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Antibiotic Sensitivity Pattern of Papogenic Bacterial Isolates From Diseased Clarias gariepinus From Selected Ibadan And Ikorodu Farms.

Byline: Nkechi C. Okeri A. Olubusola Odeniyi and K. Olanike Adeyemo

Abstract: pis study was carried out to isolate characterise and identify bacteria from diseased Clarias gariepinus and also assess pe occurrence of resistance to antimicrobial in isolated bacteria. Samples of diseased African Catfish were collected for a period of six weeks from consenting farms in Ibadan and Ikorodu in Nigeria and examined for clinical signs of disease. Pond water samples along wip organs such as gills skin intestine kidney and lungs from pese fis h were analyzed microbiologically using differential and selective media. Bacteria enumeration identification and biochemical characterization were carried out and pe physiochemical parameters of pe water samples determined. All isolates were subjected to antibiotic sensitivity test using pe standard Kirby-Bauer disc diffusion mepod. pe total bacterial load for pe organs ranged between 3.0 x 104 (lungs sample) and 6.0 x 107cfu/g (gill sample).

pe gills had pe highest average total bacterial count while lungs had pe least. Mophologically unique bacterial isolates obtained included Salmonella (14 isolates) Pseudomonas (4 isolates) Aeromonas (2 isolates) Edwardsiella (3 isolates) and Shigella (3 isolates). pese isolates displayed antibiotic resistance profile to pe following: Ceftazidime (38%) Cefuroxime (77%) Gentamicin (37%) Cefixime (73%) Ofloxacin (23%) Augmentin (66%) Nitrofurantoin (58%) and Ciprofloxacin (15%). Two Salmonella isolates had multi-drug resistance pattern. pis study showed pat indiscriminate use of unlicensed or unapproved antibiotics for aquaculture portends significant hazards to public healp perefore disease prevention is preferable prough good culture and healp management to ensure optimum yields and wholesome products.

Keywords: Aquaculture Fish disease Bacteria Antibiotics resistance Antimicrobial susceptibility. INTRODUCTION

Aquaculture is farming of high-protein aquatic organisms including fish molluscs crustaceans and aquatic plants under controlled or semi-controlled environment to enhance productivity i.e. stocking feeding and protection from predators [1]. Aquaculture is extremely valuable in developing countries. It serves as a chief source of protein to population income generation employment and collateral for obtaining loans. Over pe past pree decades aquaculture has intensified and diversified leading to heavy movement of animal and animal products such as broodstock fingerlings (seeds) and feeds which are largely responsible for pe introduction and spread of papogen and disease into aquaculture system [2].

Diseases in fish can be classified as infectious and non-infectious. Infectious diseases are contagious diseases caused by papogens (bacteria fungi virus protozoa and parasites) while pe non-infectious diseases can be broadly characterized as environmental and nutritional [3]. Bacterial infections are pe most recurrent type of disease problem in all types of aquaculture production followed closely by fungal diseases [4]. Aquaculture in Nigeria hangs essentially on catfish culture and its development which will enhance fish supply [5]. Alpough aquaculture is growing rapidly disease prevention and treatment practices are far from standardization or regulation [6]. Fish and fishery products are in pe forefront of food safety and quality improvement because pey are among pe most internationally traded food commodities.

However pe use of antimicrobial agents in aquaculture and pe possibility of antibiotic resistance among bacterial flora from fish have been identified [7]. pe use of antimicrobial agent in aquaculture can perefore result in increased prevalence of resistant bacteria which can cause a direct spread of resistance from aquatic environments to humans [8] via consumption of aquaculture food products direct contact wip culture water or aquatic organisms or prough pe handling of aquaculture food products [9]. Bacterial species of public healp importance include: Aeromonas Edwardsiella tarda Escherichia coli Plesiomonas shigelloides Salmo- nella Shigella Vibrio cholera V. Parahaemolyticus and V. vulnificus [10 11]. pe risk of administering antibiotics pat may select for not only drug-resistant

papogenic bacteria of fish but also papogens of human healp significance has led to a rising concern over worldwide aquaculture and fish healp management practices. Developed countries are especially vigilant in pis domain and pe resulting debates have led legislations to dictate requirements clearly limiting pe veterinary use of antimicrobial drugs in aquaculture [12]. Some antibiotics have been prohibited from use in food producing animals in some countries (e.g. fluoroquinolones in pe USA. pe aims of pis study were to assess pe physicochemical parameters of culture water determine microbial load of culture water and organs of diseased Clarias gariepinus from farms in Ibadan and Ikorodu: isolate and characterise bacterial papogens from pe diseased Clarias gariepinus samples and evaluate pe antibiotic susceptibility patterns of pe bacterial papogens isolated from diseased Clarias gariepinus.

MATERIALS AND MEpODS Study Area

Ibadan and Ikorodu were selected as pe study locations based on pe aggregations of fish farms in Ibadan while Ikorodu has a 34-hectare fish farm estate at Odogunyan Ikorodu. Ibadan is located in Oyo state its geographical coordinates are 723'16'' Norp 353'47'' East while Ikorodu is geographically located in Lagos State wip geographical coordinates 637'0''Norp 331'0'E in Nigeria.

Sample Collection and Processing

Samples were collected over a period of six weeks from a total of eight consenting farms depending on availability of diseased fish [five farms from Ikorodu Fish Farm estate (A-E) and pree from Ibadan (I-III)]. Samples of water (eight) were collected in sterile plastic 1L containers from each pond (transported on ice in a cooler box) while moribund diseased fish (fifty fish samples) were collected in clean sterile cooler containing ice pack and pen transported to Fish Disease Diagnostic Laboratory of pe Department of Veterinary Public Healp and Preventive Medicine University of Ibadan. Fish samples were examined for clinical disease and skin gill lungs intestine and kidney samples were obtained using sterile scalpel [13]. One gram of each sample was weighed and mixed wip 9ml of 0.1% sterile peptone water (oxoid CM9) placed in a sterile stomacher bag

(Stomacher 400 Seward medicals) and homogenized in a stomacher blender (Seward Medical United kingdom) Ten-fold dilutions of pe homogenates were made and plated [14].

Bacteria Isolation Enumeration and Identification

All pe chemicals and reagents used were of analytical grade. Media used in pis study were Nutrient Agar (NA) Nutrient brop (NB) Peptone water (PW) Salmonella- Shigella Agar (SSA) Eosin Mepylene Blue Agar (EMB) and Tryptone Soy Agar (HiMedia Lab. Pvt. Mumbai India). All media were prepared according to manufacturer's specification and sterilized at 121 for 15mins. From pe ten-fold serial dilutions of homogenates 1ml aliquots of homogenates were plated in replicates on different media using pe pour plate mepod [15]. pe plates were incubated at 37 for 18-24hrs. pe total viable aerobic bacteria count was performed on Nutrient agar. Colonies were counted using pe Lapiz(R) digital colony counter (Mumbai India) and expressed as Colony forming unit per gramme of suspension (Cfu/g). Discrete colonies were sub-cultured unto fresh agar plates by streaking aseptically to obtain pure culture of pe isolates.

Pure cultures of bacterial organisms were identified using standard colony morphological responses on pe different chromogenic media along wip oper microscopic and biochemical procedures [16].

ANTIBIOTIC SENSITIVITY TEST

Commercially available antibiotics disc from Abtek(R) Biologicals Liverpool United Kingdom was used. Pure bacteria colonies were picked from brop using a sterile wire loop and transferred to tubes each containing 5ml of sterile saline. pe suspension was vortexed and adjusted to match 0.5 McFarland turbidity standard. Sterile cotton swab was pen dipped rotated and pressed firmly on pe tube walls above pe culture to remove excess inoculums from pe swabs. pis was pen evenly swabbed on pe dried surface of Mueller- Hinton agar (HiMedia Lab. Pvt. Mumbai India) plates ensuring even distribution of pe bacterium. pe antimicrobial loaded discs were placed on pe bacteria plates using sterile forceps and incubated at 37C for 18 to 24hrs. Interpretations of results were done using pe zones diameters [17].

pe eight antibiotics used had pe following concentrations; Ceftazidime (30g) Cefuroxime (30g) Gentamicin (10g) Cefixime (5g) Ofloxacin (5g) Augmentin (30g) Nitrofurantoin (300g) and Ciprofloxacin (5g).

WATER QUALITY ASSESSMENT

Hach water quality test kit (Hach companyUSA) was used to determine levels of nitrite alkalinity dissolved oxygen pH total hardness dissolved carbon dioxide while a permometer (Donggian Instrument China) was used to determine temperature.

RESULTS AND DISCUSSION

Clinical Signs of Diseased Fish from pe Different Fish Farms

Fingerlings from Ibadan farm I were off feed for 3 days. pey had distended abdomen filled wip slightly opaque fluid (Figure 1) and pere was high mortality in pe pond from where pey were obtained.

In farm II pere were mortalities wipout visible clinical signs except pat pe fingerlings were off feed. Juveniles from farm III had ulcers at pe base of pe fins and accumulation of bloody fluid in pe abdomen (Figure 2).

At Ikorodu fish from farm A had severe hyperaemic ulcers on pe skin while pose from farm B had hyper- pigmentation severe ulceration and necrosis of pe skin and musculature. Fish from farm C presented wip pustules and ulceration of pe musculature necrosis of pe tail fin and parietal part of pe head. pose from Farm D had greenish discoloration severe cutaneous ulceration and necrosis (Figure 3).

African catfish from Farm E had accumulation of yellowish fluid in pe intestine wip ulcers at pe base of pe fins (Figure 4).

Farm II fish samples had more bacterial load pan oper farms (Table 1). pis should not be taken in isolation because furper results confirmed pat farm II pond water also had higher levels of bacterial load (Table 2). pe physico-chemical parameters of pond II revealed a high pH high dissolved CO2 high alkalinity and total hardness above pe recommended standards (Table 3). pese factors predispose fish to stress which encourages development of disease. pe poor water quality of farm II could be pe primary cause of pe mortalities observed wip pe bacterial infection

Table 1: Microbial Load of Diseased Clarias gariepinus Fingerlings from Farms I and II in Ibadan

Source###MEDIA (Cfu/g).

###SSA###EMB###NA

Farm I###2.6x10

###3.5x10

###5.3x10

Farm II###9x10###1.3x10###2.4x10

eing opportunistic. pe bacterial load of various fish organs from pe different farms is presented as Tables 4 5 6 7 and 8.

Table 2: Microbial Load of Water Samples from Ponds where Diseased Clarias gariepinus were Obtained###

###MEDIA (Cfu/ml)

LOCATION###FARM

###SSA###EMB###NA

###5###5###

###FARM I###5.5X10###9.2X10###1.4X10

###5###5###

###IBADAN###FARM II###7.4X10###5.6X10###3.5X10

###7###5###

###FARM III###1.2X10###7.6X10###2.0X10

###FARM A###1.2X10

###5

###7.4X10

###5

###3.2X10

###FARM B###3.4X10

###5

###8.5X10

###5

###4.5X10

###5###5###

IKORODU###FARM C###1.2X10###8.7X10###8.2X10

###5###5###

###FARM D###1.1X10###8.2X10###7.4X10

###FARM E###1X10

###7

###8.6X10

###5

###1.7X10

Generally in pe adult fish pe gills had more bacteria load pan all pe oper organs which is not surprising considering pe fact pat gills filter pe water to obtain oxygen and as such is exposed to environmental contaminants [18]. Also it serves as pe first point of entry for most papogens; hence it can be used as a biomarker of microbial contamination in live fish [19]. It was also observed pat water samples from Farms I and Farm II containing fingerlings had higher bacteria load pan pose of pe adult fish. Fingerlings have higher metabolic rate and are perefore fed more frequently which could lead to poor water quality hence increase in bacteria growp [20 21]. Additionally deap in fingerlings are usually more common due to pe fact pat peir immune system are not as developed as in pe adult fish so any change in environmental conditions leading to stress could be detrimental [22].

A total of 153 isolates were obtained from fish and water samples of which 57 unique cultures were selected based on peir morphology and stored on

Table 3: Physiochemical Parameters of Pond Water from the Various Farms

###Location###Farm###Temp###pH###DO###Nitrite###Dissolved###Alkalinity###Total Hardness###Age

###(C)###(mg/L)###(mg/L)###CO2 (mg/L)###(mg/L)###(mg/L)

###I###27###6.5###3.0###0.00###180.0###256.5###30.0###4weeks

###Ibadan###II###25###7.5###18.0###0.132###295.0###239.4###427.5###5weeks

###III###26###7.0###2.0###0.99###287.5###205.2###171.0###10weeks

###A###26###6.5###5.0###0.792###155.0###85.5###32.2###4months

###Ikorodu###B###26###6.0###2.0###0.594###190.0###68.4###34.2###5months

###C###26###5.5###7.0###0.99###125.0###51.3###34.2###4months

###D###25###5.5###15.0###0.66###110.0###34.4###17.1###5months

###E###26###6.0###1.0###1.32###130.0###171.0###102.6###3months

###Standard###25-30###6.5-8.5###greater than 5.0###0.1###0-15###20-200###20-100###Not

###Value###Applicable

Positively identified papogens included 14 Salmonella (24.6%) 4 Pseudomonas (7%) 2 Aeromonas (3.5%) 3 Edwardsiella (5.3%) and 3 Shigella (5.3%) species.

Antibiotics resistance is a significant public healp issue. pere have been many papers reporting a link between use in food animals emergence of antibiotics resistance in Salmonella Escherichia coli Enterococci and campylobacter in treated animals and transfer to humans via food chain [23 24]. pe antibiotics used in pe sensitivity test were mostly pird and fourp generation antibiotics wip pe exception of Gentamicin which is a second generation antibiotics. pe result revealed a very disturbing trend as pere was high resistance levels to pe antibiotics by pe isolates. pe highest resistance was to Cefuroxime where 44 of pe 57 isolates were totally resistant. Ciprofloxacin was pe antibiotic to which pe least resistance (15%) was shown (Figure 5). pis information is similar to pe findings of Akinbowale et al. [25] in which two strains of Salmonella species exhibited a multi-drug resistance pattern.

Table 4: Microbial Load of Skin Obtained from Diseased Clarias gariepinus in Different Farms

###SOURCE###MEDIA (Cfu/g)

###SSA###EMB###NA

###5###5###5

###Ibadan Farm III###2.2 x10###4.3x10###4.2x10

###4###7###6

###Ikorodu Farm A###4.0 x 10###3.7x 10###1.5x 10

###5###4###6

###Ikorodu Farm B###1.6 x10###5.0x10###2.9x10

###5###5###7

###Ikorodu Farm C###3.1x10###1.7x10###2.6x10

###5###5###7

###Ikorodu Farm D###1.0x10###1.0x10###4.1x10

###5###5###7

###Ikorodu Farm E###1.0x10###1.7x10###4.3x10

Table 5: Microbial Load of Gills Obtained from Diseased Clarias gariepinus in Different Farms

###SOURCE###MEDIA (Cfu/g)

###SSA###EMB###NA

###5###5###7

###Ibadan Farm III###5.6x10###7.8x10###1.3x10

###6###5###6

###Ikorodu Farm A###3.0x10###7.0x10###3.6x10

###5###5###6

###Ikorodu Farm B###2.0x10###7.0 x10###6.3x10

###5###5###5

###Ikorodu Farm C###4.2x10###2.5x10###4.4x10

###6###5###5

###Ikorodu Farm D###6.0x10###3.5x10###1.3x10

###7###5###7

###Ikorodu Farm E###5.0x10###7.3x10###6.0x10

Table 6: Microbial Load of Lungs Obtained from Diseased Clarias gariepinus in Different Farms

###SOURCE###MEDIA (Cfu/g)

###SSA###EMB###NA

###5###5###4

###4.5x10###2.2x10###3.7x10

###4###4###6

###Ikorodu Farm A###8.0x10###3.0x10###2.7x10

###6###6###6

###Ikorodu Farm B###3.9x10###4.8x10###5.3x10

###5###3###5

###Ikorodu Farm C###2.5x10###2.3x10###3.8x10

###5###5###4

###Ikorodu Farm D###2.7x10###4.2x10###5.0x10

###5###5###5

###Ikorodu Farm E###3.8x10###8.7x10###6.5x10

Table 7: Microbial Load of Kidney Obtained from Diseased Clarias gariepinus in Different Farms

###SOURCE###MEDIA (Cfu/g)

###SSA###EMB###NA

###5###5###5

###Ibadan Farm III###1.6x10###4.6x10###7.1x10

###5###5###6

###Ikorodu Farm A###1.7x10###7.5x10###1.6x10

###5###5###6

###Ikorodu Farm B###3.0x10###1.1x10###4.5x10

###5###5###5

###Ikorodu Farm C###3.4x10###1.7x10###2.3x10

###5###5###5

###Ikorodu Farm D###3.3x10###2.4x10###4.5x10

###5###5###5

###Ikorodu Farm E###2.6x10###5.0x10###3.3x10

Table 8: Microbial Load of Intestine Obtained from Diseased Clarias gariepinus in Different Farms

###SOURCE###MEDIA (Cfu/g)

###SSA###EMB###NA

###4###5###5

###Ibadan Farm III###7.0x10###1.5x10###4.3x10

###5###5###6

###Ikorodu Farm A###5.0x10###2.1x10###7.3x10

###5###5###6

###Ikorodu Farm B###2.6x 10###2.0x10###2.1x10

###5###5###5

###Ikorodu Farm C###1.8x10###1.9x10###1.5x10

###5###5###5

###Ikorodu Farm D###4.3x10###3.3x10###2.9x10

###5###5###5

###Ikorodu Farm E###3.6x10###4.9x10###5.4x10

Resistance to antibacterial drugs may be as a result of indiscriminate use of pese drugs in aquaculture at less pan optimum dosage and such action leads to resistance in exposed papogens. On most farms to prevent disease outbreaks chemoperapeutants of all kinds are used. pese substances are applied prophylactically under uncontrolled conditions [26]. One of pe significance of pis study is pe establishment of pe possibility of transfer of antibiotics resistant bacteria to human prough fish and fish culture. In Farm I pe bacteria isolates were highly resistant to Augmentin followed by Cefixime and Cefuroxime while pey were highly susceptible to Ciprofloxacin Nitrofurantoin Ofloxacin Ceftazidime and Gentamicin (Figure 6) which implies pat pere has been minimal abuse of pose antibiotics on pis farm. Farms II III A B C D and E had higher levels of antibiotics resistance compared to Farm I (Figures 7 8 9 10 11 12 and 13).

In a previous study by Adewoye and Lateef [27] gram-negative bacteria species isolated from pe body surfaces of C. gariepinus in Nigeria were comparably identified as Proteus Pseudomonas Serratia Enterobacter and Escherichia. pe study also reported resistance of isolated bacteria to commonly used antibiotics in Nigeria as follows: 100% (augmentin amoxicillin and cloxacillin); 85.71% (tetracycline) 80.95% (cotrimoxazole) and 71.43% (erypromycin).Fujioka [28] reported pat Escherichia coli and Salmonella can survive for very long periods in tropical waters and once introduced almost become indigenous to pe environment. pis is also possible in some oper fish papogens of food-borne importance. perefore pere is pe possibility pat some bacterial species obtained from Clarias gariepinus can serve as potential aetiological agents of infectious fish environmental or human diseases of aquatic origin [29 10].

CONCLUSION

Numerous diseases have emerged as serious economic or ecological problems in aquaculture species; meanwhile very little work has been done on fish diseases in Nigerian aquaculture [26] which pis work has addressed in pis preliminary study. It is perefore necessary to build research and diagnostic capacities in pe sub-Saharan African region to deal wip fish disease problems [30] since pe rate and extent of emergence can be reduced by pe application of biosecurity programmes designed to mitigate pe risk factors for disease emergence [31 32 33].

pe results obtained from pis study showed pat for pe control of fish bacterial diseases pe legislation of appropriate antimicrobial agent is very important to protect human healp from potential hazards associated wip antibiotic resistance from aquaculture. Disease prevention should be carried out by means of good culture and healp management to ensure optimum yield and wholesome products.

ACKNOWLEDGEMENT

pe aupors wish to show peir appreciation to: Department of Veterinary Public Healp and Preventive Medicine University of Ibadan Department of Microbiology University of Ibadan Fish farmers from Ibadan and Ikorodu sites and Mr. Yemi Okunlade. pis Project was sponsored in part by IFS Grant No. A/4933-1.

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