Globally, the development and widespread of resistance to antimicrobial in bacteria [1
] is a major challenge in drug therapy [4
] in humans and animals. Despite concerted effort to combat this evolving trend by drug combination therapy [6
] and other innovative strategies, multidrug resistance (MDR) among bacterial pathogens have posed serious threats to clinical therapy [7
]. The wide use of antibiotics in humans and animal husbandry has facilitated the increasing spread of MDR. This is particularly worrisome when it is inappropriately used as a result of its availability over the counter without prescription as practiced in most developing countries/regions. This practice and many more have made hospital, municipal, and agricultural sewages major sources of antibiotic residues within the environment [3
Until recently, the focus of antibiotic resistance has been on these sources, particularly the clinical sources of MDR development, according to a recent report, contain only a small proportion of the antibiotics resistant determinants found [11
]. Many other reports show that treated antibiotic production wastewaters from wastewater treatment plant (WWTPs) contain much higher concentrations of antibiotic residues than other aquatic environments which have been attributed to development of MDR [8
]. It is well known, however, that bacteriophage and virus DNA sequences are very common in influent waste water [14
]. In a recent study, diversity of bacteriophage and virus DNA sequences was markedly reduced in effluent water compared to influent [15
]. The authors noted that their results of viral DNA analyses obtained in the study were in agreement with other metagenomic studies, showing greater occurrence of bacteriophages compared to human virus sequences in wastewater [16
Several reports have attempted to correlate high concentrations of antibiotics within sewage treatment plants (STPs) with increased levels of resistance to antibiotics by bacteria [17
] without clarity in outcomes. Environmental bacteria in STPs have been demonstrated in many studies as carriers of antibiotics resistance determinants [18
] and potential sources of novel resistance genes in clinical pathogens [22
]. In addition, in agricultural practice, the use of treated wastewater effluent as alternative source of irrigation water may introduce active antibiotic resistant pathogens to the soil [24
] which may pose health risk to humans that come in contact with them. Owing to the limited availability of clear evidence showing the evolution of resistance and the spread of antibiotics resistance genes (ARGs) in WWTPs [11
], there is need to extend antibiotic resistance studies to WWTPs beyond clinical studies.
The dihydropteroate synthase (DHPS) genes sul
1 and sul
2 have been detected in bacterial isolates from water and other aquatic environments [26
], and even from rivers and seawater without evidence of being polluted [28
]. The sul
1 gene, as a part of class 1 integron, can be disseminated and transferred horizontally within and between species in wastewater [31
], river water [32
], and seawater [33
]. It has been found linked to other resistance genes in class 1 integrons and on large conjugative plasmids [34
] while sul
2 is usually located on small nonconjugative plasmids [35
], large transmissible multiresistance plasmids [36
], or through insertion element common region (ISCR2) element [37
]. Studies on the presence of the dihydropteroate synthase (DHPS) genes and the integron reveal the possibility of dissemination of these genes within water sources.
In this study, we investigated the antibiotic resistance profile of bacterial isolates obtained from wastewater samples collected from fourteen pharmaceutical facilities in Lagos and Ogun States Southwestern Nigeria over a twenty-six-month period. The selected pharmaceutical facilities are key players in antibiotic production at the secondary and tertiary stage of production in these regions. In Nigeria, most pharmaceutical industries produce various antibiotics and other drug types in a single production plant. In most cases, they lack wastewater treatment before the effluents are released into the environment and other bodies of water [38
]. The best practice is to hold the wastewater over a period; in some cases, the wastewater is diluted. The untreated wastewater is either deposited underground or discharged directly into nearby natural water bodies [38
]. We also investigated a wastewater treatment plant situated in an industrial Estate which receives wastewater from pharmaceutical facilities production plants and other production factories within the region. In addition, household sewages from residential quarters were collected within the treatment plant. We further studied the river water samples obtained from the site where the effluent is discharged at the end of treatment. Conventional wastewater treatment methods were employed within the system.
The results obtained in this study indicate that the pharmaceutical wastewater environment contains a large community of bacteria (Table 2
). High bacteria counts were observed in the WW, WTP and RW samples (Supplementary Materials, Table S2
). Two pharmaceutical facilities (WWi2 and WWv) showed the highest bacteria count in the study. The wastewater samples from the wastewater treatment plant (WTPi and WTPii) also have high bacterial counts. The treated wastewater WTPii have more bacteria counts than the untreated wastewater WTPi. The river water samples RWi and RWii have almost the same bacteria counts, but the upstream water RWi has more count compared to the downstream RWii sample. This is expected because the upstream source is closer to the WTP discharge point.
The result of the analysis of the 16S rRNA genes in the bacterial isolates from wastewater and surface water samples in this study showed that these environments harbor very viable bacteria population. The Gammaproteobacteria was the most frequent bacteria isolates in this study, which could be because of their favored growth in nutrient-rich culture media. This observation agrees with the work of Li et al. [8
] where the majority of the 341 bacterial isolates obtained from an oxytetracycline WTP and river water were Gammaproteobacteria. Proteus mirabilis
sp. were the most abundant bacteria isolates obtained from this study. In a similar investigation, Guardabassi et al. [46
] demonstrated that discharge of wastewater from a pharmaceutical plant was associated with an increase in the prevalence of both single and multiple-antibiotic resistance among Acinetobacter
species in the sewers.
The bacterial isolates from the sample groups showed high levels of resistance to all the tested antimicrobials. Among the 254 bacteria isolates, a large number (95.7%) of the bacteria isolates showed high resistance to Augmentin, a penicillin combination of Amoxycillin and Clavulanic Acid, which are classified as beta-lactams. The combination is expected to have very high efficacy against infectious organisms. This antibiotic is a choice therapy in Nigeria in both children and adult. This high resistance to Augmentine agrees with the work of Li et al. [47
] which shows that resistance to β-lactams antibiotics was more frequent, with much higher levels, than the other classes of antibiotics tested in isolates of a penicillin production wastewater treatment plant and receiving river. The MIC result shows a universal resistance to sulfonamide antibiotics. This result instigated the investigation of the sulfonamide resistance genes in this study.
In WW samples, antibiotic resistance phenotypes were very common. Resistance (93.4%) to more than three classes of the tested antibiotic was observed in the WW isolates. In Agbara Ogun State, WWii samples showed multi-drug resistant (MDR) strains in all the bacterial isolates, except for three of the isolates that showed resistant to less than three classes of the tested antibiotics. Samples of WWv showed all bacterial isolates to be MDR. In Sango Ota Ogun State, out of the 32 WWi wastewater samples, seven of the bacterial isolates showed resistant to all the tested eight antibiotics. On the other hand, all isolates are MDR except for one. Most of the MDR organisms showed resistant mostl to 5–7 classes of the tested antibiotics, but at least to four of the classes. In wastewater samples of WWiii in Ikeja Lagos State, all the bacterial isolates encountered are MDR. In addition, in sample WWvii, all the 38 isolates were MDR except for one. All the bacterial isolates obtained from WWx samples in Oshodi Lagos State were all MDR with resistant to at least six classes of the antibiotics tested. With exception of one non-MDR isolate, all bacterial isolates obtained from WWxi were MDR. This is in line with a recent investigation by Li et al. [8
] in oxytetracycline waste water treatment plant demonstrated that strong selective pressure was imposed by a high concentration of oxytetracycline which contributed to the proliferation of MDR bacteria strains in the wastewater environment.
The presence of antibiotics in the report above might be a single factor for the selection of MDR bacterial isolates. Wastewater treatment plants are interfaces between different environments and it has often been reported to have a high level of residual antibiotics. Li et al. [47
] investigated the bacterial characteristics of a penicillin production wastewater treatment plant and the receiving river and demonstrated that high resistance prevalence and levels could be induced by long-term penicillin exposure. This present study detected high level of antibiotics (result not presented in this report) in wastewater coming from a production line in a pharmaceutical facility (WWx) in Oshodi Lagos State. This condition might be the reason for selection of only MDR bacterial isolates in this facility. Although it is still difficult to establish clear cause effect relationships, it is widely accepted that chemical pollution contributes to antibiotic resistance dissemination [48
The WTPi bacteria isolates have six MDR, whereas the WTPii have 17 MDR bacteria isolates out of the total number of 26 isolates. This result again questions the efficacy of the treatment process, though some studies have demonstrated that wastewater treatment processes, operating according to legal recommendation, cannot reduce effectively the levels of antibiotic resistance [19
]. The increase in MDR bacteria isolates in WTPii compared to WTPi suggests that the treatment procedure in the WTP might have selected for more MDR strains. This observation may be in line with the report of Manaia [51
] that wastewater composition and the treatment process itself may pose selective pressures capable of modulating either the bacterial populations or the antibiotic resistance pool.
In the river samples, RWi (upstream) and RWii (downstream) have 18 bacterial isolates each. The upstream samples had 10 MDR, whereas the downstream bacteria isolates had 12. It is expected that more MDR will be observed in the upstream compared to the downstream samples. The number of bacteria isolates obtained from these compartments is not enough to draw major inferences in these two segments of the river water samples. The major reason for the analysis of the river water is to screen for the presence of antibiotic resistance genes or other antibiotic resistance determinants within the aquatic confinement. The presence of antibiotic in the river waters might also pose selective pressure on antibiotics resistance determinants. Although this comes in much lower concentration, reviews have it that pharmaceuticals is not only found in wastewaters, but also in surface, ground and drinking waters [52
]. The fact established in this study is that MDR bacteria are found within the river water environment.
Multi resistant bacteria are prevalent in this study and encountered in all the tested isolates. In this study, sulfonamide resistance genes sul
1 and sul
2 were detected in 31.7% and 21.7% of the bacterial isolates, respectively. This supports the high resistance to the sulfonamide antibiotics used in the antibiotics susceptibility test in this study. Since they all showed resistance to sulfonamide antibiotics, the question is: Why were sulfonamide resistance genes not found in all the bacterial isolates? There is a high chance that other sulfonamide resistance genes other than the sul
1 and sul
2 were responsible for the resistance to sulfonamide recorded, because different types of mechanisms have been found to confer resistance to sulfonamide, mostly based on changes in the sul
genes and mediation by mobile elements [55
]. There is also a possibility that beyond the sulfonamide resistance genes, there could be other factors responsible for conferring resistance of sulfonamide to the bacterial isolates. In addition, the detection of sul
ARGs does not mean that they are conferring resistance in the host; more sophisticated studies are needed to distinguish between ARG carriage in the host chromosome and ARG which confers resistance to the treatment of pathogens [11
Integrons, especially class I integrons, commonly contain antibiotic resistance gene cassettes and are closely related to MDR, generally by containing several resistance gene cassettes simultaneously [56
]. In the result presented above (Table 5
), integron 1 (Intl
1) was detected in 43.3% of the bacterial isolates while Intl2
was not detected. The presence of Intl
1 in the isolates indicates a high prevalence within the wastewater and river water medium. The relative abundance of the clinical class 1 integron-integrase gene, intI
1, is a proxy for anthropogenic pollution amongst many other factors is that they are linked to genes conferring resistance to antibiotics [56
]. This situation can pose a high possibility of dissemination of resistance determinants within the water systems from one bacterium to the other and possibly to clinical isolates in some instances.
Studies on antibiotics resistome in different environmental compartments have been carried out in many regions worldwide. However, this is the first report on the attempt to elucidate the antibiotic resistance profile of bacterial community within the pharmaceutical wastewater in the Nigerian environment, under possible range of anthropogenic influence. The overview of our findings suggests that antibiotic resistance status in the Nigerian environment is no different from what is obtainable in other regions. This result can give insight to understanding the emergence and dissemination of novel antibiotics resistance from the natural reservoirs to the clinical environments.