# Occurrence of Antibiotics and Antibiotic-Resistant Bacteria in Landfill Sites in Kumasi, Ghana

Authors: Lawrence Sheringham Borquaye; Edmund Ekuadzi; Godfred Darko; Hubert Senanu Ahor; Sarah Twumwah Nsiah; Jemima Asi Lartey; Abdul-Hakim Mutala; Vivian Etsiapa Boamah; Eric Woode
Journal: Journal of Chemistry
 (2019)

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## Abstract

The incidence of antimicrobial resistance among microbial communities is a major threat to global health care and security. Landfills, which are reservoirs for many pharmaceuticals, provide a conducive habitat for antimicrobial-resistant microbes and resistant gene transfer and are therefore a major contributor to the phenomenon of antimicrobial resistance. Hence, this study determined the levels of three widely used antibiotics, metronidazole, penicillin, and amoxicillin, and the occurrence of antimicrobial resistance amongst microbes in soil and leachate samples from active and abandoned landfill sites in Kumasi, Ghana. Soil samples were collected from one active and four abandoned landfills, while leachate specimen was collected only from the active landfill. Sonication and solid-phase extraction (SPE) were used for sample preparation, followed by analysis via an HPLC-PDA method. Isolation and characterization of bacteria were done using standard bacteriological techniques. Antibiotic susceptibility testing was determined following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. Antibiotics were detected at very high concentrations in the specimen collected from both active and abandoned landfill sites. For leachate samples obtained from Dompoase, penicillin was present at the highest concentration (67.42 ± 5.35μg/mL, p<0.05) followed by metronidazole (18.25 ± 7.92 μg/mL) and amoxicillin (10.96 ± 6.93 μg/mL). In general, the levels of antibiotics in soil samples were similar at both active and abandoned landfill sites. Nonetheless, as with leachates, penicillin levels were much higher (p<0.05) than levels of amoxicillin and metronidazole within any particular site. When screened against some antibiotics, Enterobacteriaceae and some Bacillus and Listeria species isolated from the soil and leachate samples proved to be resistant. The high levels of antibiotics coupled with the presence of resistant microbes at these landfills sites call for immediate measures to halt the disposal of pharmaceuticals in the environment so as to avert any possible public health setback.


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## Body

## 1. Introduction

Rise in the incidence of antimicrobial resistance (AMR) poses a substantial threat to public health as it results in significant reduction in the efficacy of various antimicrobial treatment regimens, leading to increased morbidity and mortality as well as a general increase in health care expenditure [1]. The economic burden of AMR is, thus, enormous and could potentially cripple the economies of developing nations like Ghana. Various reports have described the presence of multidrug-resistant strains of numerous pathogenic bacteria worldwide [2, 3]. In Ghana, resistant strains of various pathogenic bacteria have been isolated from different sources [4–8]. Isolation of antimicrobial-resistant bacteria from soils and leachates from landfills remains unexplored in Ghana, even though a number of reports from elsewhere exist [9–11].Two factors that contribute immensely to the phenomenon of AMR are the antimicrobial agents themselves and the existence of mobile resistant genes in the environment. The antimicrobial agents act as the selective agent against the microorganism in question, whereas microorganisms use the resistant genes to evade the threats posed by the antimicrobial agents [12, 13]. Some resistance genes develop as a result of mutations in the microorganisms and selection pressure due to the use of antimicrobial agents. This selection pressure, provided by the antimicrobial agents, affords competitive advantage for strains with these resistance genes. In addition, the use of sub-optimal doses of antimicrobials facilitates stepwise selection of resistance. Resistance genes can be transferred from one microbe to another by the use of extrachromosomal elements such as plasmids, bacteriophages, or by the release of naked DNA from dead bacteria into the environment (transformation) [14]. This gene transfer can be intra- or interspecies [15]. The indiscriminate disposal of unused and expired medicines into the environment has the potential of enhancing the development of resistant genes by surviving microorganisms. The mobility of these resistant genes will potentially aggravate the existence of AMR.The availability of antimicrobial agents over the counter with little or no professional control and hence potential dispensary by persons with little or no knowledge of dosage regimen, the use of sub-potent drugs (either from poor manufacturing or counterfeiting), and the presence of pharmaceuticals in the environment due to agriculture use and/or improper disposal routes all contribute to increased cases of AMR [16–19].A recent report on the fate of unused and/or expired medicines in Ghana revealed that they are disposed of as municipal solid wastes (MSWs) [20]. When these MSWs are dumped into a landfill, microorganisms in the landfill are exposed to a plethora of medicines, including antimicrobials, either intact or as metabolites. Exposure of these microbes to sublethal concentrations of the antimicrobial agents may trigger events that will lead to the acquisition of resistance genes [21, 22]. Many studies in various parts of the world have documented the presence of antimicrobial resistance genes and/or antimicrobial-resistant bacteria from leachates and soils of landfills with some being identified as pathogenic strains [23–25].β-lactam antibiotics are one of the most consumed antibiotics globally [26]. Amoxicillin, penicillin, and metronidazole represent almost 75% of all used antibiotics in human medicine in the Greater Accra region of Ghana [27]. Amoxicillin and penicillin, which are β-lactam antibiotics, function by inhibiting the synthesis of peptidoglycan. The cell wall is weakened as a result, and this leads to inhibition of cell growth and ultimately, cell death. Metronidazole, belonging to the class of antibiotics called nitroimidazoles, causes cell death by interfering with DNA synthesis [26]. The β-lactams and metronidazole are among the top classes of antibiotics detected in high concentrations in the environment [28, 29].The presence of antimicrobial agents and antimicrobial-resistant bacteria in soil and leachates of landfills in Ghana is sparingly being reported [30]. The presence of pathogenic and opportunistic strains could pose sanitary risks for landfill workers, inhabitants of the areas, and veterinary animals that graze and drink in the vicinity of the landfills. Additionally, wild birds that thrive in these landfills could potentially disperse these microorganisms. Since the presence of antimicrobial agents could influence the presence of resistant microorganisms, the levels of antibiotic residues (amoxicillin, penicillin, and metronidazole) and antimicrobial resistance patterns in leachates and soils of 5 landfill sites in Kumasi, Ghana, were evaluated.


## 2. Materials and Methods

### 2.1. Reagents

Analytical-grade chemicals were used throughout the study. Antibiotic standards, amoxicillin, penicillin, and metronidazole, were obtained from Sigma-Aldrich (St. Louis, USA). Milli-Q water was used to prepare all the reagents and calibration standards. All solutions prepared for HPLC were passed through 0.2–0.6μm polypropylene filters before HPLC analysis.


### 2.2. Sample Collection and Handling

Soil samples were collected from 5 sites: 4 abandoned landfill sites and 1 active landfill site. Leachate samples were also collected from the active landfill site. All five locations were in the Ashanti Region of Ghana. The abandoned sites were Kronum (6°45′37.926″N, 1°37′56.958″W), Ohwim (6°45′12.0″N, 1°40′20.6″W), WAEC (6°41′688.0″N, 1°36′7.704″W), and Bohyen Kropo (6°41′03.5″N, 1°37′14.2″W). Dompoase (6°37′39.51″N, 1°35′35.286″W) was the active site. A map of the sampling sites is shown in Figure1. Fifty gram soil samples and 500 mL leachate samples were collected. Samples were immediately transported under cooled conditions to the laboratory and stored in a refrigerator at 4°C prior to analysis.Figure 1
Map of Kumasi metropolis showing sampling sites.


### 2.3. Extraction and Cleanup of Antibiotics from Leachates and Soil Samples

#### 2.3.1. Leachates

A 50 mL portion of leachate sample was mixed thoroughly by swirling vigorously for 5 times and centrifuged at 3000 rpm for 10 minutes. The supernatant was then filtered using Whatman filter paper, Grade 1. Ten millilitres of the filtrate was made up to 50 mL with distilled water and used for solid phase extraction (SPE).


#### 2.3.2. Soil

Ten millilitres each of 0.2 M citric buffer (pH 4.0) and acetonitrile was added to 2 g of soil sample in a centrifuge tube, sonicated in an ultrasonicator bath (VWR USC 600T, 45 kHz, 120 W) for 15 minutes, and centrifuged at 25°C. The supernatant was then transferred into a flask. The extraction protocol was performed 3 times on each soil sample, and obtained supernatants were combined and evaporated at 55°C to remove solvent before SPE cleanup.


### 2.4. SPE Cleanup Procedure

SPE columns (Oasis® HLB 6 cm3 200 mg, 30 μm cartridges) were conditioned using 5 mL methanol (MeOH) followed by 5 mL distilled water. The samples (10 mL for soil and 50 mL for leachate) were loaded onto the cartridges using a vacuum manifold and pump. The drop-rate was adjusted to 1.0 mL/min. Antibiotics were eluted from the cartridges with 5 mL MeOH after washing with 5 mL water. The eluent was evaporated to dryness over nitrogen gas. The samples were reconstituted in 200 μL MeOH and 1500 μL water (representing concentration factors of 29.4 and 5.9 for leachate and soil samples, respectively). Samples were then transferred to 2 mL HPLC vials for analysis.


### 2.5. Chromatographic Conditions

Chromatographic separation of antibiotics was performed on WatersμBondapak™ C-18 (300 mm × 3.9 mm, 5 μm) HPLC column. Analyses were performed at a flow rate of 2.0 ml/min at the ambient temperature. A gradient flow programming with binary pumps was used, containing solvent A (0.1% aqueous formic acid) and solvent B (MeOH). The details of the flow programme are given in Table 1. The injection volume was fixed to 100 μL by using the standard volume loop. All the compounds were eluted within 13 minutes; thus, the chromatographic run was programmed for 15 minutes.Table 1
HPLC flow programme used for antibiotic residue analysis.
Step
Step type
Step time (min)
Flow (mL/min)
% A
% B
0
Equilibrate
1.0
2
99.0
1.0
1
Run
4.0
2
99.0
1.0
2
Run
3.0
2
85.0
15.0
3
Run
5.0
2
20.0
80.0
4
Run
1.0
2
95.0
5.0
5
Run
2.0
2
99.0
1.0
Solvent A: 0.1% formic acid. Solvent B: methanol.The PDA detector was used for detection, and the chromatograms were extracted at 204 nm for amoxicillin and penicillin and 320 nm for metronidazole. The chromatographic and integrated data were recorded and processed using Chromera software. Quantification was performed using external calibration and peak area measurement.


### 2.6. HPLC-PDA Method Validation

Linearity for all test samples was tested in the concentration range of 6.25–200μg/mL (6.25, 12.50, 25, 50, 100, and 200 μg/mL). The limits of detection (LOD) and limits of quantification (LOQ) for each test sample were calculated with respect to signal-to-noise (S : N) ratios.


#### 2.6.1. Standard Calibration

Each antibiotic was identified by comparing the retention time of sample with that of the standard drug. Five concentrations (6.25–200μg/mL) of a mixed standard solution were prepared and injected. Following HPLC run, a standard curve of peak area versus concentration was plotted. Quantification of analytes in samples was done using the calibration curve.


#### 2.6.2. Recoveries and Quality Assurance

To estimate SPE recoveries, samples and blanks (water and soil matrices containing none of the analytes) were spiked with known concentrations of antibiotics. Following extraction and SPE clean up (as described earlier), antibiotic levels in samples were determined from the peak areas of the analytes on the chromatogram. To ensure that the HPLC system was functioning as expected, standards were injected prior to analyses and after every 5 sample runs. Analyses of blank samples were also done after every 5 runs to monitor sample interference. The sample injections were made in triplicates. Each batch of analyses was prepared to include reagent blank to check background contamination. Recovery data are provided in Table2.Table 2
A summary of retention time, method LOD, LOQ, LR, and SPE recoveries of antibiotics from leachate and soil samples.
Antibiotic
Retention time (min)
Linear range (μg/mL)
LOD (μg/mL)
LOQ (μg/mL)
SPE recovery efficiency (%) (leachate)
Amoxicillin
7.815
6.25–200
0.097
0.295
37.92 ± 1.591
Metronidazole
6.460
6.25–200
0.003
0.010
97.86 ± 10.886
Penicillin
12.094
6.25–200
1.565
4.740
92.96 ± 5.908
LR, linear range; LOD, limit of detection; LOQ, limit of quantitation; SPE, solid phase extraction.


### 2.7. Isolation of Microorganisms from Soil and Leachate Sample

Isolation of bacteria was done by the serial dilution techniques. In this technique, 1 g of the soil sample was weighed and diluted serially up to 10−10 with sterile distilled water. These dilutions were transferred into differently labeled sterile Petri dishes. Molten agar was added onto the Petri dishes and swirled evenly to ensure homogeneity of the mixture. The plates were incubated at 37°C for 24 hours. For the leachate sample, 1 mL of leachate was introduced into 9 ml of sterile distilled water and serially diluted, as with the soil samples.Distinct colonies of different morphologies (shape, margin, elevation, and opacity) were isolated and inoculated into 10 mL nutrient broth at 37°C for 24 hours. Broth cultures were streaked onto nutrient agar plates to confirm the morphologies earlier described and then isolated into new nutrient broth as pure cultures.


### 2.8. Identification of Isolates

#### 2.8.1. Gram Staining and Biochemical Tests

Gram staining was done to determine the cellular characteristics of isolates, i.e., the Gram reaction (Gram positive or negative) and shape of isolate (cocci or bacilli). The isolated microorganisms were heat-fixed to a slide by carefully passing the slide with a small inoculum of isolate through a Bunsen burner three times. Primary stain (crystal violet) was added to the slide and left for 1 minute. The slides were rinsed with a gentle stream of water to remove unbound crystal violet. Gram’s iodine was then added for 1 minute, rinsed with alcohol first, and then with a gentle stream of water. Secondary stain, safranin, was added to the slide, allowed to stand for a few minutes and then washed gently with a stream of water. Microscopic examination of the slides was done with a microscope using the oil immersion (×100) lens. Biochemical tests such as indole, hydrogen sulphide, catalase, citrate, methyl red, and sugar fermentation tests were also performed according to established procedures [31, 32].


### 2.9. Antibiotic Susceptibility Testing

Mueller–Hinton agar was prepared and poured into Petri dish. The plates were made to dry at 35°C. Using the direct colony suspension method, a suspension of the organisms was made in saline to a density of 0.5 McFarland turbidity standard. The suspensions were used within 15 minutes after preparation. A sterile cotton swab was dipped into the suspension (excess fluid was removed by pressing and turning the swab against the inside of the test tube) and used to spread the standardized inoculum evenly over the entire agar surface ensuring that there are no gaps between streaks. Using sterile forceps, commercially prepared antibiotic discs (gentamicin, ciprofloxacin, amikacin, ampicillin, amoxycillin, cefuroxime, erythromycin, tetracycline, and penicillin from Thermo Scientific, Oxoid Antimicrobial Susceptibility Discs) with specific concentrations (5–30μg) were placed on the inoculated agar surface. This was done within 15 minutes of inoculation.Flame-sterilized forceps were used to gently press the discs onto the surface of the agar. Within 15 minutes after the disk application, the plates were incubated for 18 hours at 37°C prior to determination of results. The zones of inhibition around each of the antibiotic disks were measured to the nearest millimeter. The zone diameter of each drug was interpreted using the criteria published by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [33].


## 2.1. Reagents

Analytical-grade chemicals were used throughout the study. Antibiotic standards, amoxicillin, penicillin, and metronidazole, were obtained from Sigma-Aldrich (St. Louis, USA). Milli-Q water was used to prepare all the reagents and calibration standards. All solutions prepared for HPLC were passed through 0.2–0.6μm polypropylene filters before HPLC analysis.


## 2.2. Sample Collection and Handling

Soil samples were collected from 5 sites: 4 abandoned landfill sites and 1 active landfill site. Leachate samples were also collected from the active landfill site. All five locations were in the Ashanti Region of Ghana. The abandoned sites were Kronum (6°45′37.926″N, 1°37′56.958″W), Ohwim (6°45′12.0″N, 1°40′20.6″W), WAEC (6°41′688.0″N, 1°36′7.704″W), and Bohyen Kropo (6°41′03.5″N, 1°37′14.2″W). Dompoase (6°37′39.51″N, 1°35′35.286″W) was the active site. A map of the sampling sites is shown in Figure1. Fifty gram soil samples and 500 mL leachate samples were collected. Samples were immediately transported under cooled conditions to the laboratory and stored in a refrigerator at 4°C prior to analysis.Figure 1
Map of Kumasi metropolis showing sampling sites.


## 2.3. Extraction and Cleanup of Antibiotics from Leachates and Soil Samples

### 2.3.1. Leachates

A 50 mL portion of leachate sample was mixed thoroughly by swirling vigorously for 5 times and centrifuged at 3000 rpm for 10 minutes. The supernatant was then filtered using Whatman filter paper, Grade 1. Ten millilitres of the filtrate was made up to 50 mL with distilled water and used for solid phase extraction (SPE).


### 2.3.2. Soil

Ten millilitres each of 0.2 M citric buffer (pH 4.0) and acetonitrile was added to 2 g of soil sample in a centrifuge tube, sonicated in an ultrasonicator bath (VWR USC 600T, 45 kHz, 120 W) for 15 minutes, and centrifuged at 25°C. The supernatant was then transferred into a flask. The extraction protocol was performed 3 times on each soil sample, and obtained supernatants were combined and evaporated at 55°C to remove solvent before SPE cleanup.


## 2.3.1. Leachates

A 50 mL portion of leachate sample was mixed thoroughly by swirling vigorously for 5 times and centrifuged at 3000 rpm for 10 minutes. The supernatant was then filtered using Whatman filter paper, Grade 1. Ten millilitres of the filtrate was made up to 50 mL with distilled water and used for solid phase extraction (SPE).


## 2.3.2. Soil

Ten millilitres each of 0.2 M citric buffer (pH 4.0) and acetonitrile was added to 2 g of soil sample in a centrifuge tube, sonicated in an ultrasonicator bath (VWR USC 600T, 45 kHz, 120 W) for 15 minutes, and centrifuged at 25°C. The supernatant was then transferred into a flask. The extraction protocol was performed 3 times on each soil sample, and obtained supernatants were combined and evaporated at 55°C to remove solvent before SPE cleanup.


## 2.4. SPE Cleanup Procedure

SPE columns (Oasis® HLB 6 cm3 200 mg, 30 μm cartridges) were conditioned using 5 mL methanol (MeOH) followed by 5 mL distilled water. The samples (10 mL for soil and 50 mL for leachate) were loaded onto the cartridges using a vacuum manifold and pump. The drop-rate was adjusted to 1.0 mL/min. Antibiotics were eluted from the cartridges with 5 mL MeOH after washing with 5 mL water. The eluent was evaporated to dryness over nitrogen gas. The samples were reconstituted in 200 μL MeOH and 1500 μL water (representing concentration factors of 29.4 and 5.9 for leachate and soil samples, respectively). Samples were then transferred to 2 mL HPLC vials for analysis.


## 2.5. Chromatographic Conditions

Chromatographic separation of antibiotics was performed on WatersμBondapak™ C-18 (300 mm × 3.9 mm, 5 μm) HPLC column. Analyses were performed at a flow rate of 2.0 ml/min at the ambient temperature. A gradient flow programming with binary pumps was used, containing solvent A (0.1% aqueous formic acid) and solvent B (MeOH). The details of the flow programme are given in Table 1. The injection volume was fixed to 100 μL by using the standard volume loop. All the compounds were eluted within 13 minutes; thus, the chromatographic run was programmed for 15 minutes.Table 1
HPLC flow programme used for antibiotic residue analysis.
Step
Step type
Step time (min)
Flow (mL/min)
% A
% B
0
Equilibrate
1.0
2
99.0
1.0
1
Run
4.0
2
99.0
1.0
2
Run
3.0
2
85.0
15.0
3
Run
5.0
2
20.0
80.0
4
Run
1.0
2
95.0
5.0
5
Run
2.0
2
99.0
1.0
Solvent A: 0.1% formic acid. Solvent B: methanol.The PDA detector was used for detection, and the chromatograms were extracted at 204 nm for amoxicillin and penicillin and 320 nm for metronidazole. The chromatographic and integrated data were recorded and processed using Chromera software. Quantification was performed using external calibration and peak area measurement.


## 2.6. HPLC-PDA Method Validation

Linearity for all test samples was tested in the concentration range of 6.25–200μg/mL (6.25, 12.50, 25, 50, 100, and 200 μg/mL). The limits of detection (LOD) and limits of quantification (LOQ) for each test sample were calculated with respect to signal-to-noise (S : N) ratios.


### 2.6.1. Standard Calibration

Each antibiotic was identified by comparing the retention time of sample with that of the standard drug. Five concentrations (6.25–200μg/mL) of a mixed standard solution were prepared and injected. Following HPLC run, a standard curve of peak area versus concentration was plotted. Quantification of analytes in samples was done using the calibration curve.


### 2.6.2. Recoveries and Quality Assurance

To estimate SPE recoveries, samples and blanks (water and soil matrices containing none of the analytes) were spiked with known concentrations of antibiotics. Following extraction and SPE clean up (as described earlier), antibiotic levels in samples were determined from the peak areas of the analytes on the chromatogram. To ensure that the HPLC system was functioning as expected, standards were injected prior to analyses and after every 5 sample runs. Analyses of blank samples were also done after every 5 runs to monitor sample interference. The sample injections were made in triplicates. Each batch of analyses was prepared to include reagent blank to check background contamination. Recovery data are provided in Table2.Table 2
A summary of retention time, method LOD, LOQ, LR, and SPE recoveries of antibiotics from leachate and soil samples.
Antibiotic
Retention time (min)
Linear range (μg/mL)
LOD (μg/mL)
LOQ (μg/mL)
SPE recovery efficiency (%) (leachate)
Amoxicillin
7.815
6.25–200
0.097
0.295
37.92 ± 1.591
Metronidazole
6.460
6.25–200
0.003
0.010
97.86 ± 10.886
Penicillin
12.094
6.25–200
1.565
4.740
92.96 ± 5.908
LR, linear range; LOD, limit of detection; LOQ, limit of quantitation; SPE, solid phase extraction.


## 2.6.1. Standard Calibration

Each antibiotic was identified by comparing the retention time of sample with that of the standard drug. Five concentrations (6.25–200μg/mL) of a mixed standard solution were prepared and injected. Following HPLC run, a standard curve of peak area versus concentration was plotted. Quantification of analytes in samples was done using the calibration curve.


## 2.6.2. Recoveries and Quality Assurance

To estimate SPE recoveries, samples and blanks (water and soil matrices containing none of the analytes) were spiked with known concentrations of antibiotics. Following extraction and SPE clean up (as described earlier), antibiotic levels in samples were determined from the peak areas of the analytes on the chromatogram. To ensure that the HPLC system was functioning as expected, standards were injected prior to analyses and after every 5 sample runs. Analyses of blank samples were also done after every 5 runs to monitor sample interference. The sample injections were made in triplicates. Each batch of analyses was prepared to include reagent blank to check background contamination. Recovery data are provided in Table2.Table 2
A summary of retention time, method LOD, LOQ, LR, and SPE recoveries of antibiotics from leachate and soil samples.
Antibiotic
Retention time (min)
Linear range (μg/mL)
LOD (μg/mL)
LOQ (μg/mL)
SPE recovery efficiency (%) (leachate)
Amoxicillin
7.815
6.25–200
0.097
0.295
37.92 ± 1.591
Metronidazole
6.460
6.25–200
0.003
0.010
97.86 ± 10.886
Penicillin
12.094
6.25–200
1.565
4.740
92.96 ± 5.908
LR, linear range; LOD, limit of detection; LOQ, limit of quantitation; SPE, solid phase extraction.


## 2.7. Isolation of Microorganisms from Soil and Leachate Sample

Isolation of bacteria was done by the serial dilution techniques. In this technique, 1 g of the soil sample was weighed and diluted serially up to 10−10 with sterile distilled water. These dilutions were transferred into differently labeled sterile Petri dishes. Molten agar was added onto the Petri dishes and swirled evenly to ensure homogeneity of the mixture. The plates were incubated at 37°C for 24 hours. For the leachate sample, 1 mL of leachate was introduced into 9 ml of sterile distilled water and serially diluted, as with the soil samples.Distinct colonies of different morphologies (shape, margin, elevation, and opacity) were isolated and inoculated into 10 mL nutrient broth at 37°C for 24 hours. Broth cultures were streaked onto nutrient agar plates to confirm the morphologies earlier described and then isolated into new nutrient broth as pure cultures.


## 2.8. Identification of Isolates

### 2.8.1. Gram Staining and Biochemical Tests

Gram staining was done to determine the cellular characteristics of isolates, i.e., the Gram reaction (Gram positive or negative) and shape of isolate (cocci or bacilli). The isolated microorganisms were heat-fixed to a slide by carefully passing the slide with a small inoculum of isolate through a Bunsen burner three times. Primary stain (crystal violet) was added to the slide and left for 1 minute. The slides were rinsed with a gentle stream of water to remove unbound crystal violet. Gram’s iodine was then added for 1 minute, rinsed with alcohol first, and then with a gentle stream of water. Secondary stain, safranin, was added to the slide, allowed to stand for a few minutes and then washed gently with a stream of water. Microscopic examination of the slides was done with a microscope using the oil immersion (×100) lens. Biochemical tests such as indole, hydrogen sulphide, catalase, citrate, methyl red, and sugar fermentation tests were also performed according to established procedures [31, 32].


## 2.8.1. Gram Staining and Biochemical Tests

Gram staining was done to determine the cellular characteristics of isolates, i.e., the Gram reaction (Gram positive or negative) and shape of isolate (cocci or bacilli). The isolated microorganisms were heat-fixed to a slide by carefully passing the slide with a small inoculum of isolate through a Bunsen burner three times. Primary stain (crystal violet) was added to the slide and left for 1 minute. The slides were rinsed with a gentle stream of water to remove unbound crystal violet. Gram’s iodine was then added for 1 minute, rinsed with alcohol first, and then with a gentle stream of water. Secondary stain, safranin, was added to the slide, allowed to stand for a few minutes and then washed gently with a stream of water. Microscopic examination of the slides was done with a microscope using the oil immersion (×100) lens. Biochemical tests such as indole, hydrogen sulphide, catalase, citrate, methyl red, and sugar fermentation tests were also performed according to established procedures [31, 32].


## 2.9. Antibiotic Susceptibility Testing

Mueller–Hinton agar was prepared and poured into Petri dish. The plates were made to dry at 35°C. Using the direct colony suspension method, a suspension of the organisms was made in saline to a density of 0.5 McFarland turbidity standard. The suspensions were used within 15 minutes after preparation. A sterile cotton swab was dipped into the suspension (excess fluid was removed by pressing and turning the swab against the inside of the test tube) and used to spread the standardized inoculum evenly over the entire agar surface ensuring that there are no gaps between streaks. Using sterile forceps, commercially prepared antibiotic discs (gentamicin, ciprofloxacin, amikacin, ampicillin, amoxycillin, cefuroxime, erythromycin, tetracycline, and penicillin from Thermo Scientific, Oxoid Antimicrobial Susceptibility Discs) with specific concentrations (5–30μg) were placed on the inoculated agar surface. This was done within 15 minutes of inoculation.Flame-sterilized forceps were used to gently press the discs onto the surface of the agar. Within 15 minutes after the disk application, the plates were incubated for 18 hours at 37°C prior to determination of results. The zones of inhibition around each of the antibiotic disks were measured to the nearest millimeter. The zone diameter of each drug was interpreted using the criteria published by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [33].


## 3. Results

Linearity was tested in the concentration range of 6.25–200μg/mL (6.25, 12.50, 25, 50, 100, and 200 μg/mL). All the compounds showed good correlation with coefficients, R2, between 0.96 and 0.99. The limits of detection (LOD) and limits of quantification (LOQ) calculated with respect to signal to noise (S : N) ratios were within the ranges of 0.003–1.565 and 0.010–4.740 μg/mL, respectively, for all antibiotics. The recoveries for metronidazole and penicillin in leachate samples were high (>90%), with amoxicillin recovery lower (∼38%). No correction factor was applied in the quantitative analysis. All peaks eluted within 13 minutes, with metronidazole eluting first (6.460 minutes) followed by amoxicillin (7.815 minutes) and penicillin (12.094 minutes). The results of the analytical method development are shown in Table 2.Leachate samples were obtained from Dompoase only, since that was the only active landfill site amongst those sampled. Of the antibiotics analysed, penicillin was the most prevalent, with a mean level of 67.42 ± 5.35μg/mL. No significant difference existed between the levels of amoxicillin and metronidazole in the leachate sample. Penicillin levels were uniform in all leachate samples (upstream, downstream, or midstream), but those of amoxicillin and metronidazole varied. Amoxicillin levels decreased as one moves from upstream to downstream sections of the stream (Table 3).Table 3
Levels of antibiotics in leachates at different sections of the flow from Dompoase.
Section
Amoxicillin (μg/mL)
Metronidazole (μg/mL)
Penicillin (μg/mL)
Upstream
16.46 ± 7.41a∗
9.13 ± 3.24a∗
66.60 ± 9.9a∗∗
Midstream
10.19 ± 4.08ab∗
26.02 ± 2.41b∗∗
66.83 ± 2.27a∗∗∗
Downstream
3.88 ± 0.68b∗
19.59 ± 4.05ab∗
68.82 ± 2.65a∗∗
Mean levels
10.96 ± 6.93a
18.25 ± 7.92a
67.42 ± 5.35c
Different superscripts (letters) and different number of asterisks in a column and row, respectively, indicate significant difference (p<0.05) in concentrations.For soil samples, all three antibiotics were detected in samples from all five locations sampled. In general, penicillin levels were much higher than levels of amoxicillin and metronidazole. Kronum had the highest levels of penicillin (120.52 ± 22.33μg/g) and amoxicillin (76.62 ± 15.3 μg/g), while WAEC had the lowest levels of all antibiotics analysed (8.88 ± 1.19, 3.44 ± 2.78 and 70.36 ± 9.02 μg/g for amoxicillin, metronidazole, and penicillin, respectively). The levels of the various antibiotics in soil samples from Dompoase, Kronum, Bohyen Kropo, and Ohwin were similar. Soil samples from WAEC, however, had markedly different concentrations of antibiotics relative to the other four locations (Table 4).Table 4
Levels of antibiotics in soil samples.
Sites
Amoxicillin (μg/g)
Metronidazole (μg/g)
Penicillin (μg/g)
Dompoase
43.24 ± 28.93a
43.47 ± 41.59a
114.85 ± 19.91b
Kronum
76.62 ± 15.3ab
44.87 ± 35.86a
120.52 ± 22.33b
Bohyen Kropo
62.45 ± 20.89ac
43.76 ± 13.95a
119.08 ± 27.10c
Ohwim
56.64 ± 47.66ac
50.08 ± 48.34a
111.17 ± 28.94c
WAEC
8.88 ± 1.19a
3.44 ± 2.78a
70.36 ± 9.02b
Different superscripts (letters) in a row indicate significant difference (p<0.05) in concentrations. There was no significant difference among antibiotic levels in one town compared to the other (i.e., same column).In all, 25 pure cultures were isolated from soil (all sample locations) and leachates (Dompoase only) samples. Distinct colonies were isolated based on colour, shape, margin, elevation, and opacity (Table5). From the Gram staining reaction, 13 of the isolates were Gram positive while the remaining 12 were Gram negative. All isolates were rod-like in shape. Based on a series of biochemical tests, isolates were identified to belong to one of 9 genera: Bacillus, Citrobacter, Pseudomonas, Klebsiella, Enterobacter, Proteus, Aeromonas, Listeria, and Shigella.Table 5
Colony morphology, cellular characteristics of isolates, biochemical test, and organisms identified from soil and leachates.
Isolates
Colony morphology
Cellular characteristics
Biochemical test
Microorganism
Sugar fermentation
Colour
Shape
Margin
Elevation
Opacity
Gram reaction
Shape
Catalase
Indole
Starch
Citrate
Sucrose
Lactose
Glucose
Methyl red
H2S
DL1
Grey
Irregular
Undulate
Flat
Opaque
Positive
Rod
+
−
+
+
+
+
+
−
−
Bacillus sp.
DL3
Grey and shiny surface
Circular
Entire
Raised
Translucent
Negative
Rod
−
−
−
+
+
+G
+G
+
−
Citrobacter sp.
DO1
Grey
Irregular
Undulate
Flat
Opaque
Positive
Rod
+
−
+
+
+
+
+
+
−
Bacillus sp.
DO3
Grey
Irregular
Undulate
Flat
Opaque
Positive
Rod
+
−
+
+
+
+
+
−
−
Bacillus sp.
DO4
White
Irregular
Undulate
Flat
Opaque
Positive
Rod
+
−
+
+
+
+
+
−
−
Bacillus sp.
DO5
Cream
Circular
Entire
Raised
Translucent
Negative
Rod
+
−
−
+
−
−
+
+
−
Pseudomonas sp.
DO6
White
Circular
Undulate
Unbonate
Translucent
Negative
Rod
+
−
−
+
+
+
+G
−
−
Klebsiella pneumoniae
DO8
Cream
Circular
Entire
Raised
Translucent
Negative
Rod
+
−
−
+
−
−
+
+
−
Pseudomonas sp.
BK1
Grey
Irregular
Undulate
Flat
Opaque
Positive
Rod
+
−
+
+
+
+
+
−
−
Bacillus sp.
BK3
Grey
Irregular
Undulate
Flat
Opaque
Positive
Rod
+
−
+
+
+
+
+
+
−
Bacillus sp.
BK4
White
Circular
Undulate
Unbonate
Translucent
Negative
Rod
+
−
−
+
+
+
+G
−
−
Klebsiella pneumoniae
BK5
Grey
Irregular
Undulate
Flat
Opaque
Positive
Rod
+
−
+
+
+
+
+
−
−
Bacillus sp.
BK6
Cream
Circular
Entire
Convex
Opaque
Negative
Rod
+
−
−
+
−
+G
+G
−
−
Enterobacter sp.
BK7
Grey
Irregular
Undulate
Flat
Opaque
Positive
Rod
+
−
+
+
+
−
+
−
−
Bacillus sp.
K1
Grey and shiny surface
Circular
Entire
Raised
Translucent
Negative
Rod
−
−
−
+
+
+G
+G
+
−
Citrobacter sp.
K2
White
Circular
Undulate
Unbonate
Translucent
Positive
Rod
+
−
+
+
+
−
+
−
−
Bacillus sp.
K3
Tan
Circular
Entire
Convex
Opaque
Negative
Rod
+
+
−
+
−
+G
+G
−
+
Proteus vulgaris
K4
Grey
Circular
Entire
Raised
Opaque
Negative
Rod
+
+
+
+
+
+G
+G
+
+
Aeromonas hydrophilia
W1
Grey
Irregular
Undulate
Flat
Opaque
Positive
Rod
+
−
+
+
+
+
+
−
−
Bacillus sp.
W2
White
Circular
Undulate
Unbonate
Translucent
Positive
Rod
+
−
+
+
+
+
+
−
−
Bacillus sp.
W3
Grey
Circular
Entire
Flat
Translucent
Positive
Rod
+
−
+
+
+
+
+
+
+
Listeria sp.
W5
Cream
Circular
Entire
Raised
Translucent
Negative
Rod
+
−
−
+
−
−
+
+
−
Pseudomonas sp.
OH2
Grey
Irregular
Undulate
Flat
Opaque
Positive
Rod
+
−
+
+
+
+
+
+
−
Bacillus sp.
OH3
Grey
Circular
Entire
Convex
Translucent
Negative
Rod
+
−
−
−
−
−
+
+
−
Shigella sp.
OH5
Grey
Circular
Entire
Convex
Translucent
Negative
Rod
+
−
−
−
−
+
+
+
−
Shigella sp.
∗DL, Dompoase leachate; DO, Dompoase; BK, Bohyen Kropo; KR, Kronum; WA, WAEC; OH, Ohwim. Positive acid production (+); negative acid production (−); positive acid and gas production (+G).The results of the susceptibility tests are shown in Table6. Antibiotic susceptibility testing (AST) was based on guidelines of EUCAST. Enterobacteriaceae (Klebsiella, Enterobacter, Citrobacter, Proteus, and Shigella) isolates were resistant to ampicillin, amoxicillin, and cefuroxime but susceptible to gentamicin, ciprofloxacin, and amikacin. Pseudomonas isolates were all susceptible to all antibiotics tested (gentamicin, ciprofloxacin, and amikacin). Listeria isolates demonstrated resistance to ampicillin and penicillin but susceptibility towards erythromycin, whereas Aeromonas spp. was susceptible to both amikacin and gentamicin. For bacilli isolates, complete susceptibility was observed towards gentamicin, erythromycin, and ciprofloxacin. All Bacillus spp. were also susceptible towards tetracycline except 3 isolates which were classified as intermediate. Again, all but 2 Bacillus spp. isolates exhibited resistance to ampicillin.Table 6
Zones of inhibition (mm) of antibiotics against isolates and corresponding susceptibility and resistance of isolates to antibiotic.
Isolate
Organism
Zone diameter (mm)
CN
CIP
AK
AMP
AML
CXM
E
TE
P
BK4
Klebsiella pneumoniae
S(24)
S(34)
S(26)
R(0)
R(0)
R(0)
−
−
−
DO6
Klebsiella pneumoniae
S(24)
S(31)
S(25)
R(0)
R(0)
R(0)
−
−
−
BK6
Enterobacter spp.
S(25)
S(32)
S(26)
R(0)
R(0)
R(0)
−
−
−
DL3
Citrobacter spp.
S(24)
S(30)
S(23)
R(0)
R(0)
R(0)
−
−
−
K 1
Citrobacter spp.
S(24)
S(36)
S(23)
R(0)
R(0)
R(0)
−
−
−
K 3
Proteus vulgaris
S(26)
S(30)
S(29)
R(0)
R(0)
R(0)
−
−
−
OH5
Shigella spp.
S(19)
S(30)
S(24)
R(0)
R(0)
R(0)
−
−
−
OH3
Shigella spp.
S(27)
S(37)
S(24)
R(0)
R(0)
R(0)
−
−
−
WA5
Pseudomonas spp.
S(25)
S(36)
S(27)
−
−
−
−
−
−
DO5
Pseudomonas spp.
S(24)
S(32)
S(28)
−
−
−
−
−
−
DO8
Pseudomonas spp.
S(27)
S(39)
S(32)
−
−
−
−
−
−
WA1
Bacillus spp.
S(25)
S(30)
−
R(0)
−
−
S(30)
S(28)
−
WA2
Bacillus spp.
S(24)
S(31)
−
R(0)
−
−
S(25)
S(28)
−
K2
Bacillus spp.
S(25)
S(32)
−
R(0)
−
−
S(27)
S(30)
−
DL1
Bacillus spp.
S(24)
S(32)
−
R(0)
−
−
S(27)
I(21)
−
DO1
Bacillus spp.
S(28)
S(27)
−
R(13)
−
−
S(27)
S(31)
−
D03
Bacillus spp.
S(23)
S(38)
−
S(19)
−
−
S(25)
S(39)
−
D04
Bacillus spp.
S(25)
S(38)
−
R(0)
−
−
S(26)
S(25)
−
BK1
Bacillus spp.
S(22)
S(30)
−
S(27)
−
−
S(27)
I(21)
−
BK3
Bacillus spp.
S(23)
S(30)
−
R(0)
−
−
S(25)
S(26)
−
BK5
Bacillus spp.
S(22)
S(30)
−
R(0)
−
−
S(27)
I(21)
−
OH2
Bacillus spp.
S(23)
S(37)
−
R(0)
−
−
S(26)
S(28)
−
BK7
Bacillus spp.
S(23)
S(31)
−
R(0)
−
−
S(27)
S(27)
−
K4
Aeromonas hydrophilia
S(26)
S(27)
−
−
−
−
−
−
−
WA3
Listeria spp.
−
−
−
R(0)
−
−
S(28)
−
R(0)
CN: gentamicin; CIP: ciprofloxacin; AK: amikacin; AMP: ampicillin; AML: amoxycillin, CXM: cefuroxime: E: erythromycin; TE: tetracycline; P: penicillin. S = susceptible, I = intermediate, and R = resistant.


## 4. Discussions

In this study, the levels of twoβ-lactams (penicillin and amoxicillin) and a fluoroquinolone (metronidazole) were evaluated from soil and leachate samples collected from active and abandoned landfill sites in Kumasi, Ghana. Additionally, the occurrence of antimicrobial resistance amongst microbes isolated from the soil and leachate samples was also studied. The antibiotics examined in this study are among the most consumed antibiotics globally [26]. Amongst the landfill sites, Dompoase landfill is currently the only active site in Kumasi. The other landfill sites at Kronum, Bohyen Kropo, Ohwim, and WAEC have been covered, and the sites are used for residential, agricultural, and recreational purposes.The mean levels of antibiotics recorded in leachate samples from Dompoase were in the following order: penicillin (67.42 ± 5.35μg/mL) > metronidazole (18.25 ± 7.92 μg/mL) > amoxicillin (10.96 ± 6.93 μg/mL). For soil samples, penicillin levels were the highest amongst all antibiotics studied at all 5 locations. In general, the levels of antibiotics detected were much higher than those detected in other studies. Whereas Wu [34] and Yu [9] reported levels up to 10 μg/L, Sui and coworkers [35] also detected the presence of antibiotics and other pharmaceuticals in landfill leachates with concentrations as high as 0.3 mg/L. The levels detected in this study are much higher and suggest an indiscriminate disposal culture for these pharmaceuticals. The high levels of antibiotics recorded in both soil and leachate samples could have serious implications for the environment. Bengtsson-Palme and Larsson [36] have estimated the concentrations of antibiotics in environmental matrices that might exert selection for resistant bacteria. They concluded that for most antibiotics, environmental concentrations beyond 64 μg/L posed considerable risk for resistance selection. The levels recorded in this work are about a thousand fold higher than the limit concentration for resistance selection and could potentially be harmful to the environment. The high levels of metronidazole, amoxicillin, and penicillin at these sites can create a selective pressure which might trigger the proliferation of antibiotics resistance genes resulting in the development or acquisition of resistant genes among microbes in the absence/presence of other resistant selective agents such as heavy metals, disinfectants, and detergents [10, 34]. Increasing concentration of antibiotics at landfills has also been directly correlated to the abundance of antibiotic-resistant genes (ARGs) which are transmitted to microbes [9, 10]. Additionally, these pharmaceuticals and their metabolites could cause chronic and acute ecotoxicity on humans, wildlife, and microorganisms as well.The levels of antibiotics found in soil samples at all five landfill sites were comparable to each other, indicating that similar events probably contributed to the levels of antibiotics at these sites. Studies to collate landfill age with levels of antibiotics have been contradictory at best. In a study by Wu and coworkers [11], antibiotic concentration at landfill sites was observed to reduce with landfill age, even though ARGs increased. On the contrary, levels of antibiotics and ARGs have also been seen to increase with the age of landfills [9]. The high level of antibiotics at the landfill sites probably reflects the extent to which residents and industries dispose of pharmaceuticals via landfills. The high antibiotic levels in the abandoned landfills probably indicate that these landfills have received huge amounts of expired and/or unused antibiotics during their active years and these antibiotics may still be undergoing degradation [37].From soil and water samples, 25 pure isolates were obtained. These bacteria comprised of 13 Gram-positive and 12 Gram-negative bacteria. Bacteria belonging to the generaBacillus, Citrobacter, Pseudomonas, Klebsiella, Enterobacter, Proteus, Aeromonas, Listeria, and Shigella were amongst those isolated. Various works have also reported the isolation of bacteria belonging to these genera from different parts of the world. Flores-Tena and coworkers isolated a number of pathogenic and opportunistic bacteria from soils and leachates of a landfill site in Mexico and pointed out that the presence of these bacteria posed a significant risk for public and occupational health [24]. Most of the isolated bacteria were enteric. In a similar work carried out in Accra, Ghana, bacteria isolated included Escherichia coli, Salmonella spp., Vibro spp., and Bacillus spp. [30].Of the bacteria isolated from the sampling sites,Bacillus was the predominant genus. The enterobacteriaceae proved to be resistant to β-lactam antibiotics (ampicillin, amoxicillin, and cefuroxime), whereas resistance to ampicillin was evident in Bacillus and Listeria species. Even though most Bacillus strains are not pathogenic to humans, constant exposure to these microbes harboring resistant genes could be a recipe for disaster. They could transfer resistant genes to other pathogenic microbes and cause havoc as a result. Antimicrobial resistance by enteric microbes especially is a major public health concern globally [17]. Enterobacter organisms are responsible for numerous nosocomial infections that require prolonged hospitalization, multiple and sophisticated laboratory tests, and sometimes surgical interventions and expensive antimicrobial agents. The presence of these resistant strains in soil and leachates samples of abandoned and active landfills raises a concern that require immediate attention.Many studies have recognized the importance of the landfills as an important source of mobile genetic elements, antibiotic-resistant genes, and resistant microbes [9–11, 23, 34]. Resistant genes in bacteria could be intrinsic or could be as a result of selective pressure imposed by a particular antibiotic on the population. The high levels of the antibiotics in the study could therefore play a crucial role in the development of resistance by resident microbes and possibly in its dissemination to other microbes [9, 10, 34]. As a result, it was unsurprising that some bacteria isolated from the study samples proved to be resistant to some of the antibiotics considered in this study.There are no stringent regulations regarding the purchase of antibiotics in Ghana. As such, one can easily acquire these antibiotics over the counter. The high level of antibiotics, particularly penicillin, in all samples (both soil and leachate) is probably a reflection of the ease with which they can be purchased and the methods used for their disposal as well the frequency of their use. The disposal of unused and expired medicines is the sole responsibility of the Food and Drugs Authority (FDA) in Ghana. To access this facility, individuals or organizations apply to the FDA and pay the associated cost before making arrangement for transfer to the FDA for disposal. Because of the bottlenecks associated with this process, many pharmacies and households dispose off unused and expired medicines into dustbins, which ultimately find themselves into landfills [20].In a recent survey study carried out on the disposal practices of pharmaceuticals in Kumasi, about 80% of respondents confirmed that they disposed off used and unused medications alongside household waste. Similarly, 60% of community pharmacies and over-the-counter (OTC) medicine sellers also confirmed that unused and expired medications were routinely disposed of in a manner similar to those of household waste [20]. Whether the disposal practice is as a result of unawareness, cost, or sheer disregard of the environment, it is imperative that urgent steps are taken to reverse the trend.It is important that city authorities equip workers at the landfill sites with appropriate personal protective equipment and ensure that these are used at all times and in the correct manner. The populace that resides on lands formerly used as landfills, and in such vicinity, needs to be educated on the need to maintain high hygienic standards. Regular monitoring of such areas by city health officials needs to be in place. Finally, there is a need for an educational campaign on the disposal routes of antibiotics and other pharmaceuticals available to both households and pharmacies. Bottlenecks that are hampering the use of proper disposal routes need to be identified and removed to forestall the occurrence of a major crisis and protect the environment.


## 5. Conclusions

The levels of antibiotics (penicillin, amoxicillin, and metronidazole) present in soil and leachate samples from landfill sites in Kumasi were generally high. Penicillin levels were much higher in both soil and leachate samples in comparison to the other antibiotics studied. Bacteria isolated from the study sites proved to be resistant to some antibiotics, especially ampicillin. The irrational disposal of medicines, either used or expired, by both household and pharmacies needs to be regulated to avert any possible public health disaster.



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*Source: 6934507-2019-07-04.xml*