Antibiotic Resistance of Bacteria Isolated from Water Supplies Used in Poultry Production in Ashanti Region of Ghana

In this study, the resistance pattern of bacterial isolates from drinking water used in poultry production in the Ashanti region of Ghana from our previously published report was determined. The presence of Escherichia coli, Salmonella typhi, Staphylococcus aureus and coagulase-negative Staphylococci was determined using selective culture media (pour plate method) and confirmed through Gram staining and biochemical reactions. Antibiotic sensitivity of isolates was determined followed by detection of Extended-Spectrum Beta-Lactamase (ESBL) producing Gram-negative isolates.


INTRODUCTION
Water is one of the most important and abundant resources on earth which may either be safe or unsafe because it can potentially harbour various infective agents [1]. It has been estimated that about 1.1 billion people in the world are unable to access safe drinking water [2]. A report by Aning [3] indicated that the poultry industry in Ghana is quite extensive with many Ghanaians producing poultry and poultry products at commercial levels. Majority of poultry farms in Ghana are distributed within the Ashanti, Brong-Ahafo and the Greater Accra regions of the country. Most of these poultry farms rely on groundwater (boreholes and wells) as their main source of water supply for the poultry birds and other poultry processing purposes on the farms [4]. Water also serves as a medium for numerous microorganisms, most of which are disease-causing [5]. The use of contaminated water in poultry farming processes may serve as a source of infection in the birds.
Infections in poultry can lead to low egg production, loss of weight in poultry and death of poultry [6], and some of these infectious agents can also be transmitted from farm animals to farm workers [7]. According to Puzelli et al. [8], viral particles of avian H7 influenza viruses were identified in the sera of farm workers exposed to poultry birds infected with the viral particles in Italy. One way of preventing such occupational exposure of farm workers and others to zoonotic diseases is to control and prevent the diseases in the poultry [9]. Identification of the possible sources of infections will help to devise measures to control their transmission. Possible sources of infections in the poultry may include farm workers, water used to feed the birds, poultry feed, feeding equipment and poultry litter. Most of these infectious agents have been identified in the litter of poultry birds [4].
Ashanti region is one of the three regions in Ghana with the highest number of poultry farms, of which many farms rely on groundwater as their source of water [4]. Various pathogenic microbial organisms have been identified in groundwater (boreholes and wells) used as drinking water in various communities in the Ashanti region of Ghana [10]. Microbial assessment of water used in these farms will help control the rate of infections in the poultry birds. In our previous report, we identified bacterial isolates including Escherichia coli, Salmonella typhi, Staphylococcus aureus and coagulasenegative Staphylococci in various sources of water used in some selected poultry farms in the Ashanti region of Ghana [11]. The emergence of microbial resistance to various antibiotics calls for prompt actions to determine the susceptibility of microbial agents found in the water used in the various poultry farms and possibly determine the mechanism of resistance in order to control the infections likely to be caused by these infectious agents to poultry birds as well as poultry farmers. In this study, the resistance pattern of the isolated bacterial strains including E. coli, S. typhi, S. aureus and coagulase-negative staphylococci isolates from drinking water samples used for poultry production in the Ashanti region of Ghana from our previous study [11] and the detection of Extended-Spectrum Beta-Lactamase (ESBL) producing organisms among the resistant bacterial isolates were deter-mined.

MATERIALS AND METHODS
All the chemicals and reagents were purchased from Sigma-Aldrich, London, UK, unless otherwise stated.

Selection of Farms
Purposive sampling technique was employed in the selection of the 100 poultry farms out of the total estimated number of 820 poultry farms in the Ashanti region of Ghana. Farms with 500 birds or more were used for the study.

Sampling of Water from Poultry Farms
Samples of water were picked according to the guidelines from WHO [12] and transported to the laboratory on ice.

Isolation and Identification of Bacterial Isolates from Water Samples
The isolation and identification of the various bacterial isolates from the samples collected were performed according to the methods described by Osei et al. [11]. One millilitre (1 mL) of water sample was aseptically inoculated into 10 mL freshly prepared nutrient broth (Thermo-Fisher, London, UK) and incubated at 37˚C for 24 h. Isolates of Escherichia coli were obtained by inoculating 1 mL of the 24 h sample broth culture into 20 mL freshly prepared sterile MacConkey agar (Thermo-Fisher, London, UK) and confirmed through Gramstaining and biochemical tests including catalase activity in 3% hydrogen peroxide, indole production test in tryptone water, citrate utilization test and Methyl Red-Voges Proskauer (MRVP) test [13]. Isolates of Salmonella typhi were obtained by inoculating 1 mL of the 24 h sample broth culture into 20 mL freshly prepared sterile bismuth sulphite agar and confirmed through Gram-staining and biochemical tests including hydrogen-sulphide production test in peptone water, indole production test in tryptone water, citrate utilization test and catalase activity in 3% hydrogen peroxide solution [13].
Isolates of Staphylococcus aureus were obtained by inoculating 1 mL of the 24 h sample broth culture into 20 mL freshly prepared sterile mannitol salt agar and confirmed through Gram-staining and biochemical tests inclu-ding catalase activity in 3% hydrogen peroxide solution, coagulase test and haemolysis test on blood agar [13]. Isolates of coagulasenegative Staphylococci were obtained by inoculating 1 mL of the 24 h sample broth culture into 20 mL freshly prepared sterile mannitol salt agar (Thermo-Fisher, London, UK) and confirmed through Gram-staining and biochemical tests including catalase activity in 3% hydrogen peroxide solution, coagulase test and haemolysis test on blood agar [13].

Detection of Extended-Spectrum Beta-Lactamase (ESBL) Enzymes in Enterobacterial Isolates
The double disk synergy method was employed [14,16] using 20 mL of freshly prepared sterile Mueller-Hinton agar seeded with 10 µL of 24 h broth culture (0.5M MacFarland standard) of bacterial isolates and incubated at 37˚C for 24 h. The reference antibiotics used in the study included cef-triaxone-30 µg, ceftazidime-30 µg, cefotaxime-30 µg, aztreonam-30 µg, cefpodoxime-30 µg and cefpodox-ime +clavulanic acid-11 µg. A difference of 5 mm or more in zone diameter between cefpodoxime and cefpodoxime + clavulanic acid indicated that the ESBL enzymes acted against the individual beta-lactam antibiotics but have been inhibited by clavulanic acid with the synergistic effect of cefpodoxime and clavulanic acid [14].

Source of Water (Number of Isolates)
Total Isolates (

Detection of ESBL Producing E. coli and S. typhi Isolates
None of the bacterial isolates was found to be ESBLproducing organism.

DISCUSSION
The ubiquitous nature of microbial agents accounts for their easy contamination of substances that are used by both humans and non-humans. Depending on their rate of contamination, various infections may arise [17]. Water is an abundant resource used for farming activities such as human consumption, domestic purposes, including its use in poultry farming, etc. Physical, chemical and microbial parameters of the water may affect the various uses of water for human and non-human activities. Various degrees of contamination with total coliforms, faecal coliforms and enterococci have been found in hand-filled-hand-knotted water sold on the streets of Kumasi [18]. A study by Osei et al. [11] identified various levels of microbial contaminants in water used for poultry production in poultry farms in the Ashanti region of Ghana. Osei et al. [11] also reported that 91% of the water used on poultry farms in Ghana had various levels of microbial contamination with one or more microorganisms including E. coli, S. typhi, S. aureus and coagulase-negative Staphylococci. These microbial contaminants were found in 91.42% borehole water samples, 90.91% of well water, 90.32% of tap water and 100% of stream water samples collected. Thirty-one E. coli, 36 S. typhi, 64 S. aureus and 19 coagulase-negative Staphylococcal isolates were identified, respectively. The presence of such pathogenic organisms in water, coupled with possible antibiotic resistance, is of public health importance [19]. Table 5. Antibiotic sensitivity profile of E. coli isolates.

Source of Water (Number of isolates)
Total Isolates ( These findings confirm the report by Arhin-Sam [20], which indicated that various enterobacterial species were identified in samples of groundwater used as drinking water within the Kumasi Metropolis of Ghana. In Ghana, the quality of groundwater and surface water resources keeps deteriorating largely due to increasing levels of pollution from "galamsey" sites (illegal mining), untreated sewage and industrial waste, leachate from fertilisers and pesticides used in agriculture, and the use of chemicals in fishing. It is undeniable that Ghana's freshwater resources are seriously under threat as water resources are running dry and overwhelmingly becoming scarce day by day. This has consequently forced some managers of water treatment plants in Ghana to shut down due to pollution, which makes the cost of water treatment very expensive [ 21 ]. This has unfortunately necessitated the use of untreated water on animal farms to reduce the cost of production while increasing the risk of microbial infection among farm animals and workers, cross infection from animals to workers and increased microbial contamination of animal products [ 11 ].
All S. typhi isolates were resistant to cephalexin and ampicillin followed by cefoxitin (94.4%) and cefpodoxime (83.3%). There were low levels of resistance to ceftazidime (13.9%), aztreonam (19.4%), chloramphenicol (19.4%) and cefotaxime (19.4%), but none was resistant to norfloxacin. These findings support a report by Thung et al. [22] where S. typhi isolates obtained from raw chicken meat in retail markets in Malaysia were reported to be highly resistant to ampicillin, vancomycin and penicillin but moderately sensitive to antibiotics such as ceftazidime and tetracycline with the majority of these isolates being multi-drug resistant isolates. Almost 90% of S. typhi isolates were multi-drug resistant, which is in contrast to the findings of M'ikanatha [23] where 31% of S. typhi isolates obtained from retail poultry in the USA were multi-drug resistant. This high (88.9%) percentage of multi-drug resistant bacterial isolates can be attributed to the constant increase in the misuse of antibiotics in most developing countries, especially in animal husbandry [24].
Over 75% of S. aureus isolates were resistant to the reference antibiotics used. This high percentage is consistent with the report by Olufemi et al. [25], where over 80% S. aureus isolated from recreational waters and beach sand in eastern South Africa were resistant to the standard antibiotics used in the study. The occurrence of multi-drug resistant isolates (95%) in this current study is higher than the 52% reported by Waters et al. [26] in meat and poultry in the United States. This may be attributed to a constant increase in the misuse of antibiotics in most developing countries, especially in animal husbandry [24].
All the coagulase-negative Staphylococcal isolates were resistant to sulphamethoxazole-trimethoprim, ceftriaxone, cephalexin and cefoxitin but susceptible to norfloxacin and tobramycin. Moreover, all isolates were multi-drug resistant, with 63.2% being resistant to six antibiotics. Boamah [4] reported that Coagulase-Negative Staphylococcal (CoNS) isolates from poultry litter were resistant to similar antibiotics in Ghana. The percentage (100%) of multi-drug resistant CoNS isolates was higher compared to 65% reported by Boamah [4] and this can be attributed to the acquisition of resistant genes from strains of different organisms over the period and reported increases in the misuse and abuse of antibiotics in most developing countries including Ghana [24].
All isolates of E. coli were susceptible to tobramycin, norfloxacin and ciprofloxacin. Almost 97% of the isolates were resistant to ampicillin, cephalexin, cephalothin, cefoxitin and cefpodoxime but exhibited a low level of resistance towards ceftriaxone (6.45%), chloramphenicol (3.22%) and ceftazidime (3.22%). All E. coli isolates were multi-drug resistant. Fiftyfive percent of isolates were resistant to 6 of the reference antibiotics. The findings of this study confirm the pattern of antibiotic resistance in E. coli isolates obtained from water similar to a study by Sayah et al. [27] which reported that E. coli isolates from surface water, domestic and wild animals and faecal samples, were found to be resistant to antibacterial agents such as ampicillin, chloramphenicol, tetracycline, sulphamethoxazole/trimethoprim, cephalothin, neomycin and streptomycin. These findings are consistent with a report by Wose-Kinge et al. [28] on the antibiotic resistance profiles of E. coli isolated from different water sources in the Mmabatho locality in South Africa, where these isolates were identified to be resistant to antibiotics such as erythromycin, tetracycline, ampicillin and chloramphenicol. The occurrence of multi-drug resistant E. coli is similar to a report by Ogunleye et al. [29] where all isolates of E. coli from poultry in Abeokuta, Southwestern Nigeria were also found to be multi-drug resistant.
None of the E. coli and S. typhi isolates were found to be extended-spectrum beta lactamase producing bacteria, which confirms the report by Boamah [4] where none of the enterobacterial species from poultry litter from poultry farms in Ghana was an ESBL producing bacteria. This implies that the resistance of the isolates to beta-lactam antibiotics cannot be attributed to the production of extended spectrum betalactamases. There is a need to determine the possible mechanism(s) of resistance for these multi-drug resistant bacterial strains.

CONCLUSION
Antibiotic-resistant and multi-drug resistant E. coli, S. typhi, S. aureus and coagulase-negative Staphylococci isolates were identified and none of the isolates was found to produce extended spectrum beta-lactamases. All necessary strict measures should be taken by relevant stakeholders involved in the use, regulation and monitoring of antibiotics in animal husbandry and microbial quality assessment of water used in the poultry industry should be done to prevent the transmission of infections to both animals and humans.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE
Not applicable.

HUMAN AND ANIMAL RIGHTS
Not applicable.

CONSENT FOR PUBLICATION
Not applicable.

AVAILABILITY OF DATA AND MATERIALS
The data, including the supplementary data supporting the findings of the article, is available from the Microbiology Section of Department of Pharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana at https://pharmacy. knust.edu.gh/departments/pharmaceutics.

FUNDING
The project was mainly funded by the authors with support from DANIDA (Safe Water for Food Project) with grant number 1-058 DHI DANIDA.

CONFLICTS OF INTEREST
The authors declare no conflict of interest, financial or otherwise.