Occurrence of antibiotic-resistant bacteria in household greywater discharged into the environment in Burkina Faso's Sahel region

Greywater, consisting of wastewater from household activities like bathing, dishwashing, and laundry, comprises a significant portion of urban waste in developing nations (Maiga et al. 2014). Unlike blackwater, which contains human waste, greywater poses a potential risk of environmental contamination and public health concerns (Li et al. 2009). In developing countries such as Burkina Faso, the wastewater collection and treatment system is underdeveloped in urban areas and completely non-existent in rural areas, making wastewater treatment a major challenge (Compaoré 2022). As per the RGPH (2019), roughly 80% of generated greywater is released untreated into the environment, posing a substantial challenge for waste management in urban settings. The main consequence of the lack of greywater management infrastructure is the spreading of pathogenic microorganisms that can cause diseases such as diarrhea, cholera, typhoid fever and other gastrointestinal infections (Sorenson et al. 2011; Prüss-Ustün et al. 2014). Moreover, population growth, urbanization and climate change have pushed developing countries toward a circular economy. As such, the reuse of greywater has been proposed as an alternative to fresh water for certain uses, especially in arid regions of the world (Jahne et al. 2017). However, in addition to its potential benefits, greywater must be treated responsibly to eliminate potential environmental and health risks. The quality of raw greywater varies widely and it often contains pathogens.

In Africa, studies on the microbial load of greywater have generally focused on indicator bacteria of fecal contamination, including fecal and total coliforms, E. coli and Enterococcus faecalis (Maiga et al. 2014; Compaoré et al. 2022). The use of these fecal contamination indicators has several limitations and does not reflect the true pathogens load in wastewater because of the poor correlation between indicator bacteria of fecal contamination and pathogens in the environment (Noman et al. 2022). In effect, there may be discrepancies between the two, due to differences in their survival and behavior in the environment, which may lead to underestimates of the presence of pathogens in wastewater (Payment & Locas 2011). Recently, several studies in Israel have revealed the presence in greywater of bacteria known to cause gastrointestinal illness, such as Salmonella enterica and Staphylococcus aureus (the latter can also cause skin infections) (Benami et al. 2013; Busgang et al. 2018). Beyond its public health implications, the presence of pathogens in greywater correlates with antimicrobial resistance (Craddock et al. 2020; Noman et al. 2022), thereby presenting an additional challenge for waste management in developing countries. Increasing levels of antimicrobial-resistant bacteria in the environment due to wastewater discharge are a recent concern for human health. In fact, according to Lancet (2022), the all-age death rate attributable to antimicrobial resistance (AMR) is highest in western sub-Saharan Africa, at 27.3 deaths per 100,000 (20.9–35.3). Furthermore, in 2021, a WHO (2021) report noted an outbreak of antibiotic-resistant Salmonella spp. infections in Africa, with possible sewage contamination. Treatment of these infections requires the use of antibiotics of a last resort, resulting in high costs for local healthcare systems (WHO 2021). Despite these known concerns, the presence of antibiotic-resistant pathogens in greywater in West Africa, where this water is often discharged untreated into the environment, is poorly understood.

In sub-Saharan Africa, there is considerable research on the presence of antibiotic-resistant bacteria in hospitals as well as environmental wastewater, thus underlining the importance of monitoring antibiotic resistance in wastewater and proposing appropriate wastewater management systems in developing countries (Adegboyega et al. 2019; Adesoji et al. 2020; Kagambèga et al. 2024). However, there is a lack of studies assessing antibiotic-resistant and multidrug-resistant (MDR) bacteria in untreated greywater. Focusing on the Sahel region of Africa, where untreated greywater disposal prevails, our study aims to bridge a knowledge gap by evaluating the presence of resistant as well as multi-resistant bacteria in untreated greywater, alongside their potential implications for public health and environmental well-being. Our study endeavors to probe the presence of resistant as well as multi-resistant bacteria, including Salmonella spp. and Staphylococcus spp., in untreated greywater within Africa's Sahel region. Furthermore, we seek to scrutinize their potential contribution to antimicrobial resistance spread in the environment. These two bacteria were selected for this study as common pathogenic bacteria associated with various infections in humans (Busgang et al. 2018). In addition, they represent major public health concerns due to their ability to develop antibiotic resistance (Peleg & Hooper 2010; Graveland et al. 2011; Carattoli 2013). Here, we present the results of (i) the occurrence and isolation of Salmonella spp. and Staphylococcus spp. in greywater, (ii) the antibiotic susceptibility of presumably isolated Salmonella spp. as well as Staphylococcus spp. strains, and (iii) the biochemical characterization of bacterial strains with multidrug resistance.

Presumptive Salmonella spp. strains from greywater were isolated according to ISO 19250:2010. Strains were isolated in two steps: (i) enrichment on Rappaport Vassiliadis broth (Liofilchem srl, Italy) (37 °C for 24 h) and (ii) isolation followed by identification on Salmonella–Shigella agar (Liofilchem srl, Italy) (37 °C for 24 h). Typical Salmonella spp. colonies are colorless or very pale pink, opaque or semitransparent, usually with a black center (Nikiema et al. 2021).

Presumptive Staphylococcus spp. were isolated on Mannitol Salt Agar (MSA) (Merck, Germany) according to the method described by Akya et al. (2020). Petri dishes were incubated at 37 °C for 48 h. Gram-positive cocci isolates that formed yellow colonies with a yellow halo on MSA were considered presumptive isolates of S. aureus.

Greywater samples were subjected to cascade dilution techniques to obtain significant results. The samples were inoculated onto the specific culture media using the surface plating method. The culture plates were then incubated at the appropriate temperature for the specified time to allow bacterial colonies to grow and develop. Finally, characteristic bacterial colonies were examined for presumptive identification based on their morphological characteristics and biochemical reactions on the appropriate selective culture media.

Reference strains of Salmonella spp. and Staphylococcus spp. were used as positive controls to ensure that the isolation and identification protocols were appropriate and produced the expected results. Control samples of sterile water were used as negative controls to verify the absence of cross-contamination. In addition, analyses were carried out in duplicate for each sample to ensure reproducibility of results.

Antimicrobial susceptibility analysis was performed with the disk diffusion method on Muller–Hinton agar (Liofilchem srl, Italy) according to the recommendations of the Clinical and Laboratory Standards Institute guidelines (CA-SFM/EUCAST 2020).

Suspected Salmonella spp. isolates were tested for susceptibility to the following antibiotics: ampicillin (AMP, 10 μg), amoxicillin + clavulanic acid (AUG, 30 μg), cefoxitin (FOX, 30 μg), gentamicin (CN, 10 μg), trimethoprim-sulfamethoxazole (SxT, 25 μg), chloramphenicol (C, 30 μg), tetracycline (TE, 30 μg), ciprofloxacin (CIP, 5 μg), vancomycin (VA, 30 μg) and azithromycin (AZM, 15 μg). The reference strain Escherichia coli ATCC 25922 was used as a control for antibiotic susceptibility testing. After incubation at 37 °C for 16–18 h, the diameter of the zone of growth inhibition was measured for each antibiotic. According to the diameter of the inhibition zone, isolates were classified as resistant (diameter < reference value defined by EUCAST standards) and susceptible (diameter ≥ reference value defined by EUCAST standards). MDR strains were determined by the lack of sensitivity to at least three classes of antibiotics. In addition to measuring the inhibition zone diameter, results were interpreted according to criteria established by the European Committee on Antimicrobial Susceptibility Testing, ensuring standardized assessment of antibiotic resistance.

To determine the frequency of MDR Staphylococcus spp., suspected Staphylococcus spp. isolates were tested for susceptibility to several classes of antibiotics, including β-lactams (penicillin, 10 μg and oxacillin, 1 μg), β-lactams + beta-lactamase inhibitors (amoxicillin + clavulanic acid, 30 μg), macrolides (azithromycin, 15 μg), tetracyclines (tetracycline, 30 μg) and lincosamides (clindamycin, 2 μg) using the agar disk diffusion method according to the guidelines of the Antibiogram Committee of the French Society of Microbiology (CA-SFM/EUCAST 2020). The S. aureus strain ATCC 25 923 was used as a reference strain for quality control. After incubation at 37 °C for 16–18 h, the diameters of the growth inhibition zones were measured for each antibiotic. According to the diameter of the inhibition zone, isolates were classified as resistant (diameter < reference value defined by EUCAST standards) and susceptible (diameter ≥ reference value defined by EUCAST standards). MDR strains were determined by the lack of sensitivity to at least three classes of antibiotics. In addition to measuring the inhibition zone diameter, results were interpreted according to criteria established by the European Committee on Antimicrobial Susceptibility Testing, ensuring a standardized assessment of antibiotic resistance.

Overall, all the antibiotics used in this study were selected on the basis of their clinical or epidemiological importance for human and animal health in West Africa (Dione et al. 2011; Dissinviel et al. 2017).

MDR strains obtained from antibiotic susceptibility testing were characterized. For presumptive Salmonella spp. colonies, in addition to Gram staining and catalase testing, the API 20E gallery (BioMe rieux, Marcy l'Etoile, France) was used according to the manufacturer's recommendation, to determine their biochemical profile. The criteria used to interpret the results of the API 20E test are based on the biochemical reactions observed for each substrate. These reactions are compared to a reference table provided by the API 20E kit manufacturer. The results are then used to identify the bacterial species according to the identification schemes provided by the manufacturer.

For biochemical identification of suspected Staphylococcus spp., biochemical tests such as Gram stain, catalase test and coagulase test were performed as described by Akya et al. (2020). The criteria used to interpret the results of the coagulase test are based on the presence or absence of coagulation in rabbit plasma after incubation. The formation of a visible clot indicates a positive result, suggesting that the bacterial strain is Staphylococcus aureus. The absence of clot formation is interpreted as a negative result, suggesting that the bacterial strain is not Staphylococcus aureus (Igbinosa et al. 2016).

Statistical analysis of the results was performed in Excel and XLSTAT for Windows version 2016. Analysis of variance (ANOVA) was used to compare Salmonella spp. and Staphylococcus spp. concentrations in urban as well as rural greywater. In addition, differences between the means of the inhibition diameters of the bacterial strains were determined by ANOVA using the Fisher test (Least Significant Difference) at P < 0.05 probability level.

The results show a high concentration of presumptive pathogenic bacteria in greywater from urban and rural households. Indeed, the concentrations of Salmonella spp. and Staphylococcus spp. are very high (Table 1).

Salmonella spp. were detected in 100% of the greywater samples from urban households with high concentrations ranging from 3.65 to 7.18 Log₁₀CFU/100 mL. In contrast, Salmonella spp. were detected in 60% of samples from rural households with relatively low concentrations ranging from 1.1 to 2.08 Log₁₀CFU/100 mL (Table 1). Statistical analysis of the results showed that there was a significant difference between the concentrations of Salmonella spp. in different types of greywater from different localities (P < 0.0001). One possible explanation for this difference could be that hygiene practices can differ between urban and rural areas, which can influence the contamination of greywater with Salmonella spp. For example, waste disposal as well as toilet use practices can differ, which can affect greywater quality from a Salmonella spp. perspective (Frost et al. 2018). In addition, urban areas generally have a higher population density than rural areas, which can increase the load of Salmonella spp. contamination in greywater (Ercumen et al. 2018). Other research conducted in Ivory Coast by Coulibaly et al. (2014) has also shown the presence of multiple opportunistic pathogens such as Salmonella spp. in greywater. Katukiza et al. (2014) reported Salmonella spp. concentrations of 4.44 Log₁₀CFU/100 mL in greywater in Jordan. Contamination of greywater with Salmonella spp. usually occurs when an infected person washes or when contaminated food is washed (Birks & Hills 2007). In Burkina Faso, with the livestock sector booming, the main reservoirs of human infection could be poultry, cattle, sheep as well as pigs. Furthermore, the presence of Salmonella spp. in greywater in households is thought to be due to food handling in the kitchen (Cogan et al. 1999), and is therefore thought to enter greywater via dishwashing effluent (Al-Gheethi et al. 2018).

Furthermore, Staphylococcus spp. were detected in 100% of greywater samples from urban households with an average concentration of 4.5 Log₁₀CFU/100 mL (Table 1). Relatively high presumptive Staphylococcus spp. concentrations were also found in greywater (laundry as well as dishwashing) from rural households with mean concentrations of 4.41, 4.45, 4.44 and 3.69 Log₁₀CFU/100 mL in Pabré, Koubri, Saaba and Komki Ipala, respectively (Table 1). There were no significant differences between the concentrations of presumptive Staphylococcus spp. found in greywater from different localities (P = 0.336). Many studies have shown the presence of multiple opportunistic pathogens such as Staphylococcus spp. (Craddock et al. 2020; Nagarkar et al. 2021). Staphylococcus spp. concentrations on the order of 3.53 and 4.66 Log₁₀CFU/100 mL have been reported in greywater in England (Winward et al. 2008) and Israel (Maimon et al. 2014). Zimmerman et al. (2014) also reported the presence of Staphylococcus aureus at a lower concentration of approximately 5 Log₁₀CFU/100 mL in greywater from laundry. Staphylococcus spp. is a bacterial agent found on skin as well as mucosal tissue (Gilboa & Friedler 2008). It is then excreted in greywater during bathing and showering activities (Winward et al. 2008; Benami et al. 2013).

Salmonella spp. can cause mild to severe gastroenteritis and Staphylococcus aureus can cause skin infections through skin contact (Busgang et al. 2018). As such, the presence of these opportunistic pathogenic bacteria poses a health risk to populations (Amha et al. 2015).

Overall, the average pathogenic bacteria count in our study was relatively higher in urban environments than in peri-urban environments. In fact, 100% of urban greywater samples in this study came from a mixture of dishwashing and laundry water, whereas 40% of the rural greywater samples came from laundry water. It is therefore plausible that this may be one of the reasons for the comparatively high bacterial load in our urban samples, as the high nutrient concentrations (degraded organic matter) associated with kitchen wastewater increase biological oxygen demand (BOD) levels and favor the growth of enteric bacteria (Cogan et al. 1999; Boyjoo et al. 2013). The urban and rural households included in this study tend to have large families, often with young children, which may contribute to high bacterial concentrations in the greywater discharged from these households.

Given the wide variety of resistance to different antibiotics observed in multiple studies of human infections in Burkina Faso, the detection of antibiotics resistant bacteria (ARB) in greywater was not surprising. Although we have not studied the origin of the ARB found in the greywater tested, we hypothesize that these microorganisms may enter the greywater system when environmental or human bacteria are removed during hand washing and shower/bathing, when dirty cloth nappies are washed in the laundry or during food preparation (when poultry and other raw meats are prepared) in kitchen sinks (Maiga et al. 2014; Busgang et al. 2018).

Resistance to several antibiotics was observed among isolates recovered from urban and rural greywater samples (Table 2).

Among the 52 greywater samples, 36 Salmonella spp. isolates were detected, of which 13 (36.1%) and 23 (63.8%) were from greywater from urban households and peri-urban households, respectively.

Ten different types of antibiotics were tested on all 36 Salmonella spp. isolates. Strains showed different inhibition zones depending on the antibiotics tested (Table 2). From 52 greywater samples, a total of 28 resistant Salmonella spp. strains were isolated. Among isolates from greywater samples, resistance to several different antibiotics was observed (Table 2).

Among all isolates, the most common resistances were to vancomycin (69%), ampicillin (61%), cefoxitin (42%), trimethoprin/sulfamethoxazole (36%), amoxicillin-sulfamethoxazole (33%) and tetracycline (33%) (Table 2).

This study showed that Salmonella spp. strains isolated from greywater are highly resistant to most antibiotics commonly used in human medicine. These are mainly aminoglycosides, cephalosporins, β-lactam antibiotics and penicillins, with resistance profiles of 69.44, 61.11, 41.66, 36.11 and 33.33%, respectively. These resistance rates are higher than those obtained by Al-Bahry et al. (2007) and Coulibaly et al. (2014) in Jordan and Côte d'Ivoire, respectively. Indeed, these authors observed resistance to tetracycline, as well as third generation cephalosporin in strains (n = 11) of Salmonella spp. isolated from domestic wastewater, with resistance profiles ranging between 9.1 and 18.2%, respectively.

Several authors have identified Gram-negative bacteria resistant to antibiotics in greywater (Troiano et al. 2018; Craddock et al. 2020; Noman et al. 2022). Nuñez et al. (2012) studied antibiotic resistance in an open municipal greywater canal in Argentina. These authors observed resistance rates that were generally lower than those in our study. Indeed, the antibiotic to which their Gram-negative isolates were most frequently resistant was ampicillin, as in our study, but 34% of their isolates were resistant to ampicillin, compared with 61% of our greywater isolates (Table 2). The differences observed here suggest that there are significant variations between geographical locations in antimicrobial resistance in greywater (Craddock et al. 2020).

In general, the rates of antibiotic resistance we observed in greywater isolates were lower than those reported in previous studies of hospital effluent isolates. Indeed, a study of hospital effluent in Mekele, Ethiopia, showed simultaneous resistance to penicillin, tetracycline, doxycycline, cotrimoxazole, amoxicillin-clavulanic acid and ceftriaxone (Asfaw et al. 2017). Similarly, the study reported by Alam et al. (2013) showed concurrent antibiotic resistance in extended-spectrum beta-lactamase (ESBL)-producing enteric bacteria isolated from hospital wastewater. In addition, an increased proportion (76.2–81.5%) of MDR bacteria has been reported among hospital wastewater isolates (Asfaw et al. 2017). However, as these isolates were recovered from clinical samples, it is not surprising that the levels of resistance observed in the greywater isolates in this study are lower (Craddock et al. 2020).

Overall, multiple antibiotic resistance has been observed in several Salmonella spp. isolates. In fact, 75% (27/36) of isolates were multi-resistant, including 3 (7.14%) isolates resistant to 2 classes of antibiotics, 8 (22.22%) to 3 classes of antibiotics, 6 (16.66%) to 4 classes of antibiotics, 8 (22.22%) to 5 classes of antibiotics and 2 (5.55%) to 6 classes of antibiotics. Troiano et al. (2018) also observed that some tetracycline-resistant isolates expressed multidrug resistance, which is also consistent with our results (Troiano et al. 2018). The distribution of MDR strains in greywater could be mainly due to higher lactamase production against β-lactam antibiotics (Alam et al. 2013; Egbule 2016), horizontal transfer, exchange of resistant mobile genetic elements (Egbule 2016) and spread of resistant bacteria due to selective pressures imposed by antimicrobial residues (Jamali et al. 2015). Indeed, authors have shown the possibility of antibiotics entering greywater via personal care products, washing nappies, and urination during bathing or showering (Do Couto et al. 2013). The presence of low-dose antibiotics in greywater influences the selection of antibiotic-resistant bacteria (Craddock et al. 2020; Noman et al. 2022).

In addition, 30.35% (17/56) of isolates were multidrug resistant, of which 4 (7.14%) were resistant to 2 classes of antibiotics, 4 (7.14%) to 3 classes of antibiotics and 9 (16.07%) to 4 classes of antibiotics.

Antibiotic susceptibility testing revealed that the isolates were resistant to the beta-lactam antibiotics used clinically. In fact, the presumed strains of Staphylococcus spp. from the greywater samples were highly resistant to penicillin (55.36%) and oxacillin (48.21%). This result is lower than the 90–100% penicillin resistance rate observed in hospital-derived S. aureus in Burkina Faso, Ghana (Moirongo et al. 2020) and Malaysia (Che Hamzah et al. 2019).

The presence of Staphylococcus spp. strains in greywater is associated with their presence on human skin (Henderson et al. 2022); these germs are eliminated during hand washing and showers/baths or during laundry and dishwashing activities (Gross et al. 2007; Busgang et al. 2018). Antibiotic resistance in these isolates could therefore be explained by the extensive and uncontrolled use of antibiotics during self-medication (Sachdev et al. 2022), a common practice in West African countries, particularly Burkina Faso. Indeed, Staphylococcus are among the bacterial species most frequently observed in hospitals and have been implicated in numerous infections in humans, such as skin and soft tissue infections, wound and surgical site infections and pneumonia (Nanoukona et al. 2016). This can be a source of the spreading of these germs, which can end up in greywater. Furthermore, there has been an increase in the use of antibiotics in the livestock sector, which is expanding rapidly in Burkina Faso. Failure to follow good veterinary practice in the traditional livestock sector in Burkina is also a source of antibiotic resistance. For example, Tiseo et al. (2020) estimated the global consumption of antibiotics in chickens, cattle and pigs at 93,309 t in 2017.

Overall, this is one of the first studies to report the presence of MDR bacteria in untreated greywater discharged by domestic activities in Burkina Faso. Our results show that untreated greywater can be host to bacteria resistant to the antibiotics used in therapeutic care. Consequently, the uncontrolled discharge of untreated greywater into the environment could lead to the dissemination of resistant bacteria and resistance genes in the environment and increase the risks of human exposure to antimicrobial resistance (Porob et al. 2020). Investment in more efficient greywater treatment systems is needed to help reduce contamination by antibiotic-resistant bacteria. Studies have shown that advanced treatment systems can effectively eliminate antibiotic-resistant microorganisms (Chen et al. 2015a, 2015b). At the parallel, surveillance programs for greywater quality, in particular, for the detection of antibiotic-resistant bacteria, can enable rapid intervention in the event of a risk to public health (Gholipour et al. 2024). In addition, campaigns to raise awareness of good hygiene practices and the appropriate use of antibiotics can help to reduce the selection pressure for antibiotic resistance (Versporten et al. 2018).

Analysis of the biochemical characteristics of the presumptive Salmonella spp. strains obtained from the gallery (API 20E) showed that all the strains were catalase-negative and were unable to degrade tryptophan to indole (indole negative) or to indole pyruvic acid (IPA negative). The strains showed variable profiles for the other tests. The biochemical profiles made it possible to propose a taxonomic test for the strains using the digitized probabilistic method for the identification of Salmonella (Nikiema et al. 2021).

Remarkably, all the presumed MDR strains isolated from greywater were ESBL producers. In fact, our results show a prevalence of identified MDR isolates belonging to the genera Salmonella spp. (21.42%), Proteus spp. (14.28%), Enterobacter spp. (14.28%), Citrobacter spp. (14.28%), Klebsiella spp. (7.18%), Aeromonas spp. (7.14%), Chromobacterium spp. (7.14%), Stenotrophomonas spp. (7.14%), Serratia spp. (7.14%). Other studies reported a high prevalence of ESBL-producing Klebsiella spp. (24.9%), Enterobacter spp. (19.1%), Citrobacter spp. (11.2%), Proteus spp. (3.6%) and Serratia spp. (3%) in municipal and hospital wastewater in Côte d'Ivoire (Carole et al. 2018). In addition, recent work has shown the presence of these species in raw greywater discharged to the environment without treatment in Israel (Porob et al. 2020) and Yemen (Noman et al. 2022). The presence of these resistant isolates in greywater could contribute to the spreading of ESBL-producing bacteria in the environment (Oteng-Peprah et al. 2018). The genera isolated from our greywater samples are often recognized as opportunistic pathogens associated with urinary tract, bloodstream and respiratory infections. In this context, infections caused by ESBL-producing bacteria represent an emerging health problem in Burkina Faso, as already reported by some authors (Kpoda et al. 2017). Indeed, the presence of these MDR species in untreated greywater discharged into the environment could accelerate the transfer of ESBL genes to pathogenic bacteria, posing a clear threat to public health.

With regard to the biochemical characteristics of the 17 suspected multi-resistant isolates of Staphylococcus spp., biochemical tests showed that all isolates tested were catalase-positive and degraded mannitol. Coagulase tests showed that of the isolates (17), only 6 (35.29%) were coagulase-positive and the remaining 11 (64.70%) were coagulase-negative. Based on the biochemical characteristics, the isolates were presumptively and phenotypically identified as Staphylococcus species.

Most studies have focused on coagulase-positive (S. aureus) and coagulase-negative staphylococci from clinical (Shittu et al. 2012; Nanoukona et al. 2016) and environmental (Adegboyega et al. 2019) sources in West Africa. However, there is a paucity of data on the occurrence of these species in greywater.

Our results showing that 35.29% of isolates were coagulase-positive (S. aureus) and 64.70% were coagulase-negative are similar to observations made in previous studies. In fact, Adesoji et al. (2020) showed the presence of coagulase-positive (3.33%) and coagulase-negative (93.33%) Staphylococci in wastewater and greywater in Nigeria. Furthermore, a study conducted by Gómez et al. (2016) on wastewater in Spain showed the presence of 16.67% coagulase-positive Staphylococcus and 83.33% coagulase-negative species.

The presence of coagulase-positive and coagulase-negative Staphylococci strains in greywater could be explained by the inappropriate use of antibiotics in human medicine (Sachdev et al. 2022). In addition, research conducted by Heaton et al. (2020) showed that contact with animals or their waste can be a source of transmission of resistant strains of Staphylococcus aureus to humans, which can contaminate greywater.

Remarkably, phenotypically, the ESBL producers and Gram-positive Staphylococcus spp. were MDR. Previously, a high prevalence of ESBL-producing bacteria has been reported in urban wastewater, hospital waste and sewage (Akya et al. 2020; Porob et al. 2020), but in this study, we report their presence in urban and rural greywater in West Africa. Salmonella spp., Proteus spp., Enterobacter spp., Citrobacter spp., Klebsiella spp. and Staphylococcus spp. are often considered opportunistic pathogens associated with urinary tract, bloodstream, respiratory tract, and skin infections (Tong et al. 2015; Noman et al. 2022). In this context, infections with ESBL-producing bacteria are also an emerging health problem in West African populations (Kpoda et al. 2017). As mentioned earlier, these households are often not connected to sewerage systems, leading to higher levels of exposure to wastewater. In addition, the presence of these pathogenic MDR ESBL bacteria in urban and rural greywater could be explained by the inappropriate use of antimicrobials and the proximity of livestock and other domestic animals, which can act as a reservoir or source of ARB, including ESBL (Rousham et al. 2018). Although we cannot be certain of the origin of these ESBL-producing bacteria, it is known that ESBL genes can be transferred from one environmental source to another and, in particular, from foods of animal origin to humans via mobile genetic elements in interconnected habitats (Stalder et al. 2013; Elnasasra et al. 2017). The presence of pathogenic MDR and ESBL-producing strains in untreated greywater discharged from these households contributes significantly to the dissemination of ESBL-production genes in the environment, thus constituting a potential threat to public health.

The sample of greywater collected in rural areas is small and obtained during a limited period. This may result in inadequate representation of the diversity of sanitation practices and conditions in these areas. Future research should aim to adopt a more comprehensive approach to sample collection, including a larger number of rural municipalities and a longer collection period. This would better capture variability in sanitation conditions and greywater management practices across different rural communities. Moreover, the study relies exclusively on biochemical tests to identify MDR strains, which may result in incomplete or inaccurate characterization of antimicrobial resistance. While whole-genome sequencing is an ideal option for the detailed characterization of MDR strains, its implementation may be costly and complex. A viable alternative would be to explore more accessible molecular techniques, such as polymerase chain reaction (PCR), to detect the presence and diversity of antibiotic resistance genes (ARG). This would allow for a more comprehensive analysis of MDR strains present in greywater. Despite these limitations, the current results of our study indicate that untreated greywater may be a potential source of multi-resistant bacteria, such as Gram-negative and positive ESBL producers.

This study provides basic information on the occurrence of multi-resistant bacteria in greywater from urban and rural households in West Africa. Our results showed that the genera Salmonella spp., Proteus spp., Enterobacter spp., Citrobacter spp., Klebsiella spp. and Staphylococcus spp. were resistant to antibiotics commonly used in human medicine. This could have public health implications as greywater could play an important role as an environmental reservoir of pathogens in the development and spreading of antibiotic resistance. The common practice of discharging greywater directly into the environment without treatment is a practice that should be prohibited in this widespread context of antibiotic resistance. This situation should challenge decision-makers to formulate appropriate policies aimed at minimizing the impact of discharging untreated greywater into the environment. This study also highlights the need to propose models for treating domestic greywater before it is released into the environment or reused in horticulture, in the Sahel context.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.