phylococcus Aureus Isolated from Ready to Eat Fish in Ekiti State

Sciknow Publications Ltd.
Aquatic Biology Research
©Attribution 3.0 Unported (CC BY 3.0)
ABR 2014, 2(4):62-68
DOI: 10.12966/abr.11.01.2014
Antimicrobial Resistance Patterns and Plasmid Profiles of Staphylococcus Aureus Isolated from Ready to Eat Fish in Ekiti State,
Nigeria
C. R. Falegan*, D. O. Oguntoye, S. O. Akoja
Department of Microbiology, Ekiti State University, Ado-Ekiti, Ekiti State. Nigeria
*Corresponding author (Email: [email protected])
Abstract - Samples were taken in five different local governments in Ekiti State. For this, Total Viable Bacterial Counts (TVBC),
Total Staphylococcus Counts (TSC), and occurrence of Staphylococcus aureaus were determined for all the fish samples. The
highest TVBC (2.15 X 105 cfu/ml) was observed in dried fish sample from Ikole and lowest (1.06 X 10 5 cfu/ml) in smoked fish
sample from Ikole. Highest Staphylococcus count (2.8 X 102 cfu/ml) was found in dried fish sample from Ikole and lowest (1.0 X
102 cfu/ml) in smoked fish sample from Ado. Twenty eight isolates of Staphylococcus aureus were isolated from the entire ready
to eat fish samples. The isolates were screened against eight gram positive antibiotics, no resistance case was observed in both
gentamycin and ofloxacin, ten isolates were resistant to erythromycin, eleven isolates were resistant to ceftriaxone, twelve were
resistant to ceftazidime, fifteen isolates were resistant to cloxacillin, twenty six were resistant to cloxicillin, and all of the isolates
were resistant to augmentin. Resistance to various antimicrobial agents was not associated with presence of plasmids. This was
because no particular molecular size plasmid could be associated with any particular antimicrobial resistance. Resistance was
observed in isolates with various molecular size plasmids as well as in those that had no plasmids.
Keywords - Antimicrobial agents, Staphylococcus aureus, Plasmid Profiles
1. Introduction
Fish can be finfish, shellfish (molluscs and crustaceans), or any other form of marine or freshwater animal life that can be used
for human or domestic animal consumption. About 80% of animal protein in our diet comes from fish alone (Di Pinto et al.,
2008). However, consumption of fish may sometimes cause disease due to infection or intoxication. It is believed to be the
reflection of the general contamination in the aquatic environment. The true incidence of diseases transmitted by fish is usually
unknown. It has been estimated that as few as 1% of the diseases are actual cases of food borne diseases (Rahman, 2005). Fish are
conditioned by their environment and hence it is obvious that if the growing and harvesting environment of fish is polluted
chemically or microbiologically, the fish are also polluted. During transportation of these types of fish to market and wholesale
market, these fish may also infect associated people by handling. When the consumers purchase these fishes, the associated
microorganisms are transferred to consumers (Das, 2007).
In Nigeria, fish is eaten fresh, preserved or processed. The percentage composition of the different methods of fish disposed
for consumption in the artisanal sector according to Mouton et al. (2001) are as follows: live fish 7%, fresh fish 27%, smoke dried
45%, sun dried 20%, salted and sun dried 10%. Various traditional methods are employed to preserve and process fish for
consumption and storage. These include smoking, drying, salting, frying and fermenting and various combinations of these. In
Nigeria, smoking is the most widely practiced method. Practically all species of fish available in the country can be smoked and
it has been estimated that 70-80 percent of the domestic marine and freshwater catch is consumed in smoked form (Omojowo &
Omojasola, 2013). Smoke drying methods used in Nigeria requires low capital, investment and it is conducted in fishermen
camps and fish processing centuries in traditional smoking kilns of clay, cement blocks, drums or iron sheets. This result is in a
very short shelf life and low market value as well as inability to withstand handling and transportation by retailers. Smoked fish
constitute a major source of animal protein for a vast majority of the population in Nigeria, particularly the rural population.
These products can be kept for 2-4 weeks in market stalls with poor storage facilities (Omojowo & Omojasola, 2013).
They are prone to contamination at various stages of handling and processing and the quality is a major concern to food
processors and public health authorities (Oramadike, 2010). From the standpoint of microbiology, fish and related products are a
Aquatic Biology Research (2014) 62-68
63
risky foodstuff group. Particularly, Clostridium botulinum type E and Vibrio parahaemolyticus rank among pathogenic bacteria
associated with fish. Other potentially pathogenic bacteria associated with fish and shellfish include Clostridium perfringens,
Staphylococcus spp., Salmonella spp., Shigella spp., Vibrio cholerae and other vibrios (Di Pinto et al., 2008). Outbreaks usually
occur due to the ingestion of insufficiently heat-treated fish or products contaminated after or during their processing. There is
always a risk of deterioration of quality due to poor or unhygienic handling, transportation and storage. In Nigeria, few microbiological studies have been carried out on fish. Hence, this study attempts to assess the microbiological quality and safety of fish
in relation to environmental condition of fish markets.
2. Materials and Methods
Study Area: Microbial load of the fish samples, isolation of Staphylococcus aureus from the fish samples and antibiotics
sensitivity test of the isolated organisms are the focus of this study.
Sample Collection: In all the fish samples, one gram each were cut and collected aseptically using sterilized sampling bottles.
A total number of three different samples of processed fish were collected from five different local governments in Ekiti state.
The fifteen fish samples were immediately taken to the laboratory for experimental processing.
Isolation of the test organisms: One gram of each of the fish samples collected were aseptically macerated and introduced
into 9ml of sterilized distilled water inside test tube for serial dilution. 1ml from dilution factor 10-4 and 10-5was poured into petri
dishes followed by pouring of the molten media (Nutrient agar and Manitol Salt agar) which has already been prepared and
sterilized. Plates were incubated at 37 ºC 24 hours.
Microbial Count: An automatic colony counter counted the number of colonies from selected plates and the count was
expressed as colony forming unit (cfu/g).
Bacterial Identification: Colonies that appeared white or yellow or cream-colored on MSA plates with yellow surrounding
(indicative of fermentation) were considered as S. aureus and stock cultures were immediately prepared. Isolates were further
identified as S. aureus by testing their ability for DNAse production on agar plates. Gram stain was performed and isolates were
identified by their ability to produce catalase, coagulase and oxidase enzymes the presumptive identification was finally confirmed by Bergey’s manual.
Antibiotics Sensitivity Test of the Test Organisms: Antimicrobial susceptibility testing of Staphylococcus aureus isolates
were performed using Kirby-Bauer disc diffusion method described by the National Committee for Clinical Laboratory Standards (NCCLS) using commercially available antibiotic discs. In this study, the following antibiotic disks were tested: Ceftriaxone (30μg); Cefuroxime (30μg); Ceftazidim (30μg), Cloxicilin (5μg); Gentamicin (10μg), Erythromycin (30μg), Augmentin
(30μg) and Ofloxacin (5μg).
Plasmid Profile of Multiple Antibiotic Resistant Organisms: Plasmid DNA was isolated as described by Birnboim and
Doly, modified by use of lysostaphin for lysing the cell wall. Each Staphylococcus aureus strain was inoculated into 3 ml tryptic
soy broth and incubated overnight on a roller drum at 37°C. About 1.5 ml of each overnight broth culture was transferred into
eppendorf tubes and centrifuged for one minute at 15000 rev/min at room temperature. The supernatant was discarded and 2 μl of
lysotaphin solution (1.0 μg/ml in distilled water) added to the pellet. Tubes were capped, vortexed and placed in ice for 30 minutes. Two hundred microliters of alkaline detergent solution (0.2 N NaOH; 1% SDS) was added and tubes inverted several
times and then kept in the water bath for five minutes. One hundred and fifty microliters of 3 M Sodium acetate (pH 4.8) was
added and tubes inverted several times to mix and then kept in ice for at least 10 minutes. The tubes were centrifuged at room
temperature at 15000 rev/min for five minutes and the supernatant transferred into new eppendorf tubes. One millilitre of 95%
ice cold ethanol was added to the tubes, which were then kept at -20°C for five minutes. After five mins, they were centrifuged at
15000 rpm for three minutes, supernatant discarded and the sediment resuspended in 50 μl of Tris/ETDA (10 mMTrisHCl and 1
mM ETDA. pH 8.0). Thirty three microlitres of the contents were then loaded into wells of 1.0% agarose gels containing ethidium bromide. A 1.0 kb DNA ladder (MBI, Fermentas, Vilnius, Lithuania) was run side by side with test isolates as a molecular
size marker. Electrophoresis was carried in Tris Acetate ETDA buffer containing ethidium bromide (20 ml of 50 X TAE and 6.0
μl of 10 μg/ml ethidium bromide per litre) at 30 mA (90 V) for four hours. Plasmids were viewed on a U/V trans illuminator and
photographs taken using a Leicaflex SL-camera. Films were exposed for 90 seconds and later developed. Plasmid sizes were
estimated from a standard curve drawn of the molecular sizes of the 1.0 kb DNA ladder against their migration distance.
3. Results and Discussion
Fifteen samples of ready to eat fish were collected from five different local governments in Ekiti State (Ado, Ikole, Ikere, Ido-Osi
and Irepodun/Ifelodun Local Governments), and were analyzed.
Figure 1 shows the individual results of microbiological analysis conducted on 15 fish samples. Comparative analysis of total
viable count (TVC) shows great variation from sample to sample as well as from source to source. Dried fish samples from all the
64
Aquatic Biology Research (2014) 62-68
selected local government showed the highest TVC, while the smoked fish samples show the lowest TVC. Chart 1 show the
microbial load of the entire ready to eat fish samples. The highest microbial load was observed in dried fish sample from Ikole
local government (2.15 X 105), and the lowest microbial load was observed in smoked fish sample from Ikole local government
(1.06 X 105). High microbial counts may be due to lack of proper processing on the side of the fish processor and improper
hygiene and handling procedures adopted by the fish sellers. This is in agreement with the findings of Shena and Sanjecv (Shena
& Sanjecv, 2007) who reported that lack of proper processing and proper hygiene handling of fish products would result in a very
high microbial load. Table 2 show the total Staphylococcus count (TSC) of the entire ready to eat fish samples. Dried fish sample
from Ikole local government show the highest total Staphylococcus count (2.8 X 102), while smoked fish sample from Ado local
government show the lowest total Staphylococcus count (1.0 X 102).This may be due to lack of hygienic and sanitary measures of
processors/seller, poor hygiene/sanitary practices relating to fish products, workhouse, packaging and storage as well as the use
of inadequate and inefficient traditional processing facilities. Poor environmental sanitation and high human/vehicular traffic are
also implicated (Patterson & Ranjitha, 2009).
Twenty eight isolates of Staphylococcus aureus were isolated from the entire ready to eat fish samples. The isolates were
screened against eight gram positive antibiotics (Gentamicin, Cloxacillin, Ceftazidime, Ceftriaxone, Ofloxacin, Cefuroxime,
Erythromycin, Augmentin) and their zones of inhibition were recorded.
Table 3 is the zone of inhibition in (mm) of the Staphylococcus aureus isolates to the antibiotics and their interpretation as
compared with the standard chat. Table 4 show the numbers of susceptible, intermediate, and resistant isolates of Staphylococcus
aureus from different samples to various gram positive antibiotics. No resistance case was observed in both gentamycin and
ofloxacin. Table 5 show the numbers of resistant isolates of Staphylococcus aureus from different fish samples to different
numbers of antibiotics. (25%) of the isolates were resistant to 2 and 3 classes of antibiotics, (21.4%) of the isolates were resistant
to 4 classes of antibiotics, and 14.3% of the isolates were resistant to 5 and 6 classes of antibiotics. None of the isolates shows
resistant to 1, 7 and 8 classes of antibiotics. This may be because of acquired resistance through exchange of resistance-code
genes carried in conjugative transposons, phenomine-responsive plasmid, and other host range plasmids (Omojowo & Omojasola, 2013). It may also be as a result of reduction in drug accumulation by decreasing drug permeability and/or increasing active
efflux of the drugs across the cell surface (Aminov, 2009).
Plasmid profiles have been reported to be useful in tracing the epidemiology of antibiotic resistance (Kadlec, 2011). However,
in this study, resistance to various antimicrobial agents was not associated with presence of plasmids. This was because no
particular molecular size plasmid could be associated with any particular antimicrobial resistance (Pontes, 2009). Resistance was
observed in isolates with various molecular size plasmids as well as in those that had no plasmids. This could be attributed to the
variety of sources of fish samples from where the Staphylococcus aureus were isolated. Resistance of Staphylococcus aureus
isolates to various antimicrobial agents may be located either on chromosomes, plasmids or transposons. For example, methicillin resistance gene (mec4916) has a chromosomal locus, and is probably maintained on a mobile element (Sjostrom, 2005).
4. Conclusion
In conclusion, this study showed that the genetic basis for antibiotic resistance in the study area is not entirely plasmid mediated.
Plasmid profile analysis in conjunction with the antibiogram is valuable in differentiating various strains of Staphylococcus
aureus. Proper hygienic condition should be maintained at every processing level to reduce any chance of contamination.
Table 1. Total Staphylococcus count (TSC) of different samples of fish
Number of colonies
Source
Sample
Dilution factor
Plate 1
Plate 2
mean
Total bacterial count
(102CFU/ML)
AdoEkiti
F
D
S
1/10
1/10
1/10
16
20
08
10
16
12
13
18
10
1.3
1.8
1.0
Ire/ifeEkiti
F
D
S
1/10
1/10
1/10
15
26
15
19
22
11
17
24
13
1.7
2.4
1.3
IdoosiEkiti
F
D
S
1/10
1/10
1/10
17
23
17
19
27
15
18
25
16
1.8
2.5
1.6
IkereEkiti
F
D
1/10
1/10
18
24
14
20
16
22
1.6
2.2
Aquatic Biology Research (2014) 62-68
Ikole-Eki
ti
65
S
1/10
08
08
08
0.8
F
D
S
1/10
1/10
1/10
18
27
14
12
29
18
15
28
16
1.5
2.8
1.6
Key:
F – Fried fish, D – Dried fish, S – Smoked fish
Table 2. Resistant Pattern of the Isolates to Various Antibiotics
Antibiotics
Isolates
1
Source
ADF
CAZ
R
CRX
R
GEN
S
CTR
R
ERY
R
CXC
R
OFL
S
AUG
R
2
ADF
S
R
S
R
R
R
S
R
3
ADD
R
R
S
R
R
R
S
R
4
ADD
R
R
S
I
I
R
S
R
5
ADS
R
I
S
R
I
R
S
R
6
ADS
I
I
S
I
I
R
S
R
7
IRF
S
S
S
I
R
R
S
R
8
IRF
S
S
S
S
I
R
S
R
9
IRD
R
S
S
I
I
R
S
R
10
IRD
I
I
I
I
R
R
S
R
11
IRS
R
R
S
R
R
R
S
R
12
IDF
S
R
S
R
I
R
S
R
13
IDD
R
I
S
I
R
I
S
R
14
IDD
R
R
S
R
R
R
S
R
15
IDS
I
S
S
I
I
R
S
R
16
IDS
I
I
S
I
S
R
S
R
17
IKF
S
I
S
I
R
R
S
R
18
IKF
S
R
S
I
S
R
S
R
19
IKD
S
R
I
I
S
I
S
R
20
IKD
R
I
S
R
I
R
S
R
21
IKS
R
R
S
R
S
R
S
R
22
IKS
I
R
S
I
S
R
S
R
23
IKOF
R
R
S
R
S
R
S
R
24
IKOF
R
R
S
I
R
R
S
R
25
IKOD
S
S
S
I
S
R
S
R
26
IKOD
I
R
S
I
I
R
S
R
27
IKOS
I
I
S
I
S
R
S
R
28
IKOS
S
R
S
R
S
R
S
R
KEY:
ADF- Fried fish from Ado
ADD- Dried fish from Ado
ADS- Smoked fish from Ado
IRF- Fried fish from Ire/ife
IRD- Dried fish from Ire/ife
IRS- Smoked fish from Ire/ife
IDF- Fried fish from Idoosi
IDD- Dried fish from Idoosi
IDS- smoked fish from Idoosi
IKF- Fried fish from Ikere
IKD- Dried fish from Ikere
IKS- Smoked fish from Ikere
IKOF- Fried fish from Ikole IKOD- Dried fish from Ikole IKOS- Smoked fish from Ikole
GEN – Gentamicin
CXC – Cloxicillin CAZ – Ceftazidime CTR – Ceftriaxone
OFL – Ofloxacin CRX – Cefuroxime ERY – Erythromycin AUG – Augmentin
R- Resistance
I- Intermediate
S- Susceptible
66
Aquatic Biology Research (2014) 62-68
Table 3. Numbers of resistant isolates of Staphylococcus aureus from different samples to various antibiotics
Source (N)
ADF (2)
CAZ
S
1
I
0
R
1
ADD (2)
S
0
I
0
R
2
ADS (2)
S
0
I
1
R
1
IRF (2)
S
2
I
0
R
0
IRD (2)
S
0
I
1
R
1
IRS (1)
S
0
I
0
R
1
IDF (1)
S
1
I
0
R
0
IDD (2)
S
0
I
0
R
2
IDS (2)
S
0
I
2
R
0
IKF (2)
S
2
I
0
R
0
IKD (2)
S
1
I
0
R
1
IKS (2)
S
0
I
1
R
1
IKOF (2)
S
0
I
0
R
2
IKOD (2)
S
1
I
1
R
0
IKOS (2)
S
1
I
1
R
0
KEY: N – Number of isolates
CRX
GEN
CTR
ERY
CXC
OFL
AUG
0
0
2
0
0
2
0
2
0
2
0
0
1
1
0
0
0
1
0
0
1
0
1
1
1
1
0
0
1
1
0
1
1
0
0
2
0
0
2
0
1
1
0
1
1
2
0
0
2
0
0
2
0
0
2
0
0
1
1
0
1
0
0
1
0
0
2
0
0
2
0
0
2
0
0
1
1
0
1
0
1
2
0
0
2
0
0
2
0
0
0
0
2
0
1
1
0
1
1
1
1
0
0
2
0
0
0
1
0
0
1
0
1
1
0
2
0
0
2
0
0
1
1
0
1
1
0
1
1
0
2
0
0
1
1
0
0
2
0
1
1
0
2
0
0
1
1
0
1
1
0
0
1
0
1
0
0
0
2
1
1
0
1
0
1
1
1
0
2
0
0
1
0
1
1
1
0
2
0
0
0
0
2
0
0
2
0
0
2
0
0
2
0
0
2
0
0
1
0
0
1
0
1
1
0
0
2
0
0
2
0
1
1
0
0
2
0
0
2
0
0
2
0
0
2
2
0
0
2
0
0
2
0
0
2
0
0
2
0
0
1
0
0
0
0
1
2
0
0
2
0
0
2
0
0
2
0
0
2
0
0
2
0
0
2
0
0
2
0
0
0
0
2
0
0
2
0
0
2
0
0
2
0
0
2
0
0
1
1
0
0
0
0
2
0
0
2
0
0
2
0
0
2
0
0
2
0
0
2
0
0
2
0
0
2
Aquatic Biology Research (2014) 62-68
67
Fig. 1. Microbial load of the fish samples
250
dried
215
200
dried
174
fried
155
150
dried
135
fried
smoked
119
111
dried
158
fried
138
smoked fried
116 116
smoked
110
fried
155
dried
142
fried
smoked
112
smoked
106
dried
smoked
100
50
0
ado
ire/ife
ido osi
ikere
ikole
Fig. 2. Plasmid profile of Staphylococcus aureus after the EcoRI restriction digestion reaction set up with the selected 15 plasmid
DNA.
2500
2000
1500
1000
750
500
250
L
1
2
3
4
5
9
11
12
14
17
20
21
23
2428
68
Aquatic Biology Research (2014) 62-68
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