Antibiotic Resistance Pattern of Bacterial Pathogens Isolated from Cow Dung Used to Fertilize Nigerian Fish Ponds
Funso S. OMOJOWO
1, Folakemi P. OMOJASOLA
2*
1National Institute for Freshwater Fisheries Research (NIFFR), New Bussa, Niger State, Nigeria; [email protected]
2University of Ilorin, Faculty of Science, Department of Microbiology, P.M.B. 1515 Ilorin, Kwara State, Nigeria; [email protected] (*corresponding author)
Abstract
This study was carried out to isolate and identify antibiotic resistant bacteria from cow dung used for pond fertilization. Cow dung from over 200 cows in NIFFR integrated farms, New-Bussa, Nigeria were collected. Six bacterial pathogens; Escherichia coli, E.
coli O157:H7, Shigella dysentariae, Staphylococcus aureus, Salmonella typhi, and Aeromonas hydrophila were isolated. Antibiotic susceptibility testing by the disk diffusion method was conducted using ofloxacin, amoxicillin, tetracycline, ampicillin, erythromycin, gentamicin, nalidixic acid and chloramphenicol. All the isolated organisms were 100% sensitive to ofloxacin. The multiple resistance patterns revealed that 100% were resistant to tetracycline, ampicillin (85.6%), amoxicillin (83.3%), chloramphenicol (66%), gentamicin (47.6%), erythromycin (44.4%) and nalidixic acid (18.3%). The Public Health risks posed by the cow dung manure include proliferation of ponds with these organisms that are pathogenic to fish and man, contamination of the environment and the possible retention of these organisms in the table size fish.
Keywords: antibiotic resistance, manure, pathogenic organism, sensitivity Introduction
Integrated fish farming is widely acceptable in aqua- culture and is being practiced by the National Institute for Freshwater Fisheries Research (NIFFR), New-Bussa, Niger State, Nigeria. In this farming system, manure from livestock production is administered to fish ponds and the manure is directly consumed by fish. The release of nu- trients supports the growth of fish with low input, with the fish receiving limited, if any, supplementary feed. The pond water becomes fertile upon the application of ma- nure, resulting in more food organisms, thus a high fish production. However, organic manuring releases high con- centrations of pathogenic microorganisms into the ponds constituting a high risk to fish and fish farmers. This is espe- cially serious as an array of these bacteria has been reported to be resistant to antibiotics (Andreas et al., 2002; Olaitan et al., 2011). Antibiotics and other antibacterial drugs are the major weapons against disease-causing bacteria. They act in a number of ways to kill bacteria or suppress their activity. Antibiotics are regularly used in treating sick and diseased cow in the integrated units. Over time, however, bacteria can become resistant to antibiotics. Other prac- tices contributing towards resistance include the addition of antibiotics to livestock feeds (Mathew et al., 2007).
In addition, unsavory practices in the pharmaceutical manufacturing industry such as production of counter- feit drugs can contribute towards the likelihood of creat- ing antibiotic resistant strains (Larsson and Fick, 2009).
Emergence of bacteria resistant to antibiotics is common in areas where antibiotics are used, but occurrence of an- tibiotic resistance bacteria is also increasing in freshwater basin (Ash et al., 2002). The use of antibiotics as growth promoters in animal husbandry has been linked to certain antimicrobial resistance patterns among human bacterial pathogens (Bager et al., 1997; Wagener et al., 1999) sug- gesting that there is a possible flow of antimicrobial resis- tance genes between animal and human pathogens. Poten- tial transfer of resistant bacteria and resistance genes from aquaculture environments to humans may occur through direct consumption of antimicrobial-resistant bacteria present in fish and associated products. Few studies on an- tibiotic resistance bacteria have been carried out on dump- sites, water sources, duck droppings in the environment and hospital environment (Ikpeme et al., 2011; Olaitan et al., 2011; Omololu-Aso et al., 2011) but fewer studies have been undertaken in dynamic integrated aquaculture environment where manure is used to fertilize fish ponds.
Therefore, the objective of this study was to isolate and identify antibiotic resistant bacteria from cow dung used in fertilizing fish ponds. In addition, also examine the anti- biotic susceptibility patterns of the isolated organisms.
Materials and methods Sample collection
Samples of cow dung were randomly collected from NIFFR integrated farms in New-Bussa, Niger state, North Received 07 December 2012; accepted 12 February 2013
corded 100% susceptibility to ofloxacin and nalidixic acid; 66.7% to erythromycin and 100% resistance to tet- racycline; 66.7% resistance to gentamicin and ampicillin respectively (Fig. 2). These antibiotics are very common and are readily available as over-the-counter drugs to con- sumers in Nigeria (Omojasola and Omojasola, 2001) and may not be very useful for therapeutic purposes. The sus- ceptibility pattern of E. coli (Fig. 3) showed the organism was susceptible to ofloxacin and nalidixic acid (100%), chloramphenicol (77.8%) and erythromycin (66.7%).
However, it had 100% resistance to amoxicillin, tetracy- cline and 66.7% resistance to ampicillin. E. coli O157:H7 recorded 100% susceptibility to ofloxacin, amoxicillin and nalidixic acid with 100% resistance to tetracycline, gentamicin, erythromycin and ampicillin respectively and 50% resistance to chloramphenicol (Fig. 4). Multiple an- tibiotic drug resistance in E. coli to ofloxacin and nalidixic acid has also been reported (Aibinu et al. 2004; Olaitan et al. 2011). This agrees with earlier reports that E. coli O157 isolates had high prevalence of resistance to tetracycline, sulfamethoxazole, cephalothin, and ampicillin (Carl et al., 2002; Zhao et al., 2001). Cattle is thought to be primary reservoirs of E. coli O157 and it is highly infectious as the infective dose for humans is reported to be 10 cfu, the low- est of the common foodborne pathogens. In addition, E.
coli O157; H7 has high survival rates in as fecal counts as high as 4.2×103 cfu/mL have been reported in 100 day old manure (Kress and Gifford, 1984).
central region of Nigeria. A total of 1000 samples were col- lected from over 200 cows raised in the integrated farm.
The faecal samples were collected with sterile spatula into sterile peptone water and were analyzed within 1 hour of collection.
Isolation and identification
Isolation and identification of bacteria were investigat- ed according to Bergey’s Manual of Determinative Bacte- riology (Holt et al., 1997). Pure cultures suggestive of E.
coli were sub-cultured on Sorbitol MacConkey (SMAC) agar and incubated at 44.5°C for 24 hours to check for colonies that were colorless to pale (non- fermenting), flat and smooth, circular or serrated at the edge typical of E.
coli O157:H7.
Antibiotic Sensitivity Testing
Antibiotic resistance of bacteria was determined by the single disc diffusion method with the use of Mueller- Hinton agar, according to the Bauer-Kirby method (Bauer et al., 1979). The following eight clinical antibiotics, with their concentrations given in parentheses were used in the antibiograms as recommended by the National Commit- tee for Clinical Laboratory Standards (NCCLS, 2006):
Tetracycline (30 μg); Ofloxacin (30 μg); Gentamicin (20 μg); Erythromycin (10μg); Ampicillin (10 μg); Chloram- phenicol (30 μg); Nalidixic acid (30 μg) and Amoxicil- lin (30 μg). After incubation, a clear circular zone of no growth in the immediate vicinity of a disk indicates sus- ceptibility to that antimicrobial. Using reference tables the size of zones was related to the Minimum Inhibitory Concentration (MIC) and results recorded as whether the organism is susceptible (S) or resistant (R) to that antibi- otic. Data was statistically analyzed using SPSS Version 12, level of significance 5%.
Results and discussion
One hundred and thirty-two (132) bacteria were iso- lated from 1000 cow dung samples. These were: Escheri- chia coli: 66 (50%); Aeromonas hydrophila 30 (22.7%);
Salmonella typhi 18 (13.6%); Staphylococcus aureus 12 (9.1%) and Shigella dysenteriae 6 (4.6%) (Fig. 1). Twelve 12 (18.2%) of the sixty-six (66) E. coli isolates from cow dung were non-fermenters typical of E. coli O157:H7 which represent 9.1% of total isolates from cow dung.
Overall, there was a 100% resistance to tetracycline by all the isolates, ampicillin (85.6%), amoxicillin (83.3%), gentamicin (47.6%), chloramphenicol (66%), erythromy- cin (44.4%) and nalidixic acid (18.3%). Multiple antibi- otic drug resistance profiles have been reported in enteric bacteria from human and animal sources (Ikpeme et al., 2011; Olaitan et al., 2011; Troy et al., 2002). Resistance of a single bacterial isolate to more than one antimicro- bial drug is commonly reported. There was no resistance to ofloxacin by all bacteria isolated. Salmonella typhi re-
Fig. 1. Frequency of Occurrence of Isolated Organisms In Cow Dung used to fertilize Fish Ponds in New Bussa, Nigeria
Fig. 2. Antibiogram of Salmonella typhi isolated from cow dung used to fertilize fish ponds in New Bussa Nigeria
Tab. 1. Antibiotic Resistance profile of pathogenic bacteria isolates from Cow Dung used to fertilize fish ponds in New Bussa Nigeria
Antibiotic Susceptibility (Zone of inhibition in mm)
Isolates TET GEN OFL AMX CHL NAL ERY AMP
Salmonella typhi R(18) R(16) S(25) R(13) S(21) S(26) S(27) R(11)
E. coli R(17) R(11) S(28) R(12) S(23) S(28) S(29) R(10)
E. coli O157:H7 R(17) R(10) S(27) R(13) S(20) S(26) R(10) R(11)
Shigella dysentariae R(16) R(11) S(21) R(10) R(13) S(20) S(21) R(10)
Aeromonas hydrophila R(14) R(10) S(20) R(9) R(11) R(16) R(16) R(8)
Staphylococcus aureus R(17) S(25) S(26) R(10) S(23) S(28) S(24) R(10)
R = Resistant, S = Susceptible, TET= Tetracycline, GEN = Gentamicin, OFL = Ofloxacin; AMX = Amoxicillin; CHL= Chloramphenicol; NAL= Nalidixic acid; ERT=
Erythromycin, AMP = Ampicillin
Fig. 3. Antibiogram of E. coli isolated from cow dung used to
fertilize fish ponds in New Bussa Nigeria Fig. 4. Antibiogram of E. coli O157:H7 isolated from cow dung used to fertilize fish ponds in New Bussa Nigeria
Fig. 5. Antibiogram of Shigella dysenteriae isolated from cow
dung used to fertilize fish ponds in New Bussa Nigeria Fig. 6. Antibiogram of Aeromonas hydrophila isolated from cow dung used to fertilize fish ponds in New Bussa Nigeria
Fig. 7. Antibiogram of Staphylococcus aureus isolated from cow
dung used to fertilize fish ponds in New Bussa Nigeria Fig. 8. Percentage Multiple Antibiotics Resistance
that all the organisms were 100% sensitive to ofloxacin.
The isolates were most resistant to tetracycline, ampicillin and amoxicillin. Therefore potential role of manure fertil- ized ponds as source of antibiotic resistance in the environ- ment should further be studied. It is recommended that manure intended for pond fertilization should be treated before use. In addition, the implication of this high level of antibiotic resistance on the choice of antibiotics in relation to zoonotic infections should be noted and efforts should be made to stop indiscriminate use of antibiotics.
Acknowledgements
This data is part of an ongoing Ph.D. study, the author is grateful to the Executive Director of NIFFR, New-Bus- sa, Niger-state, Nigeria for sponsorship.
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Conclusions
From this study, pathogenic bacteria were isolated from cow dung manure. The antibiotic sensitivity testing shows
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