Salmonella spp: According to the standard method NM ISO 19250/ NM 03.7.050, the following steps were done: a) Pre-enrichment: The buffered peptone water is seeded at room temperature with a known volume of the sample or its dilutions and incubated at (36±2) °C for (18 ± 2) hours. Larger volumes can be concentrated by membrane filtration, after which the filter membrane is transferred to the buffered peptone water. b) Enrichment: Rappaport-Vassiliadis soy bean medium (RVS broth) and Muller-Kauffmann tetrathionate-novobiocin broth (MKTTn) are inoculated with the pre-enrichement culture. RVS broth is incubated at (41.5±1) °C for (24±3) hours and MKTTn broth at (36±2) °C for (24±3) hours. To detect Salmonella spp. with slow growth, incubate the enrichment broth for an additional (24±3) hours for a total incubation period of (48±4) hours at (41.5±1.0) °C. c) Isolation on selective growth medium: From the cultures obtained in enrichment, two solid selective medias are inoculated: lysine xylose deoxycholate agar (XLD agar) and Hechtoen agar. After incubation of both culture media at (36±2) °C and examine after (24±3) hours. Black-centered red colonies appear on XLD agar and black-centered green, which becomes completely black at the end of incubation on Hektoen agar.

Non enterobacteriaceae Pseudomonas aeruginosa: Standard method NM ISO 16266 was applied using cetrimide agar medium. After incubation at (41.5±0.5) °C for 48 hours, colonies (0.8 to 2.2 mm in diameter) showing flat appearance with blue or blue-green pigmentation surrounding the colonies light outer rims and brownish to greenish black-centers were selected and isolated as P. aeruginosa. These colonies were confirmed by three confirmation tests: Detection of oxidase, Pyocyanin production on King B medium, growth at 42°C in Tryptone-soy Broth.

Acinetobacter baumanii: Clinical and Laboratory Standard Institute (CLSI), using MacConkey Agar. After incubation at 30°C, colonies (2 to 3 mm in diameter) develop in 24 hours on MacConkey medium, they are convex, shiny and whitish. Aeromonas hydrophyla: The strains of A. hydrophila develop on nutrient Agar. After incubation at (36±2) °C, colonies develop in 24 hours; they are round, curved, smooth, regular and opaque.

Cocci

Staphylococcus aureus: Using Chapman-Mannitol salt agar medium (standard method NM 03.7.036/XP T 90-412). After incubation at (36±2) °C for 48 hours, Yellow colonies on a yellow background or surrounded by a yellow halo and confirmed by a tube coagulase test to give a coagulum occupies more than three quarters of the volume initially occupied by the liquid.

Enterococcus faecalis and Enterococcus faecium: According to the standard method NM ISO 7899-2/NM 03.7.006. Slanetz and Bartley Medium (Enterococcus Agar), colonies showing a red to pink color were isolated as presumptive Enterococcus, and confirmed by typical colonies will be transferred to the membrane and colonies by means of sterile tweezers, without turning, on a bile esculin-azide agar plate this has been preheated to 44 °C. After incubation at (44±0.5) °C for 2 hours, showing a brown to black color in the surrounding medium.

Identification of bacteria by Analytical Profile (APIbiochemical) strips: The bacterial strains were identified by API biochemical system for identification kits (Marcy l’Étoile, France): Api 20E for enterobacteria, Api 20NE for non-enterobacteria, Api Staph for Staphylococcus, Api 20 Strepto for Enterococcus. The API strips consists of 20 micro-tubes containing dehydrated substrates. These tests are inoculated with a bacterial suspension that reconstitutes the media. The strips were incubated for 18–24 hours at (36±2) °C. During incubation, metabolism produces color changes that are either spontaneous or revealed by the addition of reagents. The reactions were read according to the reading table and the identification was obtained by referring to the Analytical Profile Index [14].

Results

Distribution of resistant bacteria

A total of 285 strains were isolated from the different sampling points distributed as follows: Escherichia coli (N = 68), Klebsiella pneumoniae (N = 37), Salmonella spp (N = 12), Pseudomonas aeruginosa (N = 30), Aeromonas hydrophyla (N = 48), Staphylococcus aureus (N = 15), Enterococcus faecalis (N = 44), and Enterococcus faecium (N = 10), of which 201 came from P1 to P10; 26 from P11 and 58 were isolated from P12, P13 and P14

According to Table 3, eight potentially pathogenic strains isolated at the P13 sampling point are resistant to antibiotics, these strains are distributed as follows: Three strains (E. coli, K.pneumonia and A. hydrophila) resistant to ciprofloxacin, two other strains (E. coli and K. pneumonaie) resistant to cephalothin, one strain of A. hydrophila resistant to cephtazidime, a strain of E. faecalis resistant to Amoxicillin + clavulanic acid and one strain of P. aeruginosa resistant to imipenem. The secondary treatment of municipal wastewater by activated sludge technology is probably insufficient and still constitutes a health risk. Two recent studies of UV-treated wastewater in a rural sewage treatment plant in southeast Louisiana (USA) confirmed the presence of antibiotic-resistant Gram-negative bacteria [16] and the presence of methicillin-resistant Staphylococcus aureus [17]. On the other hand, the present study demonstrated that at point P14, after advanced tertiary treatment (UV disinfection, preceded by coagulation-flocculation and sand filtration), pathogenic strains were absent. This means that advanced tertiary treatment plays an important role in removing pathogenic strains and preserving the environment.

Bacterial resistance and phenotypes

These results showed that bacterial resistance to all antibiotics is dominated by Gram-negative bacteria belonging to the Enterobacteriaceae family (E. coli, K. pneumonaie) with a rate of more than 45% followed by non-Enterobacteriaceae strains (P. aeruginosa, A. hydrophila and A. baumanii) with a rate of 34% then Cocci (E. faecalis, E. faecium and S. aureus) with a rate of 21% (Figure 1).

Enterobacteriaceae isolates produced 76% high penicillinases for K. pneumoniae and 48% for E. coli and were susceptible to imipenem (Figure 2).

These results are comparable to those obtained by Guessennd et al. [18] in the Ivory Coast on hospital effluents from the university hospital centre of the city of Abidjan.

Concerning the strain of Aeromonas hydrophila which has been frequently isolated from our effluents, numerous studies showed that Aeromonas strains isolated from water and sediments were resistant to cefoxitin with a rate of 80 % [19]. In the present study, Aeromonas hydrophila strains were quinolone-resistant with a rate over 90% against 41% obtained by Varela et al. [20].

The species Acinetobacter baumannii is a frequent cause of nosocomial pneumonia and other infections, such as skin and wound infections, infective endocarditis, bacteremia, urinary tract infections, and meningitis [21]. Several studies have demonstrated that A. baumannii is resistant to many of the antibiotics used in the hospital context [22,23]. All strains of A. baumanii were resistant to beta-lactams with 82.35% resistance to imipenem, 88.23% to ceftazidime and 70.60% to gentamycin (Table 4). This bacterium is distinguished by its multi-resistant character [24].

The presence of fluoroquinolones (FQs) residues in various effluents (households, hospitals and pharmaceutical effluents) is a major cause of acute and chronic toxicity, as well as the appearance of resistant bacteria [25].

Therefore, the removal of FQs residues from the environment is a crucial problem. Regarding our study and among the eight (8) strains that we have isolated, four (4) were resistant to ciprofloxacin. The strain of E. coli alone has a rate of 88.46% (Table 4) against a rate of 34.4% found by Conte et al. [26]. Ciprofloxacin is frequently prescribed to treat urinary tract infections and it is excreted unchanged in urine and feces and remains adsorbed in sludge even after treatment in WWTPs [27]. Therefore, it is a good indicator of the presence of antimicrobials in the environment [28].

The global epidemiological situation of the resistance of E. coli strains to FQs remains variable with resistance rates of 10% in the United States and 50% in China [29]. This is probably due to the presence of emerging FQs residues in the investigated effluents which favors the acquisition of resistance of the E. coli strains and/or other strains of K. pneumoniae, A. baumanii and A. hydrophyla, which exhibits high resistance levels: 68.97%, 23.53% and 92.85% (Table 4), respectively.

The observation of this bio-resistance clearly reflects the presence of potentially pathogenic bacteria resistant to antibiotics in CHU hospital effluents and in the WWTP effluents (Raw urban wastewater and after secondary treatment) of Marrakech city. This could be explained by the selection pressure exerted by the practitioners and by the presence of low concentrations of non-metabolized antibiotics discharged from hospital effluents [30]. These different multi-resistance phenotypes could also be explained by the fact that antibiotics are among the most important molecules in Africa; and among these antibiotics, betalactams were the most frequently prescribed [31]. An 87.5% of the Pseudomonas aeruginosa were resistant to ceftazidime, 50% to ticarcillin, 43.75% to ticarcillin-clavulanic acid, 68.75% to piperacillin + tazobactam, and 62.50% to imipenem as certain strains of the hospital environment, P. aeruginosa is a ubiquitous microorganism capable of persisting in water, soil and plants, and is also responsible for a large number of common infections acquired in hospitals [32].

A study has confirmed the presence of resistant strains of P. aeruginosa in surface water bodies that received wastewaters from a hospital in Brazil [32]. All isolated Acinetobacter strains were resistant to beta-lactams and also resistant to ciprofloxacin and gentamycin with a rate of 27.3%.

The strains of Staphylococcus were recognized as having a high capacity of acquisition of resistance genes [18]. Among the aminoglycosides, the most active was gentamycin. The results obtained in this work (37.50%) are lower than those of Guessennd et al. [18] where the proportion of kanamycin, tobramycin and gentamycin (KTG) phenotype was 100% in hospital effluents. The resistance of S. aureus to many antibiotics was also observed in several studies with a rate of 90% to 95% for aminoglycosides and 90% for FQs [33]. We found a very high rate of resistance to methicillin of 37.50% compared to results obtained in hospitals 6.64% in the United States (2010), 5.97% in Canada (1999) and 0.8% in Japan (2003) [34], and in surface waters [35]. Also, the resistance rate of 75% for ciprofloxacin obtained in the present study is similar to the results obtained by Salou et al. [36] in a hospital-based study (on patients urine and pus) who demonstrated that S. aureus was resistant to ciprofloxacin 1.85 times more likely to be resistant to Methicillin resistance; methicillin resistance is also correlated with an increase in resistance to ciprofloxacin. The S. aureus resistant to methicillin is 2.54 times more likely to be resistant to ciprofloxacin. This could be explained by the emergence of resistant strains and the increase in acquired infections in hospitals resulting from the absence of good care practices and infection control guidelines.

E. faecalis and E. faecium are the most common species in hospital and urban wastewaters [37]. In this study, all strains of E. faecalis and E. faecium (Table 4) resistant to beta-lactam (amoxicillin + clavulanic acid), has very high rates of 78.26% and 100%, respectively. This may be explained by the fact that their spread in the hospital environment is due in part to their survival at high temperatures and to their resistance to chemical disinfectants such as chlorine, glutaraldehyde and alcohol [38], and in the past three decades, Enterococci has become the third most frequent nosocomial human pathogens, with a mortality rate of up to 23% [39].

Conclusion

The present study confirmed the resistance to antibiotics of the majority of bacterial strains isolated from CHU hospital sewages and the Marrakech receiving WWTP effluents (raw wastewater and even after secondary treatment). This antibiotic resistance is dominated by Gram-negative bacteria belonging to the Enterobacteriaceae family (45%) followed by non- Enterobacteriaceae strains (34%) then Cocci (21%). In addition, almost 63% of isolated bacterial strains were resistant to at least 3 families of antibiotics (beta-lactams, aminoglycosides and fluoroquinolones) except for P. aeruginosa, which revealed resistance to imipenem and ceftazidime (family of betalactams). However, the study demonstrated that the advanced tertiary treatment (coagulation flocculation + sand filtration + UV disinfection) at Marrakech WWTP removed pathogenic strains completely from the municipal wastewater. Therefore, advanced tertiary treatment is strongly recommended to ovoid dissemination of multiple resistant bacterial strains to the environment.

Acknowledgements

This work was supported by the Pole of Competences on Water and Environment (PC2E); the National Centre for Research and Studies on Water and Energy (CNEREE); the Regional Laboratory of Epidemiology and Environmental Health, Direction of Health and the Mohamed VI University Hospital Center (CHU) of Marrakech city.

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