Influence of Bizerte city wastewater treatment plant (WWTP) on abundance and antibioresistance of culturable heterotrophic and fecal indicator bacteria of Bizerte Lagoon (Tunisia)

Ecotoxicology and Environmental Safety 148 (2018) 201–210

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Influence of Bizerte city wastewater treatment plant (WWTP) on abundance and antibioresistance of culturable heterotrophic and fecal indicator bacteria of Bizerte Lagoon (Tunisia)

MARK

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Mariem Souissia, , Rached Laabidib, Patricia Aissaa, Olivier Pringaultc, Olfa Ben Saida a b c

Laboratory of Environment Biomonitoring, Coastal Ecology and Ecotoxicology Unit, Faculty of Sciences of Bizerte, University of Cathage, Zarzouna 7021, Tunisia Laboratory of Medical Analysis, Unit of Bacteriology, Regional Hospital of Bizerte, Street 13 August 1956, Bizerte 7000, Tunisia Laboratory of Coastal Marine Ecosystems, UMR 5119 CNRS-UM2-IFREMER- IRD-ECOSYM, University of Montpellier 2, France

A R T I C L E I N F O

A B S T R A C T

Keywords: Culturable fecal indicator bacteria Biodiversity Antibiotic resistance Acquired resistance ß-lactams WWTP

The waste water treatment plant (WWTP) of the city of Bizerte concentrates different types of chemical and biological pollutants in the Bizerte lagoon (Tunisia). Considering four upstream and downstream WWTP discharge stations, seventy nine, culturable bacterial strains were isolated and identified from water and sediment as fecal coliforms, fecal streptococci, pathogenic staphylococci and non-enterobacteriacea. Fecal coliforms were most abundant (2.5 105 bacteria/mg) in sediment of WWTP discharge. Leuconostoc spp (23.1%) and Chryseomonasluteola (23.1%) were the most prevalent culturable fecal indicator bacteria (FIB) isolated at the upstream discharge stations. However, Staphylococcus xylosus (13.9%) was the most prevalent culturable FIB isolated at the WWTP discharge stations. Moreover, high antibioticresistance phenotypes were present in all sampling stations, but especially in WWTP discharge station in both water and sediment. Resistance levels in water and sediment, respectively were amoxicillin (58.8%; 34.8%), penicillin (50%; 31.6%), oxacillin (60%; 33.3%), cefotaxim (55.2%; 39.1%), ceftazidim (66.7%; 50%), gentamycin (42.9%; 38.9%), tobramycin (50%; 25%), vancomycin (33.3; 71.4%), amikacin (66.7%; 0%) and ciprofloxacin (100%; 100%). Interestingly, ßlactam antibiotic resistant FIB were mostly isolated from water as well as from sediments of upstream and WWTP discharge station. Canonical correspondence analysis CCA correlating antibiotic resistance profile with the abiotic data showed that, in water column, culturable bacterial strains isolated in upstream WWTP discharge stations were interestingly correlated with the resistance to amikacin, oxacillin, cefotaxim, ciprofloxacin and gentamycin, however, in sediment, they were correlated with the resistance to amoxicillin, oxacillin, céfotaxim and vancomycin. Serious ß-lactams and aminoglycosides acquired resistance appeared mainly in fecal streptococci and pathogen staphylococci groups.

1. Introduction Aquatic ecosystems receive various types of contaminants which come mainly through WWTP discharges of urban and industrial effluents (Soudan, 1968; Stellman, 2000), with direct discharges as a secondary source (Kim et al., 2009). The presence of enteric pathogens in aquatic environments can cause human health risks and risks are aggravated when bacteria are antibiotic resistant (Servais and Passerat, 2009). Fecal bacteria can also transmit the resistance to indigenous bacteria through lateral transfer,1 thus contributing to the spread of antibiotic resistant bacteria (Davison, 1999). The sewage discharge is coupled with the direct discharge of antibiotics or their metabolites to

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the environment (Hijosa-Valsero et al., 2011), increasing selective pressure on the bacteria, and thus favouring resistance. The positive correlations between the degree of seawater contamination and frequency and variability of bacterial resistance indicate that polluted aquatic environments are sources of resistant bacteria (Fernandes Cardoso et al., 2010). Sewage effluent entering coastal waters contains a variety of harmful substances including viral, bacterial and protozoan pathogens, toxic chemicals such as antibiotics, organochlorines and heavy metals, and a variety of other organic and inorganic wastes (HMSO, 1990; Kümmerer, 2009). Moreover, hospital wastewater inflows significantly increased the prevalence of antimicrobial-resistant bacteria in WWTP and could strongly influence the toxic effects

Corresponding author. E-mail address: [email protected] (M. Souissi). The movement of genetic material between bacteria other than by the vertical transmission of DNA from parent to offspring.

http://dx.doi.org/10.1016/j.ecoenv.2017.10.002 Received 5 February 2017; Received in revised form 21 September 2017; Accepted 3 October 2017 0147-6513/ © 2017 Elsevier Inc. All rights reserved.

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mediated through transcription factor-related gene expression (Akiba et al., 2015; Guruge et al., 2015). The problems of this kind of aquatic pollution are likely to exacerbate and pose significant ecological and public health risk, especially in developing countries (Shahidul Islam and Tanaka, 2004) and recently, much attention has been paid to the safety of the treated wastewater because of water scarcity and the need to reuse waste water (Kim et al., 2009). In developing countries restricted local budgets, lack of local expertise, and lack of funding, result in inadequate operation of wastewater treatment plants (Paraskevas et al., 2002). Therefore, the problem of water pollution in the developing countries is an emergency. Organic pollution, as the most important aquatic pollution due to sewage releases, leads to disturbance of the natural environment caused by an overdevelopment of microorganisms (Bonnieux and Desaigues, 1998) and pathogenic microorganisms such as Viruses, protozoa and bacteria,which means that diseases transmitted by water remain a major safety concern throughout the world (Besassier et al., 2006). Indeed, treated and untreated wastewater, are often rejected directly in the aquatic ecosystems affecting the life of the aquatic flora and fauna and their quality that may be hazardous for the consumers (Akpor and Muchie, 2011). An environmental classification of the excreta-related diseases (Feachem et al., 1983; Mara and Alabaster, 1995) described different categories of diseases transmitted by wastewater: non-bacterial feco-oral diseases (e.g hepatitis A and E), bacterial feco-oral diseases (e.g Cholera, Salmonellosis…), geohelminthiases, taeniases, water-based helminthiases, excreta-related insect-vector disease and excreta-related rodent-vector disease. Some bacterial groups such as fecal coliform, fecal streptococci and staphylococci pathogens can be identified by specific tests and be considered as indicators of fecal contamination and the possible presence of ecological risks (Fernández-Molina et al., 2004; Tallon et al., 2005). Fecal indicators Bacteria FIB vary according by country and subnational jurisdictions (Tallon et al., 2005). The abundance of FIB is hypothesized to be correlated with the density of pathogenic microorganisms from fecal origin and is thus an indication of the sanitary risk associated with the various water utilisations (Ouattara et al., 2011). In addition, water quality is influenced by the control of other factors like the pharmaceuticals that represent a growing concern regarding their occurrence in the aquatic environment (Heberer, 2002; Smital et al., 2004; Alzieu and Romana, 2006; Budzinski and Togola, 2006; Kümmerer, 2009). These emerging chemical contaminants have been detected in river water, groundwater and drinking water samples (Halling-Sorensen al, 1998; Kanda et al., 2003). There are also several investigations showing that pharmaceuticals are not eliminated during wastewater treatment and also not biodegraded in the environment (Ternes, 1998; Daughton and Ternes, 1999; Nakada et al., 2006; Okuda et al., 2008). Antibiotics ATB, are widely used in human and veterinary medicine (Kümmerer, 2008). One of the consequences of their presence in the receiving environment is that many bacterial species develop transferable mutations, which allow them to develop antimicrobial resistance increasingly problematic for the environment. This resistance could be an aggravating factor, assuming that it would reduce the therapeutic options in case of infection. The problem is more aggravated in semi-closed aquatic environments, where the water renewal takes a relatively long time which could be long enough to make a significant impact on the environment (Harzallah, 2003). In case of sewage water discharge, simultaneous presence of FIB and ATB could be reported and therefore the problem of the spread of FIB antibiotic resistance should be an urgent concern. In a previous paper (Ben Said et al., 2016), we examined the sale of ATB pharmacies in Bizerte city in 2008 and we highlighted that ßlactams are the predominant family of consumed antibiotics. The present work puts more emphasis on the diversity of the Bizerte lagoon isolated bacterial species and their relationship with a biotic parameters prevailing in the Bay of Sabra where WWTP of Bizerte city are released.

Fig. 1. Sampling sites in Sebra Golf and Chaàara in Bizerte lagoon. →: Oued; water current direction. shellfish farm. The populated areas (dashed circles) and industrial zones (continuous circles) are indicated. S1: station upstream of the discharge; S2: discharge station; S3: downstream station of the discharge; S4: station away from discharge point (Châara). WWTP: wastewater treatment plant of Bizerte City.

2. Materials and methods 2.1. Bizerte Lagoon presentation Bizerte Lagoon is an important ecological and economic ecosystem (fishing and shellfish activities). This semi-closed aquatic environment is located in the northern part of Tunisia and extends over 150 km2 with a mean depth of 7 m and is linked to the Mediterranean Sea by an artificial channel and with the Ichkeul Lake through the Tinja oued (Zaaboub et al., 2015; Ben Salem et al., 2017). This area constitutes a receptor of several industrial sewages, aquaculture wastes, fertilizers, and pesticides through runoff and soil erosion, wastewaters from towns implanted around (Yoshida et al., 2002; Derouiche et al., 2004; Ben Said et al., 2010). In the northwest of the Bizerte lagoon, the wastewater treatment plant (WWTP) of Sidi Ahmed is situated between Bizerte and Menzel Bourguiba cities (Fig. 1). This plant treats the wastewater from the Bizerte city since 1997. Treated wastewater is discharged into the Bizerte lagoon, exactly in the bay of Sabra adjacent to the cement factory (Fig. 1). 2.2. Sampling and field measurements Water and sediment samples have been collected on September 2009 from the bay of Sabra (Bizerte lagoon) in 4 stations (Fig. 1). Sites were selected based upon the extent of WWTP discharge. The first station (S1) was located upstream of the WWTP discharge, the S2 station was located at the WWTP outflow, and third S3 and fourth S4 stations were located downstream of the WWTP discharge. Water column temperature, pH, and salinity were determined in the field with a handheld multi-parameter system (WTW Multi-197i). The water temperature was determined with a multi-parameter probe (YSI GRANT 3800). Sediments were sampled with Van Veen Grab quickly homogenized and stored at 4 °C for microbial analysis.

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Table 1 Physicochemical parameters of the stations located in the Bizerte lagoon (September 2009). S1: station upstream of the discharge; S2: discharge station; S3: downstream station of the discharge; S4: downstream station of the discharge; T: temperature, O: dissolved oxygen; Sat: salinity; Ct: conductivity; Si: Silicon; P- PO4: inorganic phosphorus; N-NH4: ammoninitrogen; N-NO2: nitrous oxide; H: humidity; NOT: total organic nitrogen; COT: total organic carbon; MOT: total organic material. Parameters

S1

S2

S3

S4

Latitude Longitude Depth (m) Hydrological parameters

N 37°15‘41.12‘‘ E 9°51‘06.71 1.58 26,3 8.22 6.7 37.8 158.1 2.17 3.64 8.27 2.07 8.68 19.796 14.98 51.33

N 37°15‘42.30“ E 9°51‘7.79 5 25.8 8.22 6.3 37.5 158.1 2.79 0.71 4.40 0.09 4.72 6.58 6.30 21

N 37°15‘42.77“ E 9°51‘09.37 6 26.1 8.22 6 38.7 158.1 0.61 0.46 2.49 0.75 9.58 13.85 8.16 20

N 37°13‘8.74“ E 9°49‘39.0 5 25.1 8.22 5.08 37.4 158.1 1.22 2.69 2.40 0.19 8.81 1.036 1.24 1.89

Sedimentological parameters

T (°C) pH O2 (mg/l) Sa (ppt) Ct (ms/cm) Si (10−3 ml) P-PO4 (10−3 ml) N-NH4 (10−3 ml) N-NO2 (10−3 ml) H% NOT % COT % MOT %

discs amoxicillin, cefotaxime, gentamycin, oxacillin, ciprofloxacin, penicillin G, vancomycin and tobramycin. The tested antibiotics were selected from the literature (Turano et al., 1994)

Chemical analyses, namely, ammonium (NH4), nitrites (NO2), orthophosphate (PO4) and Silicon (Si) were performed using standard methods (Strickland and Parsons, 1972; Sen Gupta and Koroleff, 1973). Humidity H%, total organic matter (MOT%), total nitrogen (NOT%) and total carbon (COT%) were measured in the laboratory using standard methods (Duchaufour, 1965; Hach et al., 1987).

2.6. Statistical analysis The ANOVA method was used to analyse the difference of bacterial abundance in sampling stations. Correspondence analysis (CA) was performed to assess the difference of isolated bacterial species repartition as a function of the different sampling sites in water and in sediment. Canonical correspondence analysis (CCA) was performed with bacterial antibiotic resistance of isolated bacteria and abiotic data (Ben said et al., 2015). Multivariate analyses (CA and CCA) were performed with MVSP v3.12d software (Kovach Computing Service, Anglesey, Wales).

2.3. Culturable heterotrophic and Fecal indicator bacterial quantification For water samples, 50 ml was filtered with a 0.2 µm membrane filter (Whatman®, GmbH) by vacuum filtration. The filtrate was suspended in 10 ml of physiological saline and vortexed (Bradshaw et al., 2016). Sediments were homogenized by gentle hand stirring with a large spatula, then, 1 mg of was suspended in 9 ml of physiological saline solution (Ben Said et al., 2015). Bacterial enumeration was performed by 3-tubes Most Probable Number MPN method and CFU (Colony Forming Units) method in triplicate (Oblinger and Koburger, 1975). Eosin Methylene Blue (EMB) and Tryptone Soya agar wereused, respectively, for fecal coliforms, fecal streptococci and pathogenic staphylococci. Tubes and plates have been incubated during 18–24 h at 37 °C and 44 °C.

3. Results 3.1. Site characterization

2.4. Culturable bacterial isolation and identification

The physical and chemical parameters observed in the sampling stations are summarized in Table 1. Considering hydrological parameters, no remarkable spatial variability was reported, maxima and the minima was observed for water temperature (26.3 °C (S1), 25.1 °C (S4)),salinity (38.7 psu (S3), 37.4 psu(S4)) and conductivity in water that was constant (in order of 158 ms/cmat all stations). Also, pH (8.22) was stable at the 4 stations and indicated a constant alkalinity. However, dissolved oxygen rates showed a decreasing gradient from S1 (6.7 mg/l) to S4 (5.08 mg/l) which is away from the release point. Likewise for ammonia nitrogen (N-NH4)(8.27 10−3 ml (S1) to 2.40 10−3 ml(S4)), nitrous oxide (NNO2) (2.07 10−3 ml (S1) to 0.09 μl (S2)), silicon (Si) (2.79 10−3 ml (S2) to 0.61 10−3 ml (S3)) and inorganic phosphorus (3.64 10−3 ml (S1) to 0.46 10−3 ml (S2)). To the contrary, a remarkable spatial variability was observed for sedimentological parameters. Thus, total organic matter (TOM) varied significantly between S4 (1.89%) and S1 (51.33%) with intermediate values in S2(21%) andS3 (20%). Total organic carbon contents(TOC) and total nitrogen (NOT) showed respectively a minimum of 1.24% (S4) and a maximum of14.98% (S1)and a minimum of 1.036% (S4) and a peakof19.79% for the NOT(S1). Discharge station S2 contained low levels of TOC (6.30%) and NOT (6.58%). The values of humidity H fluctuated between 4.72% (S2) and 9.58% (S4).

The isolation of fecal coliforms, fecal streptococci, pathogen staphylococci and non-Enterobacteriaceae NE was performed after plating saline suspensions (1 g sediment or 1 ml of water) on selective culture media. After incubation for 24 h at 44 °C, the morphologically different bacterial colonies were isolated and purifed by the method of transplanting streaks. Pure isolates were identified by routine microbiological tests including Gram staining, morphology, catalase and oxidase production, cell motility was examined by phase-contrast microscopy. The tests of oxidative and fermentative utilization of glucose and lactose were performed according to standard methods (Ben Said et al., 2008). Identification of isolated culturablebacterial strains was performed using API®20 Strep, API®Staph, API®20E and API®20NE galleries (BioMerieux, France) according to the manufacturer's recommendation and the results were read using an automated microbiological mini-API (Bio-Merieux, France) (Hassen et al., 2001). 2.5. Antibiotic resistance Antibiotic susceptibility was determined by the agar diffusion method as previously described (Bauer et al., 1966), using ten antibiotic 203

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3.2. Bacterial abundance

Table 2 Isolated culturable bacterial strains from Bizerte lagoon (Septembre 2009) and their percentage of isolation. S1S: station 1 sediment; S2S: station 2 sediment; S3S: station 3 sediment; S4S: station 4 sediment; S1W: station 1 water; S2W: station 2 water; S3W: Station 3 water; S4W: Station 4 water; %: percentage of bacteria specie isolation; NE: nonenterobacteriacea.

All stations were characterized by high sediment bacterial abundance compared to water samples. Two factor ANOVA statistical analysis (bacterial groups df = 3 and stations (S1, S2, S3, S4, df = 3) results showed a highly significant difference in terms of these two parameters (df = 9, p < 0.001).

References (origin)

Species

%

References (origin)

Streptococci

3.2.1. Water compartment At station S1, the bacterial abundance of culturable heterotrophic bacteria and FIB do not exceed 2 103 bacteria/ml. While at station S2, the culturable heterotrophic isolated bacteria were the most abundant (12.47 103 ± 9 103 bacteria/ml) followed by the fecal coliforms (6.65 103 ± 8103 bacteria/ml). At station S3, the fecal staphylococcus counted the most important isolated bacterial abundance (25 103 ± 6 103 bacteria/ml). But the station S4 was the most charged in fecal streptococci (15 103 ± 6 103 bacteria/ml).

(S1W)1 (S1S)2 (S1S)3 (S2W)4 (S3S)5 (S3S)6 (S3S)7 (S4S)8 (S3S)9 (S4W)10

3.2.2. Sediment compartment The fecal streptococci were most abundant in sediment samples mainly those of station S1 (~3.4 109 ± 9 106 bacteria/g) and S3(~2.3 109 ± 1,6 106 bacteria/g). The fecal coliforms were abundant in the sediment samples from the station S2 (2.5 108 ± 12 106 bacteria/g).

(S1S)11 (S2S)12 (S2S)13 (S2W)14 (S2S)15

3.3. Culturable Bacterial strains isolation

(S4S)16 (S3S)17 (S2W)18 (S2W)19

From water and sediment samples, 62culturable FIB strains [S1 (19.4% FIB), S2 (43.5% FIB), S3 (21% FIB) and S4 (15.2%)] and 17 NE bacterial strains were isolated. According to the shape and the arrangement of cells, biochemical tests and the gram stain, the isolated bacteria were identified. All bacterial isolated strains were represented in Table 2. The most abundant isolated streptococci species were Leuconostocspp then Aerococcusviridans 2, Enterococcus avium and Gemellahaemolysans and Lactococcuslactissspcremoris (7.6%; 3.8%; 3.8%; 3.8%; 3.8%) respectively. Staphylococcus xylosus then Staphylococcus haemolyticus, Micrococcus spp, Staphylococcus warneri, Staphylococcus epidermidis and Staphylococcus cohniisspcohnii (10.1%; 2.5%; 2.5%; 2.5%; 2.5%; 2.5%) were the most abundant isolated staphylococci. Coliforms were interestingly isolated mainly Chryseomonasluteola and Stenotrophomonasmaltophilia (11.4%; 2.5%). Sphingomonaspaucimobilis, Aeromonashydrophila/caviae and Ochrobactrumanthropi (6.3%; 6.3%; 3.8%) were the most abundant isolated NE species. The spatial distribution of culturable isolated species showed that Leuconostoc spp, Staphylococcus xylosus and Chryseomonas luteola strains were mainly isolated from S1 (23.1%; 15.4%; 23.1%), S2 (2.8%; 13.9%; 8.4%), S3 (11.1%; 0%; 5.6%) and S4 (0%; 8.3%; 16.7%)

2.5

3.8

1.3

(S2W)60 (S1S)61 (S1S)62

3.8

1.3

(S1W)63 (S2W)64 (S2W)65

1.3

(S2W)66

2.5 1.3

(S3S)67 (S4S)68 (S4W)69

1.3

(S3W)70

2.5

(S2S)71 (S2W)72 (S2S)73

Micrococcus spp

2.5

Staphylococcus warneri Staphylococcus hominis

2.5

(S3S)28 (S4W)29 (S3S)30 (S2S)31

(S3S)32 (S3S)33 (S4S)34 (S2S)35 (S1S)36 (S1S)37 (S2S)38 (S2S)39 (S2S)40 (S2S)41 (S2W)42 (S4W)43 (S2S)44 (S2S)45 (S2S)46 (S4W)47

(S4S)56 (S2W)57 (S2W)58 (S2W)59

(S2S)27

(S3W)24

(S1W)48 (S1S)49 (S1S)50 (S2W)51 (S2W)52 (S2S)53 (S3W)54 (S4W)55

3.8

(S2W)25 (S3S)26

(S1S)21 (S2S)22 (S3W)23

204

Lactococcus lactis ssp cremoris Aerococcus viridans 2

Streptococcus equinus Streptococcus pneumoniae Streptococcus acidominimus Gemella morbillorum Enterococcus faecium Staphylococci Staphylococcus haemolyticus

(S4S)20

3.3.1. Correspondence Analysis of the culturable bacterial species repartition based as a function of the different sampling sites In water compartment, the Correspondence Analysis CA (Fig. 2a) showed that sampling stations (S1, S2, S3) were close in terms of isolated heterotrophic and FIB species. The projection of isolated culturablebacterial species (Fig. 2a′) showed a concentration and a significant biodiversity of culturable bacterial strains at discharge station S2 (eg. Leuconostocspp,ChryseomonasLuteola,Stenotrophomonasmaltophilia, Staphylococcus haemolyticus). In sediment compartment, stations S1 and S2 were close and different from S3 and S4which are close in terms of isolated species (Fig. 2b) and they have an important concentration of culturable bacterial species. The biodiversity of streptococci and staphylococci was the most important at discharge station S2. Therefore these stations present a significant biodiversity of culturable bacterial strains (Fig. 2b′) (e.g. Enterococcus aviumAerococcusviridans 1, Staphylococcus

7.6

Aerococcus viridans 1 Gemella haemolysans

(S3W)74

1.3

(S2W)75 (S2S)76 (S3W)77 (S2W)78

Staphylococcus epidermidis

2.5

(S3S)79

Staphylococcus saprophyticus Kocuriavarians/ rosea Staphylococcus xylosus

1.3

Staphylococcus capitis Staphylococcus cohnii ssp cohnii Staphylococcus aureus

%

Coliforms

Leuconostoc spp

Enterococcus avium

Species

1.3 10.1

1.3 2.5 1.3

Chryseomonas luteola

11.4

Enterobacter cloacae Escherichia coli Burkholderia cepacia Stenotrophomonas maltophilia Shewanella putrefaciens NE Sphingomonas paucimobilis

1.3 1.3 1.3 2.5 1.3

6.3

Aeromonas hydrophila/caviae

6.3

Aeromonas shigelloides Pasteurella aerogenes Ochrobactrum anthropi

1.3

Aeromonas salmonicida ssp salmonicida Pseudomonas aeruginosa

1,3 3.8

1.3

1,3

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(a) S3

(b)

CA case scores 1.7

Axis 2

1.7

1.4

1.0

0.7

0.7

0.3

0.3 Axis 1

S1

-0.7

-0.3

0.3

0.7

1.0

1.4

1.7

-0.3

S2

Axis 2

1.3

1.0

-1.0

CA case scores S3

S1 -1.0

S4

Axis 1

-0.7

-0.3

S2

-0.7

-0.3

0.3

0.7

1.0

1.3

-0.7

1.7

S4

-1.0

-1.0

(a’)

CA variable scores Gemella morbillorum

Axis 2 (24%)

Enterococcus faecium

1.7

Pasteurella aerogenes 1.4 1.0 0.7

Aeromonas hydrophila/caviae

0.3

Chryseomonas luteola Axis 1 (30%) -1.0

-0.3

-0.7

0.3

Sphingomonas paucimobilis -0.3 Leuconostoc spp Stenotrophomonas maltophilia

0.7

1.0

1.4

1.7

Lactococcus lactis ssp cremoris Staphylococcus xylosus

-0.7

Staphylococcus warneri Staphylococcus aureus Enterococcus avium

Ochrobactrum anthropi Gemella haemolysans Burkholderia cepacia Enterobacter cloacae -1.0 Staphylococcus haemolyticus Aeromonas salmonicida ssp salmonicida

CA case scores

(b’)

Axis 2 (24%) 2.05 1.64

Leuconostoc spp

Staphylococcus warneri Staphylococcus haemolyticus Staphylococcus epidermidis Pseudomonas aeruginosa Lactococcus lactis ssp Aerococcus viridans 2

1.23 0.82

Micrococcus spp Shewanella putrefaciens Ochrobactrum anthropi -1.23

-0.82

Chryseomonas luteola Streptococcus acidominimus Staphylococcus xylosus

Axis 1 (30%) -0.41

Staphylococcus capitis Staphylococcus cohnii ssp cohnii Aerococcus viridans 1 Staphylococcus hominis Kocuria varians/rosea Aeromonas shigelloides

0.41

0.82

-0.41

Aeromonas hydrophila/caviae -0.82

1.23

1.64

2.05

Gemella haemolysans Streptococcus pneumoniae

-1.23

Staphylococcus saprophyticus

Fig. 2. Correspondence Analysis (CA) of the bacterial species repartition based as a function of the different sampling sites in water (a, a′) and in sediment (b, b′) of the Bizerte lagoon (Septmber 2009). S1: upstream of the discharge station; S2: discharge station; S3: downstream of the discharge station; S4: station away from discharge point.

3.4.1. Water compartment For all tested ATBs, a higher percentage of ATB-resistant bacterial water column isolated was reported in station S2 (Fig. 3a) which received WWTP of Bizerte city [AMX (58.8%), P (50%), OX(60%), CTX (55.2%), CAZ (66.7%), GM (42.9%), TM (50%), VA (33.3%), AN (66.7%) and CIP (100%)].

xylosus,Staphylococcus cohniisspcohni,Chryseomonasluteola).

3.4. Antibiotic resistance of the isolated culturable bacterial strains To study the ATB-resistance of culturable bacteria isolated from the Bizerte lagoon, ten antiotics were tested: amoxicillin, penicillin G, oxacillin, cefotaxime, ceftazidime, gentamycin, tobramycin, ancomycin, ciprofloxacin and amikacin.

3.4.2. Sediment compartment In all sampling stations, sediment isolated culturable bacterial 205

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Fig. 3. Frequencies of the resistance phenotype to each antibiotic (in %) among culturable fecal indicator bacteria in water (a) and in sediment (b) (September 2009). S1: upstream of the discharge station; S2: discharge station; S3: downstream of the discharge station; S4: station away from discharge point; AMX: amoxicillin; P: pénicillin G; OX: oxacillin; CXM: cefuroxime; CAZ: ceftazidime; GM: gentamicin; TM: tobramycin; VA: vancomycin; AN: amikacin; CIP: ciprofloxacin*: β-lactams.

strains were resistant to AMX, P, OX, CTX, GM and TM (Fig. 3b). Excepting for AN, resistance was observed in the WWTP discharged station (S2) [AMX (34.8%), P (31.6%), OX(33.3%), CTX(39.1%), CAZ (50%), GM (38.9%), TM (25%), VA (71.4%) and CIP (100%)]. Interestingly and similarly to the water column result, resistance to CIP was reported only at S2.

3.5.2. Streptococcus group Leuconostocsppand Enterococcus avium are ordinarily resistant to CIP. The strain S2S12 of Leuconostocsppbecame resistant only to TM. All other isolated strains of Leuconostocsppand Enterococcus avium were multiresistant and some of Leuconostocsppbecame sensitive to CIP (S1W1, S1S2 and S1S3).

3.4.3. CCA Correlation between ATB resistant profiles of isolated culturable bacterial strains and ATB tested In order to estimate how isolated culturable bacterial strains were associated with different antibiotics tested, we performed canonical correspondence analysis CCA correlating isolated culturable bacterial strains with their antibiotic resistance profile (Fig. 4). In water column (Fig. 4a), culturableS1 and S2 bacterial isolated strains were interestingly correlated with resistance to amikacin AN, oxacillin Ox, cefotaxim CTX, ciprofloxacin CIP, tobramycine TM and gentamycin GM. In sediments (Fig. 4b), culturable S1 and S2 bacterial isolated strains were correlated with the resistance to amoxicillin AM, oxacillin OX, cefotaxim CTX and vancomycin VA. At station S3, culturablebacterial isolated strains were correlated with the resistance to penicillin P andtobramycin TM. While, station S4 culturable bacterial isolated strainshad no correlation with resistance to tested antibiotics.

3.5.3. Coliforms group Chryseomonasluteola is ordinarily resistant to AMX. All strains acquired new resistances. The strains S1S50, S1S56, S2S51 and S2W55 were multi-resistant bacteria. Meanwhile, the strains S1W53(GM), S2S51(OX CTX GM), S2W52 (OX CTX), S3W49(OX CTX), S4S48 (OX CTX) and S4W54 (CTX) became sensitive to AMX.Stenotrophomonasmaltophilia is ordinarily resistant to CTX. The isolated strainS2W60 (OX CTX) acquired a resistance to OX and the strain S1S61 became sensitive to all tested ATB. 3.5.4. Non-Enterobacteriacea group Aeromonashydrophila/caviae has a natural resistance only to AMX. According to our results, multiple new ATB resistances were been acquired by all isolated strains. And Ochrobactrumanthropi has a natural resistance to CTX and CAZ. The strain S2S76 had no mutation but S2W75 (AMX CTX CAZ) and S3W77 (AMX CTX) acquired new ATB resistance profiles.

3.5. Acquired resistance 4. Discussion The acquired resistance was effectuated by comparison with the data of the natural resistance of the culturable bacterial species and the ATB resistance profile of most frequent isolated culturable bacterial (Bonnet et al., 2014). From Table 3, we noted that some isolated culturable bacteria had acquired new resistances.

We present here a study of the antibiotic resistance of culturable heterotrophic bacteria and FIB in Bizerte lagoon (Tunisia) nearby the emissary of WWTP. This is a first research in relation with distribution and antibiotic resistance of culturable heterotrophic bacteria and FIB in “Sabra bay “ of Bizerte lagoon, the semi-closed aquatic ecosystem affected by Bizerte city WWTP. It focuses on the relationship between biotic and abiotic parameters in Bizerte lagoon. Interestingly, this aquatic area is extensively exploited for shellfish since 1997. (Afli et al., 2008; Martins et al., 2016). Many studies have demonstrated affects of different polluants on the aquatic life (Roméo et al., 2006; Bouraoui et al., 2010). The bacterial growth was important mainly in sediment where the abundance was higher than that recorded in the water column. Indeed, in aquatic systems, the majority of enteric bacteria are associated with sediments (Jamieson et al., 2005) and this can be explained by the phenomenon of adhesion of bacteria to organic matter which naturally

3.5.1. Staphylococcus group Staphylococcus xylosus and Staphylococcus cohniisspcohnii are ordinary sensitives for all antibiotics. Among eight isolated strains of Staphylococcus xylosus, six strains acquired new resistance to tested ATBs. Four culturable bacterial strains isolated from discharge station were antiobiotic resistant: S2S41 (AMX P OX CTX GM VA CIP) and S2W42 (AMX P OX CTX GM VA) were multiresistant bacteria. The S4 isolated strain S4W43 (AMX P CTX) was also multiresistant. Similarly, the isolated bacterial strains of Staphylococcus cohniisspcohniiS2S45 (AMX P OX GM VA) and S2S46 (AMX P OX VA) acquired new antibiotic resistances. 206

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(a)

Fig. 4. The Canonical correspondence analysis CCA correlating the profile of antibiotic resistance (%) with the abiotic data in different sampling stations of Bizerte lagoon in water (a) and sediment (b) (September 2009). AMX: amoxicillin; P: penicillin G; OX: oxacillin; CXM: cefuroxime; CAZ: ceftazidime; GM: gentamicin; TM: tobramycin; VA: vancomycin; AN: amikacin; CIP: ciprofloxacin; S1: upstream of thereleasestation, H2: dischargestation;S3: downstream of the release station;S4: station away from discharge point.

Axis 2 (35%) 2.3

S3

1.4 0.9

AN

0.5

-1.8

-1.4

-0.9

OX

S1

S2

-0.5 CTX

0.5

0.9

1.4

Axis 1 (44%)

1.8

CAZ -0.5

2.3

S4 AMX

TM CIP

-0.9

GM

VA

P

-1.4 -1.8

(b) Axis 2 (33%) 1.7 TM

S3

P 1.4 1.0

OX

0.7 0.3

GM -1.7

CTX

-1.4

AMX

S1

CAZ

-1.0

-0.7

-0.3

0.3

0.7

1.0

Axis 1 (42%)

1.4

1.7

-0.3

S2

S4

-0.7 -1.0

VA -1.4 -1.7

contaminated with fecal staphylococci (25 103 ± 6 103 bacteria/ml) and the water column of S4 (Chaâra) was charged with fecal streptococci (15 103 ± 6 103 bacteria/ml). These virulent non-motile microorganisms could probably have been transported to the sampling site Chaâra by the intense streams of water in the channel and in the Narrows (Harzallah, 2003). In sediment compartment, station upstream of the discharge S1 was the most charged in fecal streptococci (3.4 109 ± 9 103 bacteria/g). Their abundance is extremely higher than observed in Sheldtriver sediments where the average values ranging between 102 and 1.7 105 intestinal enterococci IE/g (Ouattara et al., 2011). Fecal coliforms were abundant in sediment of discharge station S2 (2.5 108 ± 12 103 bacteria/g) that is rich of organic matter (21% MOT). Contrary, the investigated abundance of FIB in Sheldtriver sediments did not exceed 3.3 105E. coli/g (Ouattara et al., 2011). Leuconostocspp, Staphylococcus xylosus and Chryseomonasluteola were the most fecal indicator isolated bacteria in sampling stations (7.6%; 10.1%; 11.4%). The FIB isolation was mainly in stations S1 (23.1%; 15.4%; 23.1%) and S2 (2.8%; 13.9%; 8.3%). And the fecal coliforms isolated species from the discharge station S2 were various (Chryseomonasluteola, Stenotrophomonasmaltophilia, Burkholderiacepacia, Enterobacter cloacae and Escherichia coli). However, other researches demonstrated that other bacterial species (Escherichia coli, Vibrio, Enterococci, Salmonella, Shigella) were interestingly isolated from WWTP effluent (Niemi et al., 1989; Samie et al., 2009; Sidrach-

undergoes the phenomenon of sedimentation (Vatan, 1967; Cormier et al., 1984). The abundance of culturable mesophilic bacteria and FIB was highest at station of WWTP release S2. This could be related with the organic pollution of Sabra Bay as demonstrated by Limem (2007). In previous research, molecular methods (culture-independent) have been used to detect the bacterial strains in Bizerte Lagoon (Ben Said et al., 2008; Ben Said et al., 2010; Ben Said et al., 2015; Ben Salem et al., 2016). The bacterial communities were analysed at taxonomic level using molecular methods such as T-RFLP and 454-pyrosequencing targeting 16 S rDNA genes. Results obtained are in concordance with those obtained by the cultivation method (Ben Said et al., 2008; present study). According to microbiological control standards, fecal coliforms (Dansk standard, 2014), best indicators of fecal contamination (Gantzer et al., 1998), and fecal streptococci (Norme Française NFT 90–411, 1994) would be less than 100 bacteria/100 ml in water compartment. In our study, fecal coliform abundances in S2 (6.65 103 ± 8 103 bacteria/ml) was higher than 100 CFU/100 ml according to ISO 9308–1:2014 (Dansk standard, 2014). The same results were observed in Gonzaguinha beach (Brazil) that was classified as not recommended for swimming and bathing (Fernandes Cardoso et al., 2010). Similarly, the river of Sheldt (Belgium) had poor microbiological water quality with values ranging between 1.4 103 and 4.0 105E. coli/100 ml (Ouattara et al., 2011). The water column of WWTP downstream station (S3) was most 207

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Stenotrophomonas, Escherichia, Enterobacter and Shewanella. Besides, Actinobacteria phylum was detected. Similarly, using culture-independent approaches, Ben Said et al. (2010) and Ben Salem et al. (2016) proved the presence mainly of phylotypes affiliated with α-, β-, γ-, δ-, ε-Proteobacteria, Firmicutes, and Actinobacteria as well. The 16 S rRNA gene libraries were dominated by sequences related to γ- and δProteobacteria (Ben Said et al., 2010). The antibiotic resistant phenotype was predominant for the majority of ATBs in all stations, especially at the outfall station S2. Likewise, according to CCA, there is a correlation with S1 and S2 culturablebacterial isolated strains and resistance to ß-lactams (OX, CTX in water; AMX, OX, CTX in sediment) and aminoglycosides (TM, GM, AN in water; VA in sediment). Ben Said et al. (2008) payed special attention to 20 strains that exhibited the highest antibiotic resistances in Bizerte lagoon. These strains were resistant to ß-lactams and aminoglycosides. The ß-lactams represent the most important ATB family and they are the most marketed since 1946 (Essack, 2001; Kong et al., 2010). The pressure of excessive and often inappropriate use of antibiotics leads to global and epidemic spread of resistance genes (Guillemot, 1999; Struelens et al., 2007). In the discharged wastewater, a significant contribution of ATBs has promoted the selection of the ATB multi-resistance (Halling-Sorensen et al., 1998. Kümmerer, 2008; Passerat et al., 2010). According to Harzallah (2003), the water renewal in Bizerte lagoon takes a relatively long time, nearby seven months. This accentuates the bacteria antibiotic resistance problems and increases the kinetics of transmission of antibiotic resistance. The study of acquired resistance was determined by comparison with the data of the natural resistance of each bacterial genus based on the recommendations of the antibiotic susceptibility testing committee of the French Society of Microbiology (Bonnet et al., 2014). Leuconostoc spp and Enterococcus aviumare naturally sensitive to ß-lactams and aminoglycosides. Thus, they have developed a resistance to ß-lactams (AMX, P, OX, CTX) and aminoglycosides (TM, GM). The resistance in streptococci and enterococci has evolved a crucially (Pechère 1993; Leclercq, 2002b). Staphylococcus xylosus and Staphylococcus ssp cohnii cohnii species are naturally sensitive to ß-lactams, aminoglycosides, glycopeptides and ciprofloxacines. However, they developed new resistances to ß-lactams (AMX, P, OX, CTX) and aminoglycosides (GM, VA). Their antibiotic resistance profiles were varied due of a clear evolution of resistance in staphylococci (Leclercq, 2002a). Chryseomonas luteola is naturally resistant to aminopenicillins, cephalosporins (generations I, II) and to ertapenem. Thus, isolated strains have developed resistance to OX, generation III of cephalosporins (CTX), aminoglycosides (GM) and fluoroquinolones (CIP). Stenotrophomonas maltophilia is naturally resistant to CTX, while the isolated strains showed acquired resistance to OX and other strains became sensitive to CTX. The genus Aeromonas is naturally resistant to aminopenicillins (except A. trota), generations I, II of cephalosporins (except A. veronii) and ertapenem and sensitive to aminoglycosides and CIP. While, our results showed that A. hydrophila strains have developed resistance to aminoglycosides (GM, AN), others have acquired resistance to CIP. Ochrobactrum anthropi has a natural resistance to CTX and ACZ but it is sensitive to other antibiotics. But, the obtained results revealed an acquired resistance to AMX. Isolated fecal streptococci and pathogenic staphylococci were the most affected by the acquired resistance. Indeed, according to Courvalin (1994), the resistance gene flux is facilitated among grampositive bacteria. In addition, the gene transfer is extensive and polar mainly from gram-positive bacteria to gram-negative bacteria. Reported abiotic parameters of studied stations (H, NOT, TOC, TOM) could provide appropriate conditions to bacterial growth. Thus, FIB have grown up supported by the dissolved oxygen levels and nutrient salts, in particular of silicon and ammonia nitrogen (Narayana and Sunil, 2009, Béjaoui et al., 2017). The important coliform abundances in water column of the discharge point could be due to their short life affected by their sensitivity to light (Chehad and Assobhei, 2007). In sediment, These FIB have found good growing conditions

Table 3 The ATB resistance phenotypes of the most frequent isolated culturable bacteria from Bizerte lagoon (Septembre 2009). MFIB: Most frequent isolated bacteria; S1S: station 1 sediment; S2S: station 2 sediment; S3S: station 3 sediment; S4S: station 4 sediment; S1W: station 1 water; S2W: station 2 water; S3W: Station 3 water; S4W: Station 4 water; AMX: amoxicillin; P: pénicillin G; OX: oxacillin; CXM: cefuroxime; CAZ: ceftazidime; GM: gentamicin; TM: tobramycin; VA: vancomycin; AN: amikacin; CIP: ciprofloxacin. MFIB

Natural resistance

Staphylococcus xylosus, Firmicutes

Origin/ compartment (S1S3)6 (S1S)37 (S2S)38 (S2S)39 (S2S)40 (S2S)41 (S2W)42 (S4W)43 (S2S)45

Staphylococcus cohnii ssp cohnii, Firmicutes Leuconostoc spp, Firmicutes

CIP

(S2S)46 (S1W)1 (S1S)2 (S1S)3 (S2W)4 (S3S)5 (S3S)6

Enterococcus avium, Firmicutes

CIP

(S2S)12 (S2S)13 (S2W)14

Chryseomonas luteola, γProteobacteria

AMX

Stenotrophomonas maltophilia, γProteobacteria Aeromonas hydrophila/ caviae, γProteobacteria

CTX

AMX

(S1W)48 (S1S)49 (S1S)50 (S2W)51 (S2W)52 (S2S)53 (S3W)54 (S4W)55 (S4S)56 (S1S)61 (S2W)60 (S2S)71 (S2W)72 (S3W)70 (S4S)68 (S4W)69 (S4S)68

Ochrobactrum anthropi, α-Proteobacteria

CTX CAZ

(S2W)75 (S2S)76 (S3W)77

Antibiogram

AMX P CTX P OX AMX P OX CTX GM VA CIP AMX P OX CTX GM VA AMX P CTX AMX P OX GM VA AMX P OX VA AMX P OX CTX GM TM AMX P OX GM TM AMX P OX CTX GM TM AMX P OX CTX GM TM CIP AMX P OX CTX TM CIP AMX P OX CTX GM TM CIP TM AMX P OX CTX GM TM CIP AMX P OX CTX GM TM CIP GM AMX OX CTX GM AMX OX CTX OX CTX AMX OX CTX CIP OX CTX GM OX CTX CTX OX CTX OX CTX AMX CTX CAZ AMX OX CTX CAZ GM AN CIP AMX OX CTX CAZ AMX CTX CAZ AN AMX OX CTX CAZ AMX CTX CAZ AN AMX CTX CAZ CTX CAZ AMX CTX

Cardonaa and Bécaresb, 2013; Humphrey et al., 2014). The biodiversity of fecal bacterial strains in the receiving environment proved a biologic pollution at discharge station (Soudan, 1968). Firmicutes (50.7%) and Proteobacteria (40.7%) were the most dominant phyla with predominance of γ-Proteobacteria(29.3%), αProteobacteria (10.1%) and β-Proteobacteria (1.3%). Staphylococcus (25.3%) was the main genus of Firmicutes then Streptococcus, Aerococcus, Enterococcus,Gemellaand Lactococcus). Chryseomonas (11.4%)was the main genus ofγ-Proteobacteria, then Aeromonas, 208

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Shelf. Sci. 80, 269–280. Akiba, M., Senba, H., Otagiri, H., Prabhasankar, V.P., Taniyasu, S., Yamashita, N., Lee, K.I., Yamamoto, T., Tsutsui, T., Joshua, D.I., Balakrishna, K., Bairy, I., Iwata, T., Kusumoto, M., Kannan, K., Guruge, K., 2015. Impact of wastewater from different sources on the prevalence of antimicrobial-resistant Escherichia coli in sewage treatment plants in South India. Ecotox. Environ. Saf. 115, 203–208. Akpor, O.B., Muchie, M., 2011. Environmental and public health implications of wastewater quality. Afr. J. Biotechnol. 10 (13), 2379–2387. Alzieu, C., Romana, L.A., 2006. Les médicaments: nouveaux contaminants de l′environnement marin? Environ. Risq. St. 5, 320–323. Baker-Austin, C., Wright, M.S., Stepanauskas, R., McArthur, J.V., 2006. Co-selection of antibiotic and metal resistance. Trends Microbiol. 14 (4), 176–182. Bauer, A.W., Kirby, W.M.M., Sherris, J.C.A., Turck, M., 1966. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 45, 493–496. Béjaoui, B., Solidoro, C., Harzallah, A., Chevalier, C., Chapelle, A., Zaaboub, N., Aleyan, L., 2017. 3D modeling of phytoplankton seasonal variation and nutrient budget in a southern Mediterranean Lagoon. Mar. Poll. Bull. 114 (2), 962–976. Ben Said, O., Goni-Urriza, M., El Bour, M., Aissa, P., Duran, R., 2010. Bacterial community structure of sediments of the Bizerte Lagoon (Tunisia), a southern Mediterranean coastal anthropized lagoon. Microb. Ecol. 59, 445–456. BenSaid, O., Goñi-Urriza, M., El Bour, M., Dellali, M., Aissa, P., Duran, R., 2008. Characterization of aerobic polycyclic aromatic hydrocarbon-degrading bacteria From Bizerte Lagoon sediments, Tunisia. J. Appl. Microbiol. 104, 987–997. Ben Said, O., Louati, H., Soltani, A., Preud’homme, H., Cravo-Laureau, C., Got, P., Pringault, O., Aissa, P., Duran, R., 2015. Changes of benthic bacteria and meiofauna assemblages during bio-treatments of anthracene-contaminated sediments from Bizerta lagoon (Tunisia). Environ. Sci. Pollut. Res. 22 (20), 15319–15331. Ben Said, O., Souissi, M., Ben Khelil, M., Aissa, P., Beyrem, H., 2016. Antibiotic pollution pressure on Bizerte Lagoon isolated bacteria. Austin J. Environ. Toxicol. 2 (1), 1009. Ben Salem, F., Ben Said, O., Aissa, P., Mahmoudi, E., Monperrus, M., Grunberger, O., Duran, R., 2016. Pesticides in Ichkeul Lake–BizertaLagoonWatershed in Tunisia: use, occurrence, and effects on bacteria and free-living marine nematodes. Environ. Sci. Pollut. Res. 23 (1), 36–48. http://dx.doi.org/10.1007/s11356-015-4991-8. Ben Salem, F., Said, Ben, Mahmoudi, Duran, E., Monperrus, M, R., 2017. Distribution of organic contamination of sediments from Ichkeul Lake and Bizerte Lagoon, Tunisia. Mar. Pollut. Bull. http://dx.doi.org/10.1016/j.marpolbul.2017.09.024. Besassier, R., Califano, J., Carrette, M., Lombardo, M., 2006. Les pathogènes humains et leur virulence dans les eaux de distribution. Université Nice Sophia Antipolis, France. Bonnet, R., Bru, J.P., Caron, F., Cattoir, V., Chardon, H., Courvalin, P., Dubreuil, L., Jarlier, V., Lambert, T., Lefort, A., Merens, A., R, Nicolas-Chanoine, M.H., Pleisiat, P., Ploy M.C., Soussy, C.J., Varon E., Weber, P., 2014. Comité de l’antibiogramme de la société francaise de microbiologie – Recommandations 2014. Société française de microbiologie SFM., pp. 20–22. Bonnieux, F., Desaigues, B., 1998. Economie et politique de l′environnement. Dalloz. France. Bouraoui, Z., Banni, M., Chouba, L., Ghedira, J., Clerandeau, C., Jebali, J., Narbonne, J.F., Boussetta, H., 2010. Monitoring pollution in Tunisian coasts using a scale of classification based on biochemical markers in worms Nereis (Hediste) diversicolor. Environ. Monit. Assess. 164, 691–700. Bradshaw, J.K., Snyder, B.J., Oladeinde, A., Spidle, D., Berrang, M.E., Meinersmann, R.J., Oakley, B., Sidle, R.C., Sullivan, K., Molina, M., 2016. 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such as humidity (4.72%), dissolved oxygen (6.3 mg/l) and nutrients (Luna et al., 2002; Meaume et al., 2005). In spite of the usual relationship between turbidity and bacterial abundance (Cormier et al., 1984), the abundance of fecal coliforms was interesting (2.5 108 ± 12 106 bacteria/g) owing to their fimbriae that increase their adhesion to organic matter (Klemm, 1994). The Bizerte lagoon is a multi-contamination ecosystem mainly heavy metal, pesticide, hydrocarbon contamination (Ben Said et al., 2010; Zaaboub et al., 2015). Thus, bacteria have developed mechanism resistance to antibiotics which have been found regularly linked with heavy metal resistance (Baker-Austin et al., 2006; Wright et al., 2006). Often multiple metals and antibiotic resistances pathways have been found to be encoded by plasmid genes (Roy et al., 2002). These genetic elements are responsible for gene diffusion within a bacterial community by horizontal transfer (Top and Springael, 2003). Interestingly, the CCA results presented here showed that Stations S1 and S2 culturable bacterial isolated strains were correlated with the resistance to ß-lactams (OX, CTX in water; AMX, OX, CTX in sediment) and aminoglycosides (TM, GM, AN in water; VA in sediment). However, station S3 culturable bacterial isolated strains were correlated only with the resistance to P and TM. While, station S4 culturable bacterial isolated strainshad no correlation with resistance to tested antibiotics. These observations suggested that the presence of antibiotics in the discharge station and the upstream discharge station affected the abundance of the isolated bacteria from these sampling sites. Antibiotics influence the abundance and spread of benthic cocci and the sediments can be reservoirs of dormant antibiotic-resistant bacteria, which can rapidly revive in presence of new inputs of organic matter. (Di Cesarel et al., 2013; Graham et al., 2016) In conclusion, in all stations of Sabra Bay, abundance of isolated FIB exceeded the microbiological control standards with a maximum at the WWTP discharge station and supported and revealed a fecal contamination. Furthermore, some pathogenic species were considered as the most frequent: Leuconostoc spp, Enterococcus avium, Staphylococcus xylosus, Staphylococcus cohnii ssp cohnii, Chryseomonas luteola, Stenotrophomonas maltophilia, Aeromonas hydrophila/caviae and Ochrobactrum anthropi. The resistant phenotype was predominant for the majority of antibiotics in all stations, especially at discharge station. Indeed, the fecal contamination indicator bacterial strains have been more resistant to ß-lactams. Serious acquired resistance to ß-lactams and aminoglycosides appeared among the isolated culturable bacterial strains mainly the fecal streptococci and the staphylococci pathogens. We revealed a serious resistance to ß-lactams and aminoglycosides that affected the culturable bacterial strains isolated mainly from the discharge station and the upstream discharge station. The acquisition of new antibiotic resistances proved that this resistance is still developing. The ecosystem of the Bizerte lagoon is suffering from fecal contamination and an antibiotic resistance which could be seen as a health issue to report. Our results require attention so that further additional studies such as the antibiotic dosage in the Bizerte lagoon and the search for resistance genes. Acknowledgment This work was supported by a funding the Faculty of Sciences, Bizerte (FSB) and the regional Hospital of Bizerte. Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2017.10.002. References Afli, A., Ayri, R., Zaabi, S., 2008. Ecological quality of some Tunisian coast and lagoon locations, by using benthic community parameters and biotic indices. Estuar. Coast.

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