Report on Multiple Antibiotics Resistance Aeromonas hydrophila Isolated from Catfish Farms in Epe Lagos

Middle East Journal of Applied Science & Technology (MEJAST)
Vol.3, Iss.3, Pages 51-57, July-September 2020
ISSN: 2582-0974 [51] www.mejast.com
Country: Nigeria
Report on Multiple Antibiotics Resistance Aeromonas hydrophila Isolated
from Catfish Farms in Epe Lagos
Ogbonne, Fabian Chinedu1
*, Akhiromen Dorathy Iniobong2
& Wilfred-Ekprikpo, Peace Chidinma1
1
Department of Aquaculture, 2
Department of Biological Oceanography, Nigerian Institute for Oceanography and Marine
Research, P.M.B. 12729, Victoria Island, Lagos, Nigeria. Corresponding Author: ogbonnefebian@yahoo.com*
Article Received: 23 June 2020 Article Accepted: 21 August 2020 Article Published: 22 September 2020
Introduction
Fish production is one of the most significant industrial activities in Agricultural sector (FAO, 2009). However, one
of the challenges been face by fish farmers today, especially those that engage in intensive and semi-intensive
aquaculture is the menace of bacterial fish diseases. Infection of fish with microbial pathogens is considered a risk
factor in aquaculture industry because of its subsequent significant economic loss due to fish mortalities, cost of
treatment of infected fish and reduction of market value of the affected fish. Aeromonas infection in fish causes
world economic problems because of the high number of fish mortalities (Citarasu et al., 2011). Fish can be
contaminated bacteriologically by polluted water, handling, processing and unhygienic storage conditions (Sarkar
et al., 2013). Aeromonas hydrophila have been reported to cause different zoonotic disease that occurs due to
consumption of contaminated fish, sea foods and drinking water, or direct contact with recreational waters
(Yogananth et al., 2009).Aeromonas hydrophila is a Gram-negative organism that belongs to the family of
Aeromonadaceae, it is a facultative anaerobic, non-spore forming, motile, rod-shape or coccobacilli bacterium
(Martin-Carnahan et al., 2005).
Aeromonas hydrophila have been implicated as causative agent of intestinal and extra-intestinal diseases in humans
such as septic arthritis, diarrhea, gastroenteritis, skin and wound infections, meningitis, and fulminating septicemia
(Salunke et al., 2015). The principal reservoirs are fish in rivers, estuary waters and salt water. Aeromonas species
have also been isolated from chlorinated water including water supplies (Tahoun et al., 2016). The United States
Environmental Protection Agency has incorporated Aeromonas hydrophila in the contaminant candidate list of
emerging water borne pathogens (Borchardt et al., 2003). This is attributed to Aeromonas capacity to grow and
form biofilm in chlorinated water distribution systems. The pathogenicity of Aeromonads has been related to a
ABSTRACT
Many Gram negative bacteria, especially Aeromonas hydrophila are notorious for their heightened capacity to acquire and exchange antibiotic
resistance genes and consequently, are commonly targeted as indicator organism for monitoring antimicrobial resistance in aquatic milieus. This
study was aimed to investigate the prevalence and drug resistance patterns of Aeromonas hydrophila isolated from farm raised catfish at Epe fish
farm, Lagos State, Nigeria. Swabs were aseptically taken from the kidney, intestine, liver, gills, and skin after dissecting the fish samples. The
specimens were bacteriologically analyzed. After series of biochemical test, the isolated bacteria were identified presumptively according to
Bergey’s Manual of determinative bacteriology, 7th
Edition. Fifty-seven (57) Aeromonas hydrophila were recovered out of seventy-one (71) bacterial
isolated from the 35 fish samples collected from the fish farms studied. The study reveals multiple antibiotics resistance pattern among the isolates as
Aeromonas hydrophila were 100% resistant to Streptomycin, Oxytetracycline, Chloranphenicol, Pefloxacin, Ofloxacin and 70, 65 and 55% resistant
to Gentamycin, Amoxycillin and Erythromycin respectively. However, all the isolates were 100% susceptible to Ciprofloxacin and Cotrimoxazole
only. The presence of multidrug-resistant Aeromonas hydrophila in fish could be a vehicle of horizontal gene transfer to previously susceptible
bacteria and these could constitute a serious public health hazard to human and animal in the environment. Thus, the use of antibiotics in catfish
production as growth promoter or disease prevention should be discouraged and some safer, biological alternatives strategies should suffice to
mitigate bacteria drug resistance and its associated problems.
Keyword: Aeromonas hydrophila, Antibiotics Resistance, Aquaculture, Catfish, Pathogens.

number of recognized virulence factors such as aerolysin, hemolysin, proteases, lipases and DNases. These toxins
play a major role in the development of diseases either in humans or in fish (Umesha et al., 2013).
Antibiotics play an essential role in the treatment and prevention of bacterial fish diseases; however, indiscriminate
use of these antibiotics in aquaculture have contributed immensely to the surge in bacterial resistance to drugs
leading to enhanced capacity of the bacteria virulence features. Multiple antibiotic resistance (MAR) from
Aeromonas hydrophilla is a worldwide problem caused by the abuse of antibiotics (Odeyemi et al., 2012; Sharma
et al., 2015). It was reported that the resistance of Aeromonas species isolated from aquatic environment has been
increased due to the increased application of antibiotics in aquaculture treatment (Odeyemi and Ahmad, 2017).
Therefore, this study was aimed to explore the prevalence of Aeromonas hydrophila in farm raised catfish at Epe
Fish Farm in Lagos State, and to as well evaluate the antibiotic susceptibility profile of the isolates.
Materials and Methods
Sample Collection
Catfish sample were collected during surveillance at Epe fish farms in Lagos State. During sampling, the
behavioral abnormalities, gross and signs of infections on the fish were observed and recorded. Both infected, weak
fish with disease signs and healthy fish were aseptically collected and transported to Aquaculture laboratory at
Nigerian Institute for Oceanography and Marine Research, Lagos for bacteriological analysis.
Isolation and Identification of Aeromonas hydrophila
Swabs were taken aseptically from the kidney, intestine, skin, gills and liver of the dissected fish samples, and were
streaked on petridish containing Tryptone soy agar (Himedia, India) that was prepared according to manufacturers’
instructions. The plates were incubated at 35 ±2˚C for 24 hours and were observed for bacterial growth and distinct
colonies were further sub-cultured on freshly prepared Tryptone soy agar to obtain pure culture of the isolates. The
isolates were then identified using their morphological features, Gram staining reaction and battery of biochemical
test according to (Bergey’s Manual of determinative bacteriology, 7th Edition).
Antibiotic Susceptibility Testing
Some commercially available antibiotics susceptibility test single disc from Oxoid Ltd, Basingstoke, RG24 8PW,
United Kingdom, was used. Pure culture of Aeromonas hydrophila were picked from Tryptone soy broth (Himedia,
India) using a sterile wire loop and transferred to tubes each containing 5ml of sterile physiological saline. The
suspended culture was vortexed and adjusted to match 0.5 McFarland turbidity standards. Sterile swab stick was
then dipped, rotated and pressed firmly on the tube walls above the culture to remove excess inoculum from the
swab. Then, evenly swabbed on the dried surface of Mueller-Hinton agar (Oxoid Ltd., England) plates ensuring
even distribution of the bacterium. Ten (10) different antibiotics susceptibility single discs were placed on the
plates using sterile forceps and were incubated at temperature of 35±2˚C for 18 to 24 hours, after which the
inhibition zone in diameter were measured with a meter rule in millimeter. The methods described above and the
interpretations of results were done using standard (CLSI, 2016). The ten antibiotics used have the following
concentrations: Amoxycillin (25µg), Cotrimoxazole (25 µg), Erythromycin (10µg), Ofloxacin (5µg), Streptomycin

(25µg), Chloranphenicol (30µg), Gentamycin (10 µg), Ciprofloxacin (10µg), Pefloxacin (5µg) and
Oxytetracycline (30µg).
Results
The photomicrograph of the Gram staining of the isolates is presented in plate 1 showing a Gram-negative,
short-rod shaped bacterium. The bacteria were isolated from the skin, liver, kidney, gills and intestines of the
catfish.
Plate 1: Gram staining photomicrograph of Aeromonas hydrophila which is indicated by the arrow.
Table 1 shows biochemical and morphological features of the isolates which was used to confirmed the isolates as
Aeromonas hydrophila.
Fifty-seven (57) Aeromonas hydrophila were recovered out of seventy-one (71) bacterial isolated from the
thirty-five (35) fish samples collected from the fish farm studied. The isolates were identified and confirmed as
Aeromonas hydrophila through a battery of biochemical examination. Antibiotics susceptibility test shows that
Aeromonas hydrophila isolates were 100% resistant to Streptomycin, Oxytetracycline, Chloranphenicol,
Pefloxacin, Ofloxacin and 70%, 65% and 55% resistant to Gentamycin, Amoxycillin and Erythromycin
respectively. Conversely, all the isolates were 100% susceptible to Cotrimoxazole and Ciprofloxacin only as shown
in figure 1.
Table1: Biochemical and Morphological Characteristics of the isolated Aeromonas hydrophila.
S/N Biochemical Test Reaction
1 Gram stain negative
2 Morphology rod –shape
3 Catalase Positive
4 Oxidase Positive

5 Motility Positive
6 Indole Positive
7 Glucose fermentation Positive
8 Lactose Positive
9 Sucrose Positive
10 Mannitol Positive
11 Maltose Positive
12 Inositol Negative
13 Hydrogen sulphide
production
Negative
14 Urease Negative
15 Voges-proskauer Positive
16 Citrate Positive
17 Methyl Red Negative
18 Growth at 3% NaCl Positive
19
20
Resistance to ampicillin
Endospore formation
Positive
negative
Fig.1 Percentage of Antibiotics resistance and susceptibility patterns of the isolated Aeromonas hydrophila.
0
20
40
60
80
100
120
%SusceptibilityofA.hydrophila
ANTIBIOTICS
Sensitive
Resistance

Discussions
Aeromonas hydrophila isolated from the catfish investigated were observed to exhibit resistance to eight antibiotics
out of ten different antibiotics tested from this study. Multiple antibiotics resistance of Aeromonas hydrophila from
this work is in full agreement with other studies as have been reported by many researchers (Ghenghesh et al.,
2013). However, Aeromonas hydrophila resistant to Chloramphenicol, Gentamycin and Oxytetracycline from this
study was in contrast to the findings of Isoken (2014) who reported that Aeromonas hydrophila were absolutely
susceptible to Chloramphenicol, Gentamycin and Oxytetracycline. Most Aeromonas isolates are intrinsic or
chromosomally mediated resistance to Amoxycillin (Rall et al., 1998). Cotrimoxazole and Ciprofloxacin have been
reported as the most active drug against Aeromonas infection (Arslan et al., 2015). This study is in total agreement
with that report as 100% susceptibility to Cotrimoxazole and Ciprofloxacin was observed.
Emergence of multidrug-resistance among foodborne pathogens had a worldwide concern due to its public health
and economic impacts. For instances, United States Centre for Disease Control and Prevention (CDC) reported that
more than two millions of US population have suffered annually from drug resistant organisms (CDC, 2013). In
addition, this number was estimated to be 400000 in Europe (ECDC/EMEA, 2009). Development of drug
resistance among foodborne pathogens is mainly due to the abuse of antibiotics in the veterinary field including
improper usage, lack of adherence to treatment guidelines, inadequate dosing and using of therapeutic agents as
feed additives (Prestinaci et al., 2015). Nevertheless, the indiscriminate use of antibiotics for therapy in animals and
as growth promoters in fish industry have resulted in an increased resistance and consequently, resistant organisms
which can be transmitted to humans and other animals through many routes in the food chain like food
consumption and water contact even contaminated soil, livestock and pets could also be considered as sources of
resistance transfer (Odeyemi and Ahmad, 2017). Therefore, it is highly recommended to reduce the abuse of
antibiotics in veterinary field and to find alternatives to antibiotics such as probiotics and other safer biological
means to be used as feed additives in aquaculture practices.
Declarations
Source of Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit
sectors.
Competing Interests Statement
The authors declare no competing financial, professional and personal interests.
Consent for publication
We declare that we consented for the publication of this research work.
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