Diversity, Resistance Pattern and Level of Antibiotic Resistance Determinants Associated with Bacteria Isolated from Different Water Sources in Southwest Nigeria

Safe water is an essential resource for the existence and sustenance of life. The unavailability of safe water is a significant cause of water-related diseases, especially in developing countries like Nigeria. A spike in the incidence of waterborne diseases has become a public health concern. Hence, this study was designed to evaluate the diversity of bacteria, resistance pattern and level of antibiotic resistance determinants associated with bacteria isolated from different water sources in South-West Nigeria. A total of 120 water samples were collected across South-Western Nigeria from which bacteria were isolated, and identification was achieved by sequencing its partial 16S rRNA. Disc diffusion and multiplex PCR were used to determine antibiotic susceptibility and resistance determinants. A total of 50 bacterial isolates were identified, consisting of Proteus spp. (2), Bacillus spp. (8), Enterobacter spp. (8) , Klebsiella spp. (9) , Escherichia coli (7) , Citrobacter spp. (6), Pseudomonas spp. (4), and Serratia marcescens (6). All the isolates were resistant to ceftazidime, cefuroxime, cefixime and amoxicillin/clavulanate, whereas 72%, 60%, 54% and 44% were resistant to nitrofurantoin, gentamicin, ciprofloxacin and ofloxacin respectively. Quinolone-resistant genes ( qnrB , qnrA and qnrS ), and tetracycline-resistant genes (tetA and tetB) were found in the isolates. The beta-lactamase gene and class 1 integron were also detected in the isolates, with the class 1 integron genes occurring the least. The results revealed that isolates were highly resistant to antibiotics used, as evident in the presence of antibiotic genes. This reveals the unsanitary states of water consumed by the general public, and the ingestion of these antibiotic-resistant bacteria is responsible for the birth of untreatable diseases.


Introduction
Water is a critical resource for daily life's existence, sustenance, and continuance (do Nascimento et al., 2020). It is used in various domestic activities such as drinking, bathing, cooking, and industrial processes. Although water is a universally important solvent, its role in disease transmission has been reported (Sharma & Sharma, 2022). Safe water consumption is critical for maintaining and promoting good health (Raimi et al., 2019). The unavailability of water has become a dire issue in various rural areas, especially in developing countries, due to, but not limited to, factors like overpopulation (Ghaderi et al., 2022), unsanitary waste disposal processes (David et al., 2020), wars and population displacement (You et al., 2020) and open defecation (Kookana et al., 2020).
Primary sources of these water contaminants and pathogens are the discharge of domestic and industrial effluent wastes, leakage from septic tanks, marine dumping, radioactive waste and atmospheric deposition into the water. Uddin et al.
(2019) say that over 5 million people die yearly from untreated water and bad hygiene habits. However, most people living near water sources in Nigeria continue to drink from rivers, streams, and other water bodies, regardless of their state or treatment (Raimi et al., 2019).
Several pathogens have been identified as waterborne, including Proteus spp., Bacillus spp., Enterobacter spp., Klebsiella spp., Escherichia coli, Citrobacter spp., Pseudomonas spp. and Serratia marcescens (Barrantes et al., 2022). A limited supply of potable water in developing nations like Nigeria is a significant reason for the occurrence of some waterborne diseases like typhoid, cholera, traveller's diarrhoea, or shigellosis, which are associated with significant morbidity and mortality (Okpasuo et al., 2020), especially in this era of bacterial multidrug resistance.
Antibiotic resistance is a global concern that is wreaking havoc on the economy and inflicting financial hardship on patients (Ventola, 2015). Several studies have connected antibiotic abuse and the presence of resistance genes to the development of antibiotic resistance (Cepas et al., 2019;Richard et al., 2022). Antibiotic-resistant microorganisms have caused enormous death and morbidity worldwide (Murray et al., 2022). It was recorded that 671,689 illnesses were caused by bacterial antibiotic resistance (Innes et al., 2020), and 495 million fatalities were caused by bacterial antibiotic resistance in 2019 (Murray et al., 2022). The six most prevalent pathogens responsible for mortality due to antibiotic resistance in 2019 were Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa, accounting for 929,000 deaths (Murray et al., 2022). Acquisition of resistance determinants by bacteria can potentially equip them to resist as many as nine different antibiotic classes (He et al., 2020). Antibiotics, biocides, metals, pharmaceuticals, and personal care items released into the environment can promote antimicrobial resistance, thereby increasing selective pressure on bacteria in the aquatic environment, either directly or indirectly (Heß et al., 2018). With the increasing unavailability of safe drinking water in Nigeria and the threat of antibiotic resistance in microorganisms globally, it becomes imperative to determine the diversity, resistance pattern and level of antibiotic determinants associated with microorganisms isolated from different water sources in South-West Nigeria. This study was designed to expose the increasing danger of consuming unsafe water and the implication of ingesting these resistant organisms.

DNA Extraction & Polymerase Chain Reaction
The genomic DNA was extracted according to the manufacturer's instructions using the Quick-DNA mini prep plus kit (Zymo Research, Biolab, USA). Exactly 200 µl of a physiologically young culture of the isolates (16-24 hours old) were inoculated into Eppendorf tubes, and an equivalent volume of biofluid cell buffer was added, and the contents of the tubes were vortexed for 10-15 seconds before being incubated for 10 minutes at 55 o C. After digestion, a volume of Genomic binding buffer (420µl) was added and vortexed for 10-15 seconds. The mixtures were transferred into a Zymo-Spin collection tube and centrifuged for one minute at 12000 rpm. The spin columns were pipetted with exactly 700µl of gDNA wash buffer and spun at 12000 rpm for 1 minute; the collecting tubes with the flow-through were discarded. The spin columns were transferred into clean Eppendorf tubes, and 50µl of DNA elution buffer was poured directly onto the matrix. It was incubated for 5 minutes at room temperature before being centrifuged for 1 minute at a speed of 12000 rpm to elute the DNA. The eluted DNA was immediately used for Polymerase Chain Reaction (PCR).

Gene Detection by Multiplex PCR
A PCR mixture consisting of 15 µl of OneTaqQuck load 2X Master Mix Buffer (New England Biolabs), 0.5 µl of 10 mM of both the forward and reverse sequence selected primer (qnrA, qnrB, qnrS, tetA, tetB, SHV, HEP) was added to each PCR tube containing 3.0 µl of DNA and the reaction mixture was rounded up to 30 µl with sterile water. DNA amplification was done with miniPCR (USA) with thermal cycling profiles appropriate for the primer combinations employed ( Table 1). The amplified DNA was verified by gel electrophoresis; the gel mixture was prepared according to the manufacturer's instruction, heated and poured into the gel tank, and allowed to solidify after insertion of the comb to create wells. The bands were viewed using a trans-illuminator.

Species Barcoding
Isolate representatives (19) were chosen for sequencing. Prior to sequencing, Extracted PCR amplified DNA of selected representatives; PCR mixture consisting of 12.5 µl of OneTaq Quick load 2X Master Mix Buffer (New England Biolabs), 0.5µl of 16SrRNA primers; 341F: 5'-CCTACGGGAGGCAGCAG3' and R806: 5'GGACTACHVGGGTWTCTAAT-3', and an aliquot of 2.0 µl of DNA suspension were added to sterile PCR tubes. With sterile water, the reaction mixture was rounded up to 25 µl. The following thermal cycling profile was used for DNA amplification using miniPCR (USA): Initial denaturation at 95°C for 5 minutes; 30 cycles of denaturation (30 seconds at 95°C), annealing (1 minute at 48°C), and extension (the 30s at 68°C) and a final extension at 68°C for 5 minutes. Standard techniques were used to generate unidirectional sequence reads combined using the Bioedit sequence tool. Molecular evolutionary genetics analysis (MEGA 11) was used to undertake evolutionary analysis.

Susceptibility Testing
The Kirby-Bauer disc diffusion method was used for the antibiotic susceptibility test, as recommended by the Clinical and Laboratory Standards Institute (CLSI, 2021), to test for the sensitivity or resistance of isolates to the antibiotics. In a test tube containing 1ml of nutrient broth (Oxoid, UK), a single colony of the pure isolate was inoculated and cultured overnight at 37°C. The overnight broth was standardised to match the McFarland standard of 0.5. A sterile swab stick was dipped in the standardised suspension and spread across the surface of Mueller Hilton agar plates (Oxoid LTD, Basingstoke, Hamshire, England) that had already been prepared. Antimicrobial susceptibility discs (Abtek Biologicals Ltd.) containing different antibiotics: ceftazidime (CAZ, 30 µg), cefuroxime (CRX, 30 µg), gentamicin (GEN, 10 µg), cefixime (CXM, 5 µg), ofloxacin (OFL, 5 µg), amoxicillin/clavulanate (AUG, 30µg), nitrofurantoin (NIT, 300 µg), ciprofloxacin (CPR, 5 µg) were used for the disc diffusion test according to clinical laboratory standard institute (2021).

Species Distribution and Diversity
Fifty (50) bacterial isolates were obtained from the total of 120 samples collected. Different species were identified; tap water sample (15), well water (16) and river water (19)  The evolutionary history was inferred using the Neighbour-Joining method, and distances were computed using the Jukes-Cantor method. All isolates were grouped into 9 clusters.

Discussion
The bacteria species distribution in this study was similar to the results of Sirisha et al. (2017). The abundance of microorganisms in the water samples can be attributed to a variety of factors, including faulty or inadequate tube-well structures, proximity to sources of contamination (dump sites and toilet facilities, bathing in rivers), unsanitary practices and activities near the source (indiscriminate use and poor handling of fetchers used for the wells) (Agbabiaka & Olofintoye, 2019). In this study, most isolated bacteria belong to the worrisome family Enterobacteriaceae. This is due to its pathogenic potential in healthcare and community settings, especially multidrug resistance. The Enterobacteriaceae family is the most common cause of urinary tract infection, ellulitis, wound infections, septicemia, ear infections, meningitis, abscess, pneumonia, bacteremia, endocarditis, sinusitis, sore throat, gastroenteritis, necrotic enteritis and poisoning and diarrhoea (Babalola et al., 2021;Cristina et al., 2019;Otorkpa, 2019;Paudyal et al., 2017;Manhique et al., 2020). Bacillus spp. is infamous for causing food poisoning and eye infections; nevertheless, in hospitals, Bacillus spp. is usually treated as an environmental pollutant by doctors (Ikeda et al., 2015). Pseudomonas spp. is a pathogen that causes bacteremia, ventilatorassociated pneumonia, urinary tract infections, and skin and soft-tissue infections; it is also known for developing drug resistance that affects practically all antibiotic classes (Bassetti et al., 2018).
It was observed from the phylogenetic relationship that there is an evolutional similarity among isolates from Lagos, Osun, Oyo and Ogun State. This similarity could be attributed to the proximity in geographical areas, frequent interstate trade and the confluence of shared water bodies. The study by Ezeamagu et al. (2021) reported that the isolates from farther locations showed more similarities than isolates from closer locations as opposed to the result from this study. This study indicated that the evolutionary relationship might not be a distance function.
Isolates in this study were found to have high levels of resistance to ceftazidime, cefuroxime, cefixime, amoxicillin/clavulanate, ciprofloxacin, and gentamicin. Iliyasu et al. (2018) and  found similar results, possibly due to the proximity of sampling locations in the studies. This study showed a significant resistance level to the antibiotics utilised, possibly due to antibiotic pressure in the environment. While the overuse and misuse of antibiotics in humans are undoubtedly a primary driving force for this severe problem, antibiotic-resistant genes have also been recognised to play an essential role in the emergence and spread of antibiotic resistance (Bengtsson-Palme et al., 2018). Efflux pumps, bypass of antibioticinhibited metabolic pathways, alteration of target sites, and, most critically, degradation of antibiotics by microbial enzymes have all been found to allow bacteria to survive antibiotic therapy and develop resistance (Munita & Arias, 2016).
Quinolone-resistant genes (qnrA, qnrB, and qnrS), tetracycline resistance genes (tetA and tetB), and ESBL genes (SHV, TEM) have all become a global public health concern (Sharma et al., 2016). In this investigation, the qnrA, qnrB, and tetA genes were found in 24 per cent, 16 per cent, and 10% of the isolates, respectively. The results were similar to the study by Hossain et al. (2018), who reported that qnrB (26.15%) and tetA (16.92%) were present in their study except for qnrA, which was absent. The absence of qnrA could result from the difference in geographical location. The slight variation in gene prevalence could also result from the difference in sample size. In this study, SHVresistant genes were detected in 40% of the isolates, similar to the study by Haller et al. (2018), with 41.1% occurrence. Extended-spectrum betalactamases (ESBLs) are enzymes widely employed by Gram-negative bacteria, particularly the Enterobacteriaceae, and are responsible for carbapenem resistance in most antibiotic classes (Coque et al., 2008;Pitout & Laupland, 2008;Abdelhady et al., 2016). Temoneira (TEM), sulfhydryl-variable (SHV), and cefotaximase (CTX-M) genes are found on mobile genetic elements (plasmids), which can be passed from one bacterial strain to another. Although over 150 ESBLs were detected worldwide, class A betalactamases have received the most attention (Rupp & Fey, 2003).
The high level of integron gene pollution is evident in this study and studies by Ghazalibina et al. (2019), McKinney et al. (2018 and Pormohammad et al. (2019), where significant levels of integrons were detected in clinical samples tallied with the high occurrence of Class 1 integron genes in this study. This has a significant health implication because it poses a threat to consumers of unsafe water as these genes can be transferred between organisms by horizontal gene transfer, thus increasing the risk of treatment failure in the event of infection. It is more likely that integrons Class 1 are frequently detected among clinical isolates than environmental isolates in Nigeria.

Conclusion and recommendation
The results revealed that the bacteria isolated were highly resistant to antibiotics due to prevailing associated antibiotic resistance determinants, which negatively affect treatment outcomes in the event of infection. Therefore, the urgent need for a safe water supply to various communities, awareness of the proper use of antibiotics and an antibiotic surveillance system is necessary to curtail the spread of antibiotic-resistant bacteria in our community.