Emergence of Multidrug-resistance Bacteria Isolated of Drinking Water, Groundwater and Hospital Wastewater in Southern Benin: Antibiotic Resistance Prole and Resistomes Determination

Background: The environment plays an important role in the dissemination of multidrug resistant bacteria, especially through the aquatic ecosystem, which is made up of liquid hospital euents, rivers, but also spring water and drinking water. This study aims to determinate resistomes in some aquatic matrices in southern Benin. Methods: These samples were ltered through a membrane 0.22 µm thick. After ltration, the membrane was deposited on Muëller Hinton agar. Then the colonies resulting from this subculture were subjected to a microbiological examination by the conventional method. The antibiotic sensitivity test was carried out by the Kirby Bauer method according to the recommendations of the French Society of Microbiology. Resistance genes were looked for by standard PCR and the PCR products were used for agarose gel electrophoresis. Results: Of the 222 water samples collected, 265 bacterial strains were isolated, the majority of which were strains of Coagulase Negative Staphylococcus (CNS) with 37.74% (n = 100), followed by strains of Klebsiella pneumoniae (21.89%; n = 58), Escherichia coli (10.57%; n = 28). All isolated gram-negative bacilli strains are multidrug resistant with resistance of all strains to amoxicillin, ampicillin and amoxicillin + clavulanic acid. Of the 15 resistance genes sought in the genome of Gram-negative bacilli strains, only 8 were detected, namely the TEM, SHV, CTX-M15, VIM, NDM, SUL1, SUL2 and AADA genes. Strong resistance of CNS strains to amoxicillin, oxacillin and cefoxitin was observed. The Mec A gene was detected in all CNS strains. While the Van A and Van B genes were only detected in strains isolated from drinking water in sachets collected from producers and street sellers. Conclusion: These results show the dissemination of resistance genes in the aquatic ecosystem of Benin and once again conrms the urgency of a global ght against antimicrobial resistance.


Background
Antibiotic resistance is a major problem of public health in the world. This phenomenon affects all industrialized and non-industrialized countries [1]. The major causes of this phenomenon of antibiotic resistance are the inappropriate and fanciful use of antibiotics in human health [2], the joint use of antibiotics in animal health, agriculture and human health [1][2][3]. In addition to these causes, the role of the environment in the spread of resistance to antibiotics has been increasingly questioned in recent years [4]. Numerous studies have shown the role of the environment as a source or reservoir of resistant bacteria, antibiotic resistance genes and antibiotic residues that contribute to the selection of multidrug resistant germs [5,6]. Many matrices have been targeted as a source of dissemination of multidrug resistant bacteria in the environment and to humans, but water resources constitute an important source of dissemination of these multidrug resistant bacteria [7]. E uent hospital liquid, passing by piping waters and drinking water have been implicated as a vehicle for the spread of multidrug-resistant bacteria [8]. Integrated management of water resources would reduce the spread of pathogens both in hospitals and in the community [9]. However, the implementation of these management policies for both wastewater and drinking water is still precarious in developing countries like Benin.
Indeed, numerous studies showed the presence of multidrug resistant bacteria in the environment in Benin [10][11][12][13][14]. The vast majority of hospitals do not have a treatment and puri cation system for hospital liquid e uents which are in the majority of cases discharged into the environment and into the great lakes. The drinking water is produced without any hygienic quality control and sold in the streets, sometimes under the sun. However, it is well known that at certain temperatures microorganisms can multiply in drinking water. These are all problems that led to the implementation of this study, which aims to identify the ecologies of multidrug resistant bacteria in liquid e uents and in drinking water in the knowledge of Benin.

Material And Methods
Zone and Period of Study The study was carried out in southern Benin in the municipalities of Cotonou, Abomey-Calavi and Seme-Kpodji. It was carried out during the period from July to September 2020. In the municipalities of Cotonou and Abomey-Calavi, samples of drinking water in sachets were taken both from producers and from street sellers. As for the samples of wastewater (hospital environment) and groundwater (well water) intended for the consumption of the populations, they were taken in the commune of Seme-Kpodji.

Sample collection
Four different brands of drinking water in sachets (33 ml of water / sachet) were selected and their producers were identi ed in the communes of Cotonou and Abomey-Calavi. 30 samples of this water were taken from each of the four producers. Randomly, two street sellers of each brand of drinking water were chosen and 10 water samples were purchased from them in the communes of Cotonou and Abomey-Calavi. 10 samples of wastewater were then taken in the Seme-Krake Health Center in the commune of Seme-Kpodji. 6 wells were randomly selected from the houses surrounding the Health Center and 2 groundwater samples were taken per well. Thus, a total of 172 water samples were taken as part of this study (Table 1). 1000 ml of each of the hospital wastewater and groundwater samples were collected in a sterilized vial at each site. The collected samples were transported directly to the laboratory in a cooler containing cold accumulators. The samples were handled on the same day. If necessary, they were stored at + 4 °C while awaiting their handling.

Bacteriological identi cation
Once in the laboratory, each sample was ltered twice using a ltration pump through a 0.22 µm membrane. The volume of each ltered sample was 300 ml (2 x 150 ml). After ltration, the two membranes obtained from each sample were removed and placed respectively on Manitol Salt agar plates and on Eosine Methylene Blue agar.
These plates were incubated at 37 °C for 18 h. The Gram stain was carried out on the colonies obtained after 18 h. Then these colonies were seeded on Mueller Hinton agar plates and incubated for 18 h at 37 °C to obtain young pure colonies. Each colony obtained was used for identi cation of bacterial species by the classical method of microbiology based on Gram control and biochemical characteristics (catalase, oxidase, seeding of the API 20 E gallery (for only Gram-negative bacilli), free staphylocoagulase and Dnase tests (for only Grampositive cocci).

Antibiotic susceptibility test
The antibiotic susceptibility test of the isolated strains was carried out by the method of Kirby Bauer which consists of the diffusion of the discs of antibiotics on the Mueller Hinton II agar according to the recommendations of the French Society of Microbiology [16].

Statistical analysis
The data were entered into an excel spreadsheet and analyzed with SPSS software. The graphics were made using Graphpad Prism 7 software.

Resistance of Gram-negative bacilli strains to the antibiotics used
The test of antibiotic resistance showed that all isolated gram-negative bacilli strains are multidrug resistant with resistance of almost all strains to amoxicillin, ampicillin and amoxicillin + clavulanic acid. Moderate resistance was noted to ceftriaxone and very low resistance to imipenem ( Figure 1). However, we noted that all strains of K. pneumoniae isolated from groundwater were sensitive to amikacin, nalidixic acid and gentamicin.
While the strains of Aeromonas spp. isolated from the same samples were only sensitive to amikacin. As for the P. aeruginosa strains isolated from the same samples, they were sensitive to imipenem, amikacin and gentamicin ( Figure 1A). The strains isolated from the samples of hospital wastewater showed very strong resistance to the antibiotics used. Thus, the strains of K. pneumoniae showed strong resistance to amoxicillin (100%) and ampicillin (100%) and moderate resistance to omoxicillin + clavulanic acid (50%). All strains of Aeromonas spp. showed complete resistance to amoxicillin, ampicillin, amoxicillin + clavulanic acid and nalidixic acid and 50% resistance to ceftriaxone, imipenem, amikacin and gentamicin. As for the E. coli strains, they were all resistant to amoxicillin, ampicillin, amoxicillin + clavulanic acid and ceftriaxone ( Figure 1B). The resistance of strains isolated from drinking water in sachets collected from producers showed that all strains of Shigella spp. isolated were resistant to all antibiotics tested except amikacin. Strains of Enterobacter spp. isolated from the same samples also showed complete resistance to amoxicillin, ampicillin, amoxicillin + clavulanic acid, ceftriaxone and gentamicin. Strains of K. pneumoniae, on the other hand, showed strong resistance to amoxicillin (100%), ampicillin (100%) and amoxicillin + clavulanic acid (60%), and low resistance to ceftriaxone (40%) and imipenem (20%) ( Figure 1C). All the strains isolated from drinking water in sachets collected from street vendors showed total resistance to amoxicillin, ampicillin and amoxicillin + clavulanic acid, except for the strains of Enterobacter spp. which showed resistance of 66.67% to amoxicillin + clavulanic acid and strains of Serratia spp which showed low resistance of 33.33% to amoxicillin, ampicillin and amoxicillin + clavulanic acid ( Figure 1D).  Table 4 shows the association of resistance pro les and resistance genes of the bacterial strains (Gram-negative bacilli) isolated according to the different types of samples. Thus, out of the 58 strains of K. pneumoniae isolated in this study, 4 resistance pro les were established. These 4 pro les are associated with 5 resistance genes (SUL1, SUL2, AADA, SHV and VIM). Of the 28 strains of E. coli isolated in our study, 3 resistance pro les were established and then associated differently with 3 resistance genes (CTX-M15, TEM and SHV). Only one resistance pro le was not associated with any resistance gene. This strain was isolated from drinking water in sachets collected from producers. 5 resistance pro les were established after the susceptibility test of the strains of Aeromonas spp. antibiotics. These 5 pro les were associated with 4 resistance genes (SHV, VIM, AADA and SUL2). However, 3 strains of Aeromonas spp. isolated from hospital wastewater and having the same resistance pro le were not associated with any resistance gene. Regarding the P. aeruginosa strains isolated in this study, 3 resistance pro les were established with an association of 5 resistance genes (TEM, SHV, SUL1, SUL2, AADA) ( Table 4).

Resistance genes detected in
Resistance of Gram-positive cocci strains to the antibiotics used All Gram-positive cocci strains isolated in this study are all coagulase-negative staphylococcus (CNS). A total of 100 strains of CNS were isolated ( Table 3). The susceptibility test of the CNS strains to the antibiotics used showed that all the CNS strains isolated from hospital wastewater were resistant to all antibiotics tested except fosfomycin and erythromicin. Strains isolated from groundwater showed high resistance to amoxicillin (100%), oxacillin (100%) and cefoxitin (80%) and low resistance to vancomycin (40%), fosfomycin (20 %) and clindamycin (20%). As for the strains isolated from drinking water in sachets collected from producers, the highest resistance is to amoxicillin (72.73%), oxacillin (72.73%) and cefoxitin (63, 64%). While, the resistance of strains from drinking water in sachets collected from street vendors is much higher to amoxicillin (72.73%), oxacillin (54.55%), and fosfomycin (54.55%) (Figure 3).

Resistance genes detected in coagulase-negative staphylococcus (CNS) strains
Three resistance genes (Mec A, Van A and Van B) were detected in the genome of the CNS strains isolated during this study. The Mec A gene was detected in all strains isolated from groundwater (100%) and hospital wastewater (100%). This same gene was detected at 71.67% and at 41.67% in strains isolated respectively from drinking water in sachets collected from producers and street vendors. As for the Van A gene, it was detected in strains isolated from drinking water in sachets collected from producers and street vendors with frequencies of 33.33% and 8.33% respectively. Whereas, the Van B gene was detected only in the strains isolated from drinking water in sachets collected from producers with a frequency of 33.33% (Figure 4).
Out of the 100 CNS strains, we were able to establish with the results of the susceptibility test of these strains to the antibiotics used, 17 different resistance pro les. These resistance pro les were associated with the resistance genes Mec A, Van A and Van B. All this according to the types of samples analyzed (Table 5).

Discussion
Public health problem around the world. The presented study carried out on drinking water, wastewater and groundwater around the hospital shows the presence of bacteria such as Aeromonas spp., Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis.
The presence of Aeromonas spp. in aquatic ecosystems was noted by numerous studies [25]. For the authors [26,27], the presence of this genus in the aquatic environment is natural because they are ubiquitous and especially present in the soil, on plants and in water. However, this genus can be involved in human and animal infections, especially in the contamination of sh [28]. The absence of this bacterial genus in drinking water is contrary to numerous studies which have isolated this genus in many drinking waters sources [29][30][31]. The absence of this kind in our drinking water samples is due to the preliminary treatment with molecules based on alum before marketing. These molecules therefore have an action on the genus Aeromonas which is known for its involvement in human infections by many toxins that it produces [32]. The presence of this kind in hospital wastewater were also documented by authors [33]. Bo ll-Mas et al. [34] showed in their studies that bacteria of the genus Aeromonas can be found in groundwater and municipal pipes near hospitals by irrigation of hospital wastewater.
The presence of other bacteria such as Enterobacter spp., Klebsiella pneumoniae, Proteus mirabilis in our samples corroborates the results obtained by Olaoye and Oniludé [35] who carried out their studies in the southwest of Nigeria, part of the border with our study area. Indeed, our study area shares a strong practice with the study area of the latter. This shows that the problem of contamination of drinking water in sachets has not been resolved over time. On the contrary, the practice of selling these waters without control continues and spreads from day to day.
In our study, we noted a weak diffusion of bacteria between the wastewater and the surrounding groundwater. This shows the e ciency of the water treatment system in the health center. This should serve as a model for other health centers which generally do not provide for any treatment strategy for these hospital liquid e uents. These observations join those of Buelow et al. [36] who have shown the limitation of the in uence of liquid hospital e uents on the resistome and on the microbiome. The strong presence of coagulase negative staphylococcus in our study may re ect poor hygiene in the packaging of bags consumption waters and the risk of cross-transmission through the hands in hospital environments mismanagement of hospital wastewater. It is well known that the coagulase negative staphylococci, commensal of the water are strongly involved in the cross transmission of the germs by the hand in both community and hospital settings [37].
The study of resistance to the antibiotic showed the presence of many resistance genes to beta-lactams whose resistance genes (TEM, SHV, CTX-M 15, NDM, VIM for Gram-negative bacilli and Mec A for Gram Positive Cocci), aminoglycosides (AADA) and glycopeptides (VAN A and Van B), sulfonamides (SUL 1 and SUL 2). Numerous studies have shown the presence of these resistance genes in bacteria isolated from water sources such as hospital wastewater and groundwater [8,38], drinking water [30,39,40]. The presence of certain multi-resistant bacteria and the absence of others in drinking water following the street sellers is the problem of distribution conditions of these drinking water bags. Indeed, this water generally in contact with heat (Sun or non-refrigerated collector contributes to the multiplication of certain multidrug resistant bacteria. This sector must therefore be supervised for better consumer safety [35]. Similarly, the results obtained show that surrounding groundwater have certain genes that do not come from the hospital wastewater. Indeed, the environment in general present a risk of spreading of multidrug resistant bacteria except those contributed by the wastewater. An effective ght against the has antimicrobial resistance is integrated a more comprehensive "One Health" approach includes the environment, animal health and human health [4].

Conclusion
These results, which took stock of the presence of multidrug resistant bacteria in sources of drinking water, hospital wastewater and groundwater, show the important role that the environment plays in the dissemination of multidrug resistant bacteria. and resistance genes. For an effective ght in this context, it will be necessary to adopt strategies taking into account all the elds concerned, which are animal health, human health and the environment. In this study we did not use samples of human origin. Our samples were water samples exclusively. However, we have also received the approval of drinking water producers and leads of households before the respective sampling of drinking water in sachets and groundwater (in wells). Likewise, the management of the Seme-Krake health center gave us the approval for the collection of hospital wastewater. This approval was noti ed to us by the noti cation letter n ° 95/08 / MS / DDSOUE-PLA / CH-SEME-KRAKE / SAAE / DGAP / SGA of June 15, 2020.

Consent for publication
In our manuscript we did not include data from individuals. Because we did not handle a sample of human origin. Thus, we do not need to request consent for publication. However, all authors have consented for publication.

Availability of data and material
The datasets used and/or analyzed during the current study and material used are available from the corresponding author upon reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
This work was not supported by any grants from funding agencies in the public or commercial sectors.
Authors' contributions HK, VD, CL and AJA wrote the protocol. HK, VD, CL and GA processed the samples. VD and AJA did the statistical analyses and produce the graphs. HK, VD, AJA and SS wrote the draft of the manuscript. VD, HB and LB-M reviewed the manuscript. All authors read and approved the nal version of the manuscript.