One health approach: Isolation, Antibiogram and Factors Associated with Staphylococcus Aureus from Fish at the Landing Site and Selected Restaurants in Central Gondar, Ethiopia
ABSTRACT

In developing countries, food-borne infections constitute a major public health problem. Food-related illnesses are caused by various factors, with Staphylococcus aureus being a significant contributor to both veterinary and public health issues. This study aimed to isolate Staphylococcus aureus, determine its antimicrobial resistance patterns, and assess its prevalence and associated factors in unloading sites and restaurants in central Gondar, Ethiopia. A cross-sectional study was conducted from December 2023 to September 2024, involving the collect (Preprint)
Authors: Gondar university , Kenubih Ambaye , mokennen misganaw 
Published: February 2025

BACKGROUND

Fish plays a vital role in the human diet, with an ever-growing global demand. Over the past 60 years, world fish production has dramatically increased, reaching approximately 179 million tons in 2018, with a value of $401 billion. Similarly, global fish consumption rose from 9.0 kg per capita in 1961 to 20.5 kg in 2018, marking a significant transformation in the fishery industry (Hussein, 2014; FAO, 2018). In Africa, fish contributes 19% of animal protein and essential micronutrients, particularly fatty acids that cannot be replaced by other food commodities (Quinlan, 2013). Fish consumption in Africa averages 10.8 kg per person per year, whereas in Ethiopia it is significantly lower at just 0.2 kg per person per year (Breuil and Grima, 2014).
Ethiopia, being a landlocked nation, relies entirely on its inland lakes, reservoirs, and rivers for fishing resources (Seo and Bohach, 2007). The country’s annual fish production potential is estimated at 51,400 tons (Mainous et al., 2006). However, the domestic fish market is relatively small outside major fishing regions. Most lakes are located within the East African Rift Valley system, with Lake Tana being the largest, accounting for over half of Ethiopia’s inland water area (Alazar, 2016).
Fish is an ideal dietary option due to its high nutritional value and easy digestibility (Adugna et al., 2019). However, fish meat is highly susceptible to various bacterial infections, many of which are pathogenic, while others are saprophytic in nature (Bujjamma and Padmavathi, 2015). Bacterial pathogens in fish include zoonotic and pathogenic bacteria such as Edwardsiella, Salmonella, Escherichia coli, Staphylococcus aureus, Vibrio, and Aeromonas. These pathogens have been isolated from fish in various parts of Ethiopia (Otte et al., 2021). Fish diseases caused by bacterial infections include dropsy, epizootic ulcerative syndrome (EUS), swim bladder disease, scale loss disease, fin rot, and tail disease (Adugna et al., 2019). Most pathogenic bacteria are naturally occurring saprophytes and opportunistic pathogens that invade fish tissue under favorable conditions (Hussein, 2014).
Zoonotic diseases are estimated to cause 2.5 billion cases of human illness globally every year (Salyer et al., 2017). More than 60% of existing and 75% of emerging or re-emerging human diseases are zoonotic, with 36% of these diseases linked to food-producing animals (Otte et al., 2021).

Staphylococcus aureus is a major cause of foodborne illnesses, primarily through the consumption of preformed staphylococcal enterotoxins. These enterotoxins are highly heat-stable and often associated with staphylococcal foodborne intoxication (SFI) (Dabassa et al., 2019). Prepared foods containing more than 10³ colony-forming units per gram (cfu/g) of S. aureus are considered unsatisfactory, and counts exceeding 10⁴ cfu/g render the food potentially harmful for consumption. Consuming food contaminated with staphylococcal enterotoxins in amounts as small as nanograms to micrograms can cause severe illness, ranging from mild skin infections to life-threatening conditions (Seo and Bohach, 2007).
Improper refrigeration or exposure to elevated temperatures during food processing often creates conditions favorable for the growth of S. aureus (Dabassa et al., 2019). The bacterium colonizes 30–50% of the healthy human population, with the anterior nares of the nose being the most common carriage site (Wertheim et al., 2005). According to the National Health and Nutrition Examination Survey (2001–2002) in the United States, approximately 32.4% of the non-institutionalized population, including children and adults, were nasal carriers of S. aureus (Mainous et al., 2006).
Preventing staphylococcal food poisoning can be challenging, as carriers often exhibit no symptoms. A cross-sectional study conducted in Gondar revealed that 16.5% of fingernail samples from 127 food workers in cafeterias tested positive for S. aureus (Andargie et al., 2008). Similarly, in Botswana, 57.5% of 200 food workers tested positive for S. aureus (Loeto et al., 2007).
Globally, antimicrobial-resistant S. aureus poses a significant threat to public health. Unhygienic and improper food processing practices are major contributing factors to the emergence of resistant strains (Quinlan, 2013). In developing countries like Ethiopia, where raw fish consumption is common, antimicrobial-resistant S. aureus strains are an emerging concern (Dabassa et al., 2019). The objective of the current research is to provide baseline data on the status of S. aureus along the fish value chain in Gondar town and contribute, if possible, to the development of national food safety strategies (Mulder et al., 2020).

OBJECTIVE

1.3. Objective of the Study

1.3.1. General objective

	To isolate S. aureus and assess its antibiogram and factors associated for its occurrence in unloading sites and selected restaurants of Gondar city North west Ethiopia
1.3.2. Specific objectives

	To isolates S. aureus from fresh and ready to eat fish at unloading sites and restaurants. Respectively.
	To assess the antimicrobial susceptibility pattern of S. aureus.
	To assesses factors associated with fishing activity and food safety in study sites.

METHODS

3. MATERIALS AND METHODS

3.1. Study Area

The study was conducted in Gondar city (restaurants) and in Lake Tana unloading sites (miterha, sheha and gorgora). The city of Gondar is situated in North-western parts of Ethiopia, Amhara Regional State. It is at 120 3‟ N latitude and 370 28‟E.Longitude Gondar is located at 727 km from Addis Ababa, the capital city of federal government of Ethiopia, and 120 km from Bahir Dar, the capital city of Amhara National Regional State. Gondar has five sub cities and a total area of 192.3 km2 with undulating mountainous topography. According to the 2023 National Population and Housing Census estimation Gondar consists of a total of 675651peoples. Gondar is the center of political and economic activities of the North Amhara region and it is main city of the central Gondar Zone. It stands at an elevation of 7,500 feet (2,300 meters) on a basaltic ridge from which streams flanking the town flow to Lake Tana, 21 miles (34 km) Gondar City Administration. (2015).
Lake Tana is the head quarter water source of Blue Nile River and is the largest fishing sites in the region and the country which is almost dominated by artisanal fishermen. This lake is found in Amhara Region and has a surface area of 32,000 km2 with a maximum and minimum depth of 14m and 8m respectively. The Lake provides commercially important fish groups; namely, African Cat fish (Clarius gariepinus) locally called “Ambaza”, Nile tilapia (Oreochromis niloticus) locally called “Kereso” and Labeobarbus spp (cypernidie) locally called “Nech Asa”.

Figure 1: Map of the study area and Geographical location of Gondar city and Lake Tana
Source (GIS software, 2020).

3.2. Study Population and Sample Type

The study population consists of raw fish (both fresh unfilleted and filleted), including species such as Nile Tilapia (Oreochromis niloticus), Labeo Barbus (Cyprinidae), and Catfish (Clarias gariepinus), which were collected by fishermen from Lake Tana. Additionally, frozen and cooked fish (ready-to-consume) were included, such as: Asa Tibs (fried fish): A dish prepared by gutting or removing inedible parts of the fish, followed by thorough cooking in oil at high temperatures for human consumption. Asa Lebleb (undercooked fish): A dish made by cutting the muscle into pieces, adding spices, and cooking at low temperatures. Asa Wot (fish stew): A dish prepared by mincing the fish muscle, adding spices, and cooking at high temperatures for human consumption. The types of samples included in the study were: Fish meat swabs from raw and prepared fish, Hand swabs from fisheries and restaurant workers, and Knife swabs from knives used for fish processing.

3.3. Study Design and sample size determination

A cross-sectional study was conducted from December 2023 to September 2024. In addition, an observational checklist and a pre-tested questionnaire were administered to workers along the value chain, including restaurants, to determine possible sources and sites of contamination within the value chain. The sample size was determined using the Thrusfield formula (Thrusfield, 2005), with a calculated total sample size of 384. However, since the fish population in Lake Tana was unknown, the sample distribution was based on data from the Gondar Zuria Wereda Fishery Sector. According to this data, fisheries collect 10% Catfish, 30% Labeo Barbus, and 60% Nile Tilapia. Therefore, a total of 301 samples were investigated.

3.4. Sampling Method
Twelve restaurants were selected using purposive sampling from five sub-cities. These five sub-cities, identified based on Gondar City Administration data (2015), include Arada Kefle Ketema, Azezo Teda Kefle Ketema, Zobel Kefle Ketema, Facile, and Maracie Kefle Ketema.
In Lake Tana, three landing sites—Mitreha, Sheha, and Gorgora—located in Gondar Zuria Wereda and West Denbia Wereda were also selected through purposive sampling. At the fishing sites, fishing activities occurred primarily twice per day (morning and afternoon). Fish samples from unloading sites were collected using a simple random sampling method, based on the number of fishing activities per day and the number of trips made to unloading sites by the fishery.
A total of 301 samples were collected, including 201 samples from unloading sites and 100 samples from restaurants. These were gathered over a total of 18 observations: 12 observations for collecting swab samples from unloading sites and 6 observations for collecting swab samples from restaurants in Gondar City.
3.5. Data Collection Procedure

3.5.1. Sample Collection

Hand swab and knife swab samples were collected from selected restaurants using sterile sampling bottles containing buffered peptone water and kept in an icebox with ice packs. Similarly, swab samples from raw or fresh fish at selected fishing sites were collected and placed in sterile sampling bottles containing buffered peptone water. Sterile cotton swabs were used to transfer the fish swab from the uncooked fish meat plate to the sampling containers.
After labeling and coding with all necessary information, the samples were immediately transported to the University of Gondar College of Veterinary Medicine and Animal Sciences, Veterinary Microbiology Laboratory, using an icebox with ice packs. The samples were processed within 24 hours of collection, and all inoculums were incubated overnight at 37°C for the isolation and identification of Staphylococcus aureus from fish meat swabs and contact surface sampling swabs.
3.5.2. Questionnaire Survey and Observation
A pre-tested questionnaire was designed to collect separate data (Annex V and VI) for fish processors in restaurants and fish harvesters at unloading sites. Questionnaire surveys were conducted to assess factors associated with cross-contamination in raw and ready-to-eat fish-derived foods. The questionnaire also included socio-demographic factors, transport-related issues, educational status, and personal hygiene practices.
Respondents were purposively selected based on their significant roles in food processing and handling. The questionnaire and observational checklists were administered in accordance with the standard guidelines of the Codex Alimentarius Commission of the Food and Agriculture Organization (Annex VII) (FAO, 2009).
3.6. Isolation and Biochemical Tests

The assignment of Staphylococcus aureus species and final identification of staphylococcal organisms were performed using various culturing methods, followed by Gram staining and biochemical tests. The biochemical tests included the catalase test, tube coagulase test, slide coagulase test, Voges-Proskauer test, and mannitol sugar fermentation test. Both coagulase tests, using rabbit plasma, were conducted in parallel to further confirm the identification of S. aureus.
Gram's staining
Gram staining was performed on all suspected Staphylococcus species cultures, and the sizes, shapes, and cell configurations of the cultures were examined under a light microscope. Presumptive Staphylococcus species were identified based on Gram-stained smears of typical colonies, which revealed Gram-positive cocci arranged in irregular grape-like clusters.
Catalase test
Using a bacteriological loop, pure cultures of the isolates to be tested for catalase were removed from the agar plate and mixed with a drop of 3% hydrogen peroxide on a sanitized slide. Within a few seconds, bubbles of oxygen were released, indicating a positive reaction and the presence of Staphylococcus aureus (Quinn et al., 2002).
Mannitol salt Agar (Mannitol fermentation)
The colonies were streaked onto Mannitol Salt Agar plates and incubated at 37°C. Growth was checked after 24 to 48 hours. These colonies were confirmed through Gram staining, hemolysis on blood agar, colony characterization, and a positive catalase test. The presence of growth and a pH shift from red to yellow in the medium indicated the presence of coagulase-positive Staphylococcus aureus. Fermentation of mannitol by S. aureus causes the medium to turn yellow within 24 hours of incubation (Quinn et al., 2002).
Coagulase test
Both slide coagulase and tube coagulase tests were used as coagulase assays. Staphylococcus aureus presumed to be identified from Mannitol Salt Agar was subcultured onto a nutrient agar plate. After 24 hours, the culture colonies of S. aureus were selected using a bacteriological loop, placed on a clean slide, and emulsified. A drop of rabbit plasma was added to the test suspension, and it was thoroughly mixed with a wire loop for five to ten seconds.Clumping of the cocci was interpreted as a positive result (Quinn et al., 2002). For the tube coagulase test, 0.5 ml of selected Staphylococcus isolates cultured in tryptic soy broth at 37°C for 24 hours was added to 0.5 ml of rabbit plasma in sterile tubes. This test was conducted for those isolates that were negative in the slide coagulase test. Any visible clotting inside the tube, ranging from a loose to a firm clot that remained immovable when the tube was inverted (tilted), was considered a positive result. No clotting at all was interpreted as a negative result (Quinn et al., 2002).

Vogues proskauer test

The Voges-Proskauer test is a biochemical test that detects the ability of bacteria to metabolize pyruvate into a neutral intermediate product called acetylmethylcarbinol or acetoin. The test is performed by adding alpha-naphthol and potassium hydroxide to the Voges-Proskauer broth. This test is conducted on Gram-positive, catalase-positive species to identify coagulase-positive Staphylococcus aureus (Quinn et al., 2002).

3.7. Antimicrobial Susceptibility Profile
All isolates of S. aureus were subjected to an antibiotic susceptibility test using the Kirby-Bauer agar disc diffusion method, following the Clinical Laboratory Standards Institute (CLSI) guidelines of the USA, on Mueller-Hinton agar (MHA). The antibiotics were selected based on their availability and relevance for routine testing and reporting on non-fasidious organisms. One representative antibiotic from each subclass of commonly used and widely available antibiotics for treating staphylococcal-related diseases in both animals and humans was chosen. Based on these criteria, seven antibiotics were selected for this study: chloramphenicol (30 µg), ciprofloxacin (5 µg), vancomycin (30 µg), erythromycin (15 µg), gentamicin (10 µg), tetracycline (30 µg), and penicillin (10 units) (CLSI, 2020).
For the susceptibility test, three to five well-isolated colonies of the same morphological type were selected from a nutrient agar plate culture and transferred into test tubes containing sterile saline. The suspension was mixed thoroughly, and the density was adjusted to 0.5 McFarland using saline or additional S. aureus colonies. A sterile swab was dipped into the suspension, and the excess inoculum was removed by pressing it against the sides of the tube to prevent over-inoculation of the plates. The inoculum was spread evenly over the entire surface of the agar plate by swabbing in three directions. Antibiotic discs were applied firmly onto the agar surface, and the plates were incubated for 24 hours at 37°C. The diameter of the zone of inhibition around each disc was measured using a ruler in millimeters (mm) and interpreted according to the CLSI standards as susceptible, intermediate, or resistant. Isolates showing resistance to three or more antibiotics were considered multiple drug-resistant (MDR) (Beyene et al., 2017).
3.9. Data Analysis

All data collected during the study period were checked, coded, and entered into an Excel spreadsheet before being analyzed using STATA software version 16 (Texas 77845, USA). Descriptive statistics, such as percentages and proportions, were used to compute the number of fish samples positive for S. aureus. A univariable logistic regression model was employed, and variables with a p-value of <0.05 were exported to a multivariable logistic regression model to assess the effects of potential confounders. The degree of association between risk factors and the occurrence of S. aureus in fish samples was quantified using the adjusted odds ratio obtained from the multivariable logistic regression models. In all analyses, the confidence level was set at 95%, and a p-value of less than 5% (P < 0.05) was considered statistically significant.

RESULTS

4. RESULTS

4.1. Occurrence of Staphylococcus aureus

From a total of 301 samples taken, 79 (26%) were found positive for S. aureus. Out of this 53(26%) on fish and 26(26%) on hand and knife swabs were found positive for S. aureus. The occurrence of S. aureus on fish and both contact surfaces have statistically significant difference (OR=2.2, p=0.024) (Table 2).

Table 1: Occurrence of Staphylococcus aureus from fish and contact surfaces
Study Site	No. of samples	No of positives (%)	OR	P-value 95%CI	(n=301)	(n=79)
On fish	201	53(26%)
On hand and knife	100	26(26%)	2.2 0.024	1.10304-1.4739
Total	301	79(26%)
Key: OR= Odds Ratio, statistically significant at p<0.05, CI=Confidence Interval

4.2. Isolation of Staphylococcus aureus Related to species.

From a total of 201 fish samples, Nile Tilapia 24% (n=30), Labeobarbus 27% (n=13) and African Cat fish 36% (n=10) were identified positive for S. aureus. Based on the above occurrence of S. aureus on different fish species at different swab sites are statistically non-significant (OR= -1.281561, P=0.272) Table 3 shows the occurrence of S.aureus from different fish species.

Table 2: Isolation of Staphylococcus aureus related to species
Risk factors	No. of samples No. of positive (%) OR	P-value	95%CI
(n=201)	(n=53)
Species
Cat fish	28	10(36%)	1.281561	0.272	0.823-1.996
Labeobarbus	48	13(27%)
Nile Tilapia	125	30(24%)
Total	201	53(26%)
Key: OR= Odds Ratio; statistically significant at p<0.05, CI=confidence interval
4.3. Isolation of Staphylococcus aureus from Fish Samples at different sampling sites

From a total of 201 samples examined the proportion of S.aureus was 36% (n=24) from Miterha, 19% (n=13) from Sheha, and 24% (n=16) from Gorgora were positive for S.aureus showed that the prevalence had statistical insignificant (OR=1.237537 and p= 0.157) but the occurrence of S. aureus in unloading sites 26%(n=53) and in restaurants 26%(n=26) had statistically significant differences (OR=2.2 and p=0.024) among the sampling sites below Table 4
From a total of 201 raw fish samples; 97 fresh unfilleted and 104 fresh filleted fish were sampled at three fish unloading sites. From collected fresh unfilleted fish samples 13 % ( n=13) and fresh filleted fish samples 39% (n=40) of S. aureus were positive. These differences were found statistically significant (OR=2.980519 and p=0.026) (Table 4).

Table 3: Isolation of Staphylococcus aureus from samples at different sampling sites
Sample type and	No o	f samples	No of positives	OR	P-value	95% CI
Sample site	(n=301)	(n=79)
Raw unfilleted	97	13(13%)
Raw fillted	104	40(39%)	2.980519	0.026	1.139-7.797
Total	201	53(26%)
Miterha	67	24(36%)
Sheha	67	13(19%)	1.237537	0.157	0.921-1.663
Gorgora	67	16(24%)
Gondar	100	26(26%)
Total	301	79(26%)
In unloading sites	201	53(26%)	2.2	0.024	1.109-4.366
In restaurants	100	26(26%)
Total	301	79(26%)
Key: OR= Odds Ratio; statistically significant at p<0.05, CI=confidence interval

4.4 The Occurrence of S. aureus From Different Swab Sites

The present study result revealed that the prevalence of S. aureus was on hand swabs (from fish handlers) is 20% (n=10), fish meat swabs (from fishes) is 26% (n=53), and 32% (n=16) on Knife swabs (from knives used for processing) Were positive by S.aureus However, there is no statistically significant difference between hand swab, knife swab and fish meat swab (OR=0.9795685, P-value=0.921) as Table 5 shows the occurrence of S.aureus from different swab sites.

Table 4: The occurrence of S. aureus from different swab site
Swab site	No of Samples	No of positives	OR	P-value	95%CI
(n=301)	(n=79)
Hand Swab	50	10(20%)
Knife Swab	50	16(32%)	0.9795685	0.921	0.652-1.472
Fish meat swab	201	53(26%)
Total	301	79(26%)

Key: OR= Odds Ratio; statistically significant at p<0.05, CI=confidence interval

4.5. Isolation and Identification of Staphylococcus aureus Species

The present study result revealed that from a total of 301 fish, hand and knife swab samples 120 (40%) samples have beta hemolysis, 102 (85%) samples were gram positive, 87 (85%) samples were catalase positive 80 (92%) samples shows mannitol fermentation (yellow zone around colonies, 79(99%) samples were coagulase positive and 79(100%) samples were positive for vogues proskauer.

Figure 2: Image of S. aureus Slide Coagulase Test on Rabbit Plasma

4.6. Questionnaire Survey Results

4.6.1. Demographic Characteristics of the participants

From a total of 90 respondents 66 fishermen and 24 restaurant workers engaged in fishing activity and fish origin food processers were interviewed in the study area. Out of participants 76(84.44%) were males and also 58(64.44%) were literate (Table 6).

Table 5: Demographic characteristics of the participants (n=90).
Variables	Description	No. of Respondents
Sex	Male	76(84.44%)
Female	14(15.55%)

Age	20--30	26(28.88%)
31—40	41(45.55%)
>40	23(25.55%)

Educational-	Literate	58(64.44%)
Status	Illiterate	32(35.55%)

Years of business-	1-2 years	20(22.22%)
Experience	3-5 years	24(26.66%)
6-10years	24(26.66%)
Above 10 years	22(24.44%)

4.6.2. Questionnaire for Fish Harvesters at unloading Sites

Total number of the respondents transport fishes without ice by using a plastic bag and most of them harvest Nile Tilapia fish 41(62%). above 60(90%) of the respondents did not wash or clean their boats before and after starting of fishing activity and 54(81.8%) of the respondents had known on improper transportation of fish and improper use of hooks and filleting boards can be a source of fish food contamination. Most of the fishery men were sold the caught fishes within 6hrs–12hrs (Table 7).

Table 6: Questionnaire about food safety for fish harvesters and filleters at unloading sites (n=66).
Statements	Value	No of respondents (%)
Improper transportation,	Yes
improper use of hooks
and filleting boards	No	54(81.8%)

12(18.2%)
0(0%)
66(100%)
38(57.57%)
28(42.42%)
16(24.24%)
18(27.27%)
32(48.48%)
45(68.18%)
21(31.81%)
66(100%)
0(0%)
Transportation of fish
to the next chain
Time to market all
Harvested fish
Type of containers
Used to carry fish

Wash hands before and
after handling of fish
Wash hands after
Using toilet	with ice
Without ice
6 to 12 hours
2 to 6 hours
plastic bag
wooden basket
Others
Yes
No
Yes
No

4.6.3. Data Obtained by Direct Observation on Fish Handlers

To evaluate the hygienic practices and status of the fish handlers operating in the kitchens of several restaurants. From a total of 24 workers 14(58.33%) wash hands before starting work,100% workers no discharge, workers wear 19(79%) hair covers %16(67%) over coat and 18(75%) workers were clean. Table 8 shows data obtained by direct observation.

Table 7: Direct observation on fish handlers in restaurants. (n=24)
Observational points	Value	Frequency	Percentage (%)
Washing of hand before
Starting work	Yes
No	14
10	58.33%
41.66%
Discharge from nose, eye,	Observed	0	0%
Ear and coughing	Not observed	24	100%
Wear of jewelry or ring	Observed
Not observed	21
3	87.5%
12.5%
Wear of appropriate	Yes	19	79.16%
Hair covers	No	5	20.83%
Wear of appropriate overcoat	Yes
No	16
8	66.66%
33.33%
Cleanness of overcoat
and visible body part	Clean
Not clean	18
6	75%
25%

4.7. Antimicrobial Susceptibility Profile

All 79 S. aureus isolates were tested to seven selected antimicrobial agents on Muller Hinton agar by disc diffusion methods. In the present study, all isolates of S. aureus were susceptible to ciprofloxacin (100%), Chloramphenicol (92%), Vancomycin (84%), and Gentamycin (68%) However Erythromycin (55%), Tetracycline (72%) and Penicillin G (87%) isolates were the highest levels of resistance as shown in table 9.

Table 8: Susceptibility of S. aureus isolates against some selected antimicrobials.
Antimicrobial	Antimicrobial	Susceptibility pattern of Staphylococcus aureus
drugs	concentration
Susceptible	Intermediate	Resistant
Ciprofloxacin	5 μg	79 (100%)	0 (0%)	0 (0%)
Chloramphenicol	30 μg	73 (92%)	2 (3%)	4 (5%)
Vancomycin	30 μg	66 (84%)	8 (11%)	5 (6%)
Gentamycin	10 μg	54 (68%)	11 (14%)	14 (18%)
Erythromycin	15 μg	22 (28%)	13 (17%)	44 (55%)
Tetracycline	30 μg	14 (17%)	8 (11%)	57 (72%)
Penicillin G	10 Unit	7 (9%)	3 (4%)	69 (87%)

Figure 3: The antimicrobial susceptibility profile of S. aureus to the selected antibiotic discs.
According to the current investigation 57% (n=45) of S. aureus samples tested positive for multidrug resistance (MDR). The results of the antibiotic susceptibility tests indicated that the isolates exhibited traits of a general pattern of multidrug resistance. The highest MDR of drugs which used during susceptibility test (Penicillin G, Tetracycline, and Erythromycin).

Table 9: Patterns of drug resistance of S. aureus isolated from fish and fish contact surfaces.
Frequencies	Antimicrobial’s resistance pattern	No of resistant	%
Three	P,TET,ER	29	37%
P,TET,VAN	1	1%
P,TET,GEN	3	4%
P,ER,GEN	2	3%
Total	35	44%

Four	TET,P,ER,GEN	2	3%
P,ER,TET,VAN	1	1%
P,TET,VAN,GEN	1	1%
Total	4	5%

Five	P,TET,ER,GEN,CHL	3	4%
TET,P ER,GEN,VAN	2	3%
P,TET,ER,GEN,CHL	1	1%
Total	6	8%
TET- Tetracycline, P- Penicillin G, ER- , Erythromycin, GEN- , Gentamycin, VAN- Vancomycin , CHL- Chloramphenicol.

CONCLUSIONS

6. CONCLUSION AND RECOMMENDATION

The present study revealed that more than one-fourth of the samples were positive for S. aureus. S. aureus was found in large quantities on knives and hands that came into contact with fish meat. The presence of S. aureus was detected in samples taken from hand swabs, fish meat swabs, and knife swabs. A statistically significant difference was observed between the study sites, with samples from Lake Tana unloading sites and restaurants in Gondar City. Furthermore, a significant difference was found between fresh unfilleted and fresh filleted fish samples. The study revealed that respondents had the habit of handling fish with bare hands at the landing sites, which may explain the high number of S. aureus isolates in fresh filleted fish. This contamination could be attributed to the direct contact of fish handlers' hands and knives during the processing or filleting of fish. It is also likely a result of the poor personal hygiene and sanitation practices of workers at fish meat vendors, as most did not clean their tools with detergents after use.
All S. aureus positive samples were tested using various biochemical tests. Additionally, the S. aureus isolates were tested for susceptibility to seven selected antimicrobial agents on Mueller-Hinton agar using the disc diffusion method. Penicillin and tetracycline exhibited high resistance.
Based on the findings of this study, the following recommendations are made:
1.	Improve the awareness of fish meat sellers and workers about safe fish meal preparation, handling, and distribution. Attention should be given to the cleanliness of fish meat seller areas.
2.	Regularly monitor the status of antibiotic resistance in S. aureus.
3.	The treatment of choice for diseases caused by S. aureus in humans and animals should be ciprofloxacin and chloramphenicol.
4.	Further research should be conducted on antimicrobial resistance-producing genes in S. aureus.
5.	Continuous training must be provided to fishery societies and restaurant workers on hygiene and food safety practices.