATCAGGTTGCAGA	Dihydrotase
		pyrC-rev	GGTCTTCTTCGTGAACGGCAA
	recN	recN-for	AGGGAAATTCAATCGTGATGGC	DNA repair protein
		recN-rev	TCTTCAAAAGTTCGCGAGCTTG
	570
	571

Table 2 .

 2 Isolation of thermophilic lactic cocci (growth at 42°C, Gram+, catalase-)

	572
	No.	Product	Fermented milk	Origin	Isolates
		code			number
	1	LB1	Lben	Milk seller 1	10
	2	Rb1	Rayeb	Milk seller 2	11
	3	LbFb2	Lben	Milk seller 3	17
	4	RbFb1	Rayeb	Milk seller 3	18
	5	LbFb1	Lben	Milk seller 4	18
	6	RbFb2	Rayeb	Milk seller 4	16
	7	LbG1	Lben	Milk seller 5	18
	8	RbG1	Rayeb	Milk seller 5	17
	9	LbG2	Lben	Milk seller 6	19
	10	RbG2	Rayeb	Milk seller 6	16
	11	LbK1	Lben	Milk seller 7	18
	12	Rbk1	Rayeb	Milk seller 7	17
	13	Lbk2	Lben	Milk seller 8	16
	14	Rbk2	Rayeb	Milk seller 8	17
	15	Lbk3	Lben	Milk seller 9	18
	16	Rbk3	Rayeb	Milk seller 9	16
	17	Lbk4	Lben	Milk seller 10	17
	18	Rbk4	Rayeb	Milk seller 10	18
	19	LbS	Lben	Milk seller 11	19
	20	RbS	Rayeb	Milk seller 11	16
	21	LbA	Lben	Milk seller 12	13
	22	RbA	Rayeb	Milk seller 12	16
	23	LbW	Lben	Milk seller 13	17
	24	LbN1	Lben	Farm 1	16
	25	RbN1	Rayeb	Farm 1	18
	26	LbN2	Lben	Farm 2	15
	27	RbN2	Rayeb	Farm 2	17
	28	LbN3	Lben	Farm 3	15
	29	RbN3	Rayeb	Farm 3	18
	30	LbN4	Lben	Farm 4	16
	31	RbN4	Rayeb	Farm 4	19
	32	Lbchek	Lben	Farm 5	17
	33	RbMR1	Rayeb	Farm 6	18
	34	RbMR2	Rayeb	Farm 7	18
	35	RbFz	Rayeb	Farm 8	19
	36	Rb1FP	Rayeb	Farm 9	18
	37	Rb2FP	Rayeb	Farm 9	17
	38	RbJ	Rayeb	Farm 10	18
	39	RbM	Rayeb	Farm 11	16
	40	RbC	Rayeb	Farm 12	17
	Total 40	Rayeb (23),	Farms (12),	670
			Lben (17)	Milk sellers (13)
	573

Table 3 .

 3 Genotypic identification of presumptive S. macedonicus and S. thermophilus isolates

	574
	575	(no growth on BEA, NaCl (6,5%) and pH 9,6)
		No. Isolate	Fermented milk	Origin	RNAr 16S	ITS 16S-23S
		1	LK13	Lben (LbK1)	Milk seller 7
		2 3	LS5 PA	Lben (LbS) Rayeb (Rb1FP)	Milk sell. 11 Farm 9	S. macedonicus	S. macedonicus
		4	P2	Rayeb (Rb2FP)	Farm 9
		5	LF9	Lben (LbFb2)	Milk sell. 3		S. thermophilus ITS I
						S. thermophilus
		6	LA14	Lben (LbA)	Milk sell. 12		S. thermophilus ITS II
	576	ITS types were attributed according to (Mora et al. 2003).
	577
	578

Table 4 .

 4 Genetic diversity at S. macedonicus loci

	579
	Locus	Length of	No. of variable	No. of	Maximal % divergence within
		sequence (bp)	sites	alleles	S. macedonicus
	pyrC	477	7	3	1.5
	ilvC	776	3	3	0.4
	pta	466	2	3	0.4
	recN	400	3	2	0.7
	ddlA	842	6	2	0.7
	dpr	363	2	2	0.5
	gmK	415	2	2	0.4
	parC	462	2	2	0.4
	rpoD	435	1	2	0.2
	serB	490	1	2	0.2
	580
	581
	582

Table 5 .

 5 MLST data of the 4 S. macedonicus strains isolated in this study and the ACA-DC 198 strain

	583
	584	(Papadimitriou et al. 2012)
	Strain	Origin		Allele no.
		pyrC ilvC	pta	recN ddlA dpr	gmK	parC rpoD serB
	PA	Rayeb (Rb1FP)

Table 6 .

 6 Synthetic table representing some studies about acid and bile salt tolerance and adhesion to IEC of S.

	586
	587	macedonicus
		Source	Methods	Results
		Acid tolerance
		Zoumpopoulou et al.	S. macedonicus ACA-DC 198 in PBS	No survival after exposure 1 h at pH
		(2008)	solution pH 2.5; incubation at 37°C for	2.5.
			0.5, 1, 2 and 3 h.
		Cho et al. (2010)	LC743 strain in solution at pH 2, 3 and	No effect at pH 4. Slight decrease
			4; incubation at 37°C for 1, 2 and 3 h.	after 3 h at pH 3 (7.14 to 7.05 log-
				value); at pH 2, decrease from 7.14 to
				5.33 log.
		Ripamonti et al.	SB230 and SB330 strains in artificial	At pH 7, decrease from 8.58 to 8.36
		(2011)	gastric fluid (NaCl, 0.72 g/L; KCl, 0.05	log-value after 8 h.
			g/L; NaHCO3, 0.37 g/L; pepsin, 0.3 g/L)	At pH 3, no viability after 90 min.
			pH 3 and 7 for 0, 90 or 180 min.
		Blaiotta et al. ( 2013)	Ten S. macedonicus strains in solution	Highly variable (from 5 to 95%
			with NaCl 0.85% at pH 2.5 and 6 for 30	survival) depending on the strain; only
			min at 37°C.	two strains with a survival rate higher
				than 60%
		Zoumpopoulou et al.	S. macedonicus strains in PBS solution	Strong reduction in viability (decline
		(2017)	pH 2.5; incubation at 37°C for 2 h.	> 3 log)
		Our study	Four S. macedonicus strains in HBSS	Our strains tolerate acidity until pH
			solution pH 7, 4, 3 and 2 for 2 h at 37°C.	value of 3.
		Bile salt tolerance
		Zoumpopoulou et al.	Strain ACA-DC 198 in PBS solution,	Resistante: no viability loss after
		(2008)	pH 8 with 1% or 2% Oxgall, incubation	exposure 4 h to bile salts.
			at 37 °C for 1, 2 and 4 h.
		Cho et al.(2010)	LC743 strain in MRS broth with 0.05%	Good bile salts tolerance (decrease
			L-cysteine and 0.3% Oxgall for 7 h at	from 9 to 8.4 log-value).
			37 o C.
		Ripamonti et al.	3 h incubation in artificial gastric fluid	Reduction of population from 8.36 to
		(2011)	pH 7, then 3 h in artificial intestinal	7.63 log-value.
			fluid (0.1% pancreatin and 0.3% Oxgall
			bile salts, pH 8).
		Blaiotta et al. (2013)	Ten S. macedonicus strains on MRS or	From 1 to 100% of survival: only two
			M17 agar plates with 0.3% Oxgall,	strains out of 10, survived at 100%
			incubation at 37°C for 24-48 h.	and 60%; the 8 other strains less than
				46%.

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Conflicts of interest

354

The authors declare that they have no competing interests.

Ethics statement

356

This article does not contain any studies with human participants or animals performed by any of the authors. 4 Lben (strains LS5, LK13, LF9 and LA14) and 2 Rayeb (strains PA and P2) BEA (-), NaCl 6.5% (-), pH 9.6 (-)

Growth at 42°C on LM17, cocci Grampositive, catalase negative, facultative anaerobic Figure