Antibiotic sensitivity patterns of microbial isolates from fish ponds : A study in the Greater Accra Region of Ghana

Intensive use of antimicrobial agents in aquaculture provides a selective pressure creating reservoirs of drug-resistant bacteria and transferable resistance genes in fish pathogens and other bacteria in the aquatic environment. From these reservoirs, resistance genes may disseminate by horizontal gene transfer and reach human pathogens, or drug-resistant pathogens from the aquatic environment may reach humans directly. This study aims at identifying the antibiotic sensitivity patterns of Pseudomonas aeruginosa, Salmonella typhi, and Escherichia coli isolates from fish ponds in two localities (Ashiaman and Dawhenya) in the Greater Accra region of Ghana. A total of 43 isolates were tested using the KirbyBauer agar disc diffusion method against Ciprofloxacin (5 μg), Erythromycin (15 μg), Cefuroxime (30 μg), Gentamicin (10 μg), and Tetracycline (30 μg).P. aeruginosa was the most isolated organism with 90% prevalence, followed by E. coli (75%) and S. typhi (50%). All the P. aeruginosa isolates were resistant to Erythromycin and Cefuroxime while 90% of the S. typhi isolates were resistant to Tetracycline and Erythromycin. E. coli isolates showed 100, 93.33 and 66.66% resistance to Erythromycin, Cefuroxime and Tetracycline respectively. Gentamicin, Tetracycline and Ciprofloxacin were sensitive to 66.66, 61.10 and 50% of P. aeruginosa isolates respectively. Ciprofloxacin was sensitive to 90% of the S. typhi isolates whiles Gentamicin was sensitive to 70% of the S. typhi isolates. Ciprofloxacin and Gentamicin were more sensitive to E. coli isolates (80 and 66.66% sensitivity respectively). Multi-drug resistant strains were obtained in 77.78% of P. aeruginosa and 70% of S. typhi isolates whiles 66.67% of E. coli isolates were also resistant to more than two classes of the antibiotics tested. High levels of microbial resistance were observed in the isolates with 72.09% of isolates being multidrug resistant strains.


INTRODUCTION
With the rise in public awareness about the loss of effectiveness of antibiotics due to over use; consumer groups, public health experts and environmentalists have begun to challenge antibiotic usage in livestock, poultry *Corresponding author.Email: j.a.apenteng@gmail.comor japenteng@central.edu.ghTel: +233 249 449 249.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License and fish farming.Antibiotics in fish farming and other animal food production is widely believed to contribute to the dramatic increase in numbers of antibiotic-resistant bacterial strains now threatening human health (Benbrook, 2002).As a result of the non-hygienic and stressful conditions present in aquaculture facilities, the risk of bacterial infections among aquaculture fish is high, therefore, heavy amount of antimicrobials are used in fish feed for preventive and curative purposes in aquaculture facilities worldwide (Sapkota et al., 2008).
The heavy use of antimicrobial agents in aquaculture has resulted in increase of strains resistance to these agents.Potentially, these resistant strains can have impact on the therapy of fish and human diseases or the environment of the fish farms (Smith et al., 2003).It is therefore necessary to study the presence of clinically important bacteria in the environment and their antibiotic resistance pattern.This study shall focus on resistance strains found in fish farms and the specific isolates will be Salmonella typhi, Escherichia coli and Pseudomonas aeruginosa.
P. aeruginosa is a clinically important gram negative bacterium, which is responsible for a variety of systemic infections like urinary tract infections, respiratory system infections, gastrointestinal infections, dermatitis, soft tissue infections, bone and joint infections (Favero et al., 1971).The overall prevalence of antibiotic resistant P. aeruginosa is increasing, with up to 10% of global isolates found to be multidrug resistance strains (Gales et al., 2001).It is recognized as the second leading cause of gram negative nosocomial infection and a major treatment challenge for P. aeruginosa infections (Carmeli et al., 1999).
Salmonella is a Gram-negative bacterium belonging to the family Enterobacteriaceae, and known as "enteric" bacteria.Salmonella are found in the intestinal tract of animals and humans.Some serotypes of Salmonella, such as S. typhi and S. paratyphi are only found in humans (Miller and Pegues, 2005).
In humans, Salmonella are the cause of two diseases called salmonellosis: enteric fever (typhoid), resulting from bacterial invasion of the bloodstream, and acute gastroenteritis, resulting from a food borne infection or intoxication (Foley et al., 2006).Salmonella bacterium is one of the commonest causes of food poisoning worldwide.
E. coli is the most prevalent facultative anaerobic species in the gastrointestinal tract of human and animals, usually a harmless microbe, but it is also a medically important bacterium causing a number of significant illnesses.E. coli can be easily disseminated in different ecosystems through the food chain and water.One of the possible ways of entry of various microbes could be by the adoption of improper hygienic measures during handling and processing of harvested fishes.Antimicrobial resistance in E. coli has been reported worldwide.
The aim of this study is to isolate and assess the prevalence of S. typhi, E. coli, and P. aeruginosa in fish ponds and to determine their respective antibiotic resistance patterns against some clinically used antibiotics.

Collection of samples
A total of 20 fresh water samples were collected from Ashaiman and Dawhenya Fish Ponds.The samples were collected into a sterile sample container at four different sites of the fish ponds.They were then transferred to the Microbiology Laboratory of Central University Pharmaceutics department immediately and kept refrigerated for 24 h.A volume of 1.0 mL of the collected samples were then transferred into 10 mL peptone water and incubated at 37ºC for 48 h.

Isolation of bacteria
The initial isolation of bacteria (P.aeruginosa, S. typhi and E. coli) was done using various selective, differential isolation media such as Cetrimide, MacConkey and Bismuth sulphite agar.Further isolation were performed on the initial isolates using biochemical methods using fermentation, gram staining and other microbe specific media such as brilliant coliform E. coli media, Methyl Red Vogues Proskauer (MRVP) tests and Citrate utilization tests.A total of 43 isolates comprising the organisms of interest were obtained.All microbial media used were purchased from Oxoid, UK.

Antibiogram study
A total of 43 isolates were tested for antimicrobial drug susceptibility against five commonly used antibiotics belonging to the following antibiotic classes;(Aminoglycosides, Floroquinolones, Macrolides, Cephalosporins and Tetracyclines) by Kirby-Bauer agar disc diffusion method (Bauer et al., 1966).The isolated bacteria (P.aeruginosa, S. typhi and E. coli) in the nutrient broth were spread on the surface of sterile nutrient agar.Antimicrobial discs (Oxoid, UK) were placed on the surfaces of inoculated nutrient agar plates using a multidisc dispenser (Oxoid, UK).The plates were kept on the bench for 30 min prior to incubation at 37ºC for 24 h.After 24 h incubation, the plates were examined and the diameters of the inhibition zones were measured from the edge of the disc to the edge of the zone using a zone reader.Susceptibility and resistance were determined according to the interpretation criteria described in the Clinical and Laboratory Standard Institute (CLSI) Guidelines (2011) (Table 1).

Data analysis
All graphs were plotted and analysed using Microsoft Excel 2013.

Overall prevalence of isolates according to location
A total of 43 isolates were obtained out of which 18 were P. aeruginosa, 10 were S. typhi and 15 were E. coli.The  2.

Pseudomonas aeruginosa
Antibiotic sensitivity test of 18 isolates of P. aeruginosa showed different sensitivity and resistance patterns according different sources.Out of the five antibiotics tested, Gentamicin (CN) was sensitive to 66.66% of isolates.Tetracycline (TE) and Ciprofloxacin (CIP) were sensitive to 61.1 and 50% of isolates respectively.Ciprofloxacin (CIP) and Tetracycline (TE) were however intermediate resistant to 5.56% of isolates.Ciprofloxacin (CIP) was resistant to 44.44% of isolates.Erythromycin (E) and Cefuroxime (CXM) showed no antimicrobial activity against the isolates Figure 1.

Salmonella typhi
Antibiotic sensitivity test of 10 isolates of S. typhi showed different sensitivity and resistance patterns.Results from the five antibiotics tested showed that, Ciprofloxacin (CIP), Gentamicin (CN) and Cefuroxime (CXM) were sensitive to 90, 70 and 30% of isolated S. typhi respectively.Tetracycline (TE) and Erythromycin (E) were sensitive to 10% of isolates.Gentamicin (CN) on the other hand was intermediate resistant to 10% of isolates.The highest resistance was found with Tetracycline (TE) and Erythromycin (E), showing resistance to 90% of isolates, followed by Cefuroxime (CXM), which showed resistance to 70% isolates.Gentamicin (CN) was resistant to 20% of isolates, whereas, Ciprofloxacin (CIP) to 10% of the isolated S. typhi shown in Figure 2.

Antibiogram profile of Escherichia coli
Out of the five antibiotics tested on the 15 isolates, Erythromycin (E) was resistant to 100% of isolates, followed by Cefuroxime (CXM), which was resistant to 93.33% of isolates.Ciprofloxacin (CIP), Gentamicin (CN), and Tetracycline (TE) showed resistant to 13.33%, 26.66%, and 66.66% of isolated E. coli respectively.Ciprofloxacin (CIP), Gentamicin (CN), and Cefuroxime (CXM) were intermediate resistant to 6.66% of isolates.Ciprofloxacin (CIP) on the other hand showed the highest sensitivity against 80% of isolates.Gentamicin (CN) and Tetracycline (TE), showed sensitivity to 66.66 and 33.33% of isolates respectively which is shown in Figure 3.

Multidrug resistant organisms
The presence of multidrug resistance strains were also studied among isolates.Of the isolates, 31 (72.09%) were resistance to more than two different classes of antibiotics out of which 14 (77.78%)isolates of  P. aeruginosa, 7 (70%) isolates of S. typhi and 10 (66.67) isolates of E. coli were all found to be multidrug resistant strains.P. aeruginosa had the highest number (14 out of 18 isolates) of multidrug resistant strains, followed by S. typhi.

DISCUSSION
Aquaculture is growing rapidly in many regions of the world, and aquaculture products constitute an important food supply with increasing economic importance.Use of  antimicrobial agents in aquaculture has resulted in the emergence of reservoirs of antimicrobial-resistant bacteria in fish and other aquatic animals, as well as in the aquatic environment (Akinbowale, 2006;Schmidt et al., 2000).The three bacteria studied in this project are of public health importance (Bangtrakulnonth et al., 2004;Nadeem et al., 2009;Ministry of Health, 2002) and presence of resistance strains in fish pond water should be of much concern because, the people around and the environment are exposed to these microorganisms.A total of 43 isolates were obtained in this study which 18(41.36%) were P. aeruginosa, 15(34.88%)were E. coli and 10(23.26%)were S. typhi.Among the organisms isolated from Ashaiman and Dawhenya Fish Ponds, P. aeruginosa was the mostly found organism with 90% prevalence followed by E. coli (75%) and S. typhi (50%).Uğur et al. ( 2012) reported in their study in Turkey that, Pseudomonas species was the most frequently detected microorganism in fish ponds.All the P. aeruginosa isolates obtained in this study were resistance to Erythromycin and Cefuroxime.This is similar to a study conducted by Sivanmaliappan and Sevanan (2011) in Coimbatore, India where 100% of P. aeruginosa isolates were resistant to Erythromycin.A study conducted in Egypt by Gad et al. (2008) also showed that 100% of P. aeruginosa isolates were resistant to Cefuroxime.Resistance to Cefuroxime is largely due to the production of extended spectrum βlactamase (ESBL) enzymes by the bacteria.Resistance to Cefuroxime by P. aeruginosa could also be due to a combination of mechanisms such as the expression of chromosomal AmpCcephalosporinases and over expression of active efflux systems (McGowan, 2006).Resistance to Erythromycin may be due to the use of this agent in fish farming.Gentamicin, Tetracycline and Ciprofloxacin were potent against P. aeruginosa isolates with 66.66, 61.1% and 50% susceptibility, respectively.This is comparable to a study carried out in Dhaka, Bangladesh where both Gentamicin and Tetracycline were found to be sensitive to 93.70% of P. aeruginosa isolates (Nasreen et al., 2015).A similar observation has also been reported in Bangladesh where Ciprofloxacin was highly sensitive to P. aeruginosa isolates (Hossain et al., 2013).Gentamicin and Ciprofloxacin resistance to P. aeruginosa isolates is due to the ability of P. aeruginosa to acquire further resistance mechanisms to these agents (Streteva and Yordanov, 2009).This could be the reason why P. aeruginosa isolates were resistant (33.33%) to Gentamicin and (44.44%) to Ciprofloxacin, even though Gentamicin and Ciprofloxacin are hardly used in fish farming.P. aeruginosa isolates resistance to Tetracycline (33.33%) could be due to its frequent use in aquaculture facility.
Infection by Salmonella is a common cause of food poisoning in humans (Hobbs and Robert, 1993).In this study S. typhi was the least isolated organism compared to P. aeruginosa and E. coli.Erythromycin and Tetracycline exhibited the highest resistance to S. typhi (90%) isolates whiles Cefuroxime showed resistance against 70% S.typhi isolates.According to Chanda et al. (2011), most farms use tetracycline and erythromycin to eliminate pathogenic problems.Antibiotic resistance develops when microorganisms are exposed to effective doses of an antibiotic within a shorter period or when the microorganisms are exposed to smaller concentrations or residues of the antibiotic over a longer period of time (Todar, 2008).These theories may support the high resistance observed for Tetracycline and Erythromycin as a result of prolong exposure to microorganisms.It was observed that the most sensitive antibiotics to S. typhi isolates were Ciprofloxacin and Gentamicin.A similar result was obtained in a study in Bangladesh where S. typhi was mostly sensitive against Ciprofloxacin and Gentamicin ( Mannan et al., 2014).A total of 100, 93.33, and 66.66% of the E. coli isolates exhibited resistance to Erythromycin, Cefuroxime and Tetracycline respectively.A similar result was obtained by Kibret and Abera (2011), where Erythromycin and Tetracycline were resistance to 89.4 and 72.6% to E. coli isolates respectively.A study conducted by Ahmed et al. (2015) in Islamabad showed complete resistance of E. coli isolates to Cefuroxime.Ciprofloxacin and Gentamicin revealed the highest sensitivity against the E. coli isolates with 80 and 66.66% sensitivity, respectively.This in accordance with a study carried out in Nigeria where 78.9% of E. coli isolates showed sensitivity to Ciprofloxacin and same percentage was obtained for Gentamicin (Reuben and Owuna, 2013).
In general, the relatively higher proportion of susceptible responses of E. coli, S. typhi, and P. aeruginosa isolates to Ciprofloxacin and Gentamicin suggest the effectiveness of these agents in the treatment of infections caused by E. coli, S. typhi and P. aeruginosa.P. aeruginosa was the most isolated organism from the fish ponds.This is because P. aeruginosa thrives very well at habitats with adequate amount of moisture (Hardalo et al., 1997).Out of the 43 isolates, 31 (72.09%) were resistant to more than two different classes of antibiotics.P. aeruginosa had the highest percentage of multidrug resistance strains accounting for 77.78%.According to Gales et al. (2001), up to 10% of global P. aeruginosa isolates are found to be multidrug resistance.The high number of resistance of E. coli, S. typhi, and P. aeruginosa found in the two fish ponds serves as a reservoir which can be easily transferred to other pathogens and even humans.

Conclusion
The study has shown the presence of resistant strains in the fish ponds at Dawhenya and Ashiaman.All the P. aeruginosa isolates were resistant to Erythromycin and Cefuroxime whiles 90% of the S. typhi isolates were resistant to Tetracycline and Erythromycin.Cefuroxime, Erythromycin and Tetracycline respectively showed 100, 93.33 and 66.66% resistance to E. coli isolates.Ciprofloxacin and Gentamicin showed the highest sensitivity when tested on all the isolates.P. aeruginosa had the highest number of multi-drug resistant strains.This study has therefore proved the need for the monitoring of antibiotics usage in fish farming and the adoption of proper hygienic measures in aquaculture facilities.

Table
CLSI inhibition zone diameter for P. aeruginosa, S. typhi and E. coli.

Table 2 .
Summary of prevalence of P. aeruginosa, S. typhi and E. coli according to location.
aeruginosa, S. typhi and E. coli in Dawhenya and Ashaiman Fish Ponds were recorded as shown in Table