antibiotic susceptibility of pyogenic streptococci in catfish and tilapia from fresh water...

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Continental J. Pharmaceutical Sciences 8 (1): 7 - 15, 2014 ISSN: 2141 4149 Wilolud Journals, 2014 http://www.wiloludjournal.com Printed in Nigeria doi:10.5707/cjpharmsci.2014.8.1.7.15

ANTIBIOTIC SUSCEPTIBILITY OF PYOGENIC STREPTOCOCCI IN CATFISH AND

TILAPIA FROM FRESH WATER DEMONSTRATION FISH PONDS, MANDO, KADUNA.

Chonoko, U. G., Sule, E and Ado, A. Department of Applied Sciences, College of Science and Technology, Kaduna Polytechnic, Kaduna, Nigeria.

ABSTRACT

A total of three hundred (300) fish samples comprising of 150 samples each of Catfish (Clarias gariepinus Trewavas) and Tilapia (Oreochromis niloticus Teugels) were obtained from six fish ponds located in Demonstration Fish Ponds and Training Center, Mando, Kaduna. The samples were screened for pyogenic streptococci between August 2012 and January 2013. Physico-chemical parameters such as temperature, pH, conductivity, turbidity, nitrate, and biochemical oxygen demand were assessed in wet and dry seasons and the values were compared. The values obtained were further subjected to statistical analysis. The difference in values for temperature was only significant in pond 6 (p < 0.05) but not significant in ponds 1-5 (p > 0.05). The pH values in the six ponds were significantly different (p < 0.05). There was no significant difference (p > 0.05) in conductivity of pond 5 but ponds 1-4 showed significant difference (p < 0.05). Likewise there was no significant difference (p > 0.05) in values for nitrate, turbidity, and B.O.D across the ponds. Of the three hundred fish samples screened for pyogenic streptococci, 9 (42.2%) Streptococcus pyogenes were isolated from the body of catfish and tilapia in ponds: [P2, 2 (7.7%); P3, 3 (14.3%); P4, 2 (9.1%) and P6, 2 (11.1%)]. Streptococcus pyogenes isolates were resistant to ampiclox, amoxicillin and erythromycin but sensitive to pefloxacin, gentamycin, cotrimoxazole, cefuroxime, ceftriaxone, ciprofloxacin and streptomycin. Minimum antibiotic resistant (MAR) index was high (0.3). The implications of the findings are discussed. KEYWORDS: Catfish, Tilapia, physico-chemical, Antibiotic, Susceptibility, Pyogenic Streptococci.

Received for Publication: 15/07/14 Accepted for Publication: 26/10/14 Corresponding Author:

INTRODUCTION Streptococci are gram-positive, non-motile, non-spore forming, catalase-negative cocci bacteria that occur in chains. They are mostly facultative anaerobes, and some are obligate (strict) anaerobes, but most require enriched media for proper growth (NCCLS 1999). Streptococci differ from the staphylococci in that they are arranged primarily in chains rather than in clusters, and are often associated with many mixed infections with staphylococci. Streptococci can also separately cause diseases such as pneumonia, meningitis, endocarditis, pharyngitis, erysipelas, and glomerularnephritis (Alharbi, 1994). Several species of streptococci are normal inhabitants of the pharynx. They can also be isolated from surfaces of the skin, teeth, saliva, colon, rectum and vagina. The type of heamolytic reaction displayed on blood agar has long been used to classify the Streptococci (Kenneth, 2002). Beta haemolysis is associated with complete lyses of red cells surrounding the colony. Whereas alpha


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Chonoko et al.,: Continental J. Pharmaceutical Sciences 8 (1): 7 - 15, 2014

heamolysis is a partial or green heamolysis associated with reduction of red cell hemoglobin. Non hemolytic colonies have been termed gamma hemolytic. Heamolysis is affected by the species and age of red cells as well as other properties of the base medium. Group A Streptococci are nearly always beta haemolytic. Infections caused by Group A Streptococci are of particular concern because serious complications such as glomerulonephritis, rheumatic fever, and scarlet fever may result if left untreated (Ogbulie, 1998). Group B streptococci can manifest alpha, beta or gamma heamolysis. Most strains of S. pneumoniae are alpha haemolytic but can cause beta haemolysis on anaerobic incubation. Most of the oral Streptococci and enterococci are non-hemolytic. The property of haemolysis is not very reliable for the absolute identification of streptococci, but it is widely used in rapid screen for identification of S. pyogenes and S. pneumoniae (Kenneth, 2002). The streptococci of great medical significance are S. pyogenes, S. agalactiae and S. pneumoniae. While those of lesser importance are S. faecalis, S. faecium, and S. bovis. Among the pyogenic streptococci, S. pyogenes (Lancefield Group A) remains the major pathogen of man (Cheesbrough, 2000). Usually the first step in its recognition is the presence of -heamolytic colonies on blood agar plates due to the oxygen-stable S lysine. Many isolates however are only weakly hemolytic and cut the agar during inoculation of plates or anaerobic incubation often the extent of heamolysis with such strains. Some isolates lack the S lysine completely and are therefore non-heamolytic (Cheesbrough, 2000). Streptococcus pyogenes is one of the most frequently isolated pathogens of humans (Kenneth, 2002). It is estimated that between 5-15% of normal harbor the bacterium usually in the respiratory tract, without signs of disease. As part of normal flora, S. pyogenes can infect humans when defenses are compromised. When the bacteria are introduced or transmitted to vulnerable tissues, a variety of types of suppurative infections can occur (Eyo, 2001). Group A Streptococci are universally susceptible to penicillin G, a fact that precludes antimicrobial susceptibility testing for this organism unless the patient is allergic to penicillin. Over ninety percent of all streptococcal infections are caused by Group A streptococcus (Finegold and Martin, 1982). Asymptomatic carriers colonized in the nasopharynx, skin, vagina, or rectums are thought to transmit this organism through close person to person contact (Miller, 1984). Contaminated food may also be a source of transmission of infections in humans (Facklam and Carey, 1985). The aim of this research is to isolate three species of pyogenic streptococci (i.e. Streptococcus pyogenes, Streptococcus pneumoniae and Streptococcus agalactiae) in fish samples collected from various ponds located in Fresh water Demonstration Fish Farm and Training Centre, Mando, Kaduna, Nigeria.

MATERIALS AND METHODS Sample Collection A total of 300 fishes, namely 150 Catfish (Clarias gariepinus) and 150 Tilapia (Oreochromis niloticus) were collected from six fresh water earthen ponds measuring 200m2 each at Federal Department of Fisheries, Mando, Kaduna, between August 2012-January 2013. Sampling was done on weekly basis. Identification of the two species was at the Federal Department of Fisheries Mando, Kaduna. The fish were caught with nets. Using sterile swab sticks, the surface of the fish was swabbed and brought to the laboratory for analysis. Media Preparation Each medium was prepared following the manufacturers instructions. Sterilization was done by autoclaving at 15lb/sq inch at 121oC for 15 minutes. All glass wares (pipettes and Petri dishes) were packed in canisters and


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Chonoko et al.,: Continental J. Pharmaceutical Sciences 8 (1): 7 - 15, 2014 sterilized in the hot air oven at 160oC for 90 minutes as described by Ogbulie et al. (1998). Aseptic conditions were maintained throughout media preparation and sample analysis. Blood agar, Nutrient agar, Nutrient broth, Mueller Hinton agar were used in this study. Carbohydrate Fermentation Tests Carbohydrate fermentation tests employing 1% sugars were done according to (Cheesbrough, 2000). The sugars included mannitol, sorbitol, adornitol, sucrose, and lactose. Primary Isolation of Pyogenic Streptococci The inocula were separately inoculated using radiant streaking method on to different plates of Blood Agar (Oxoid) and incubated in 5% carbon dioxide at 37oC for 24 hrs. -Hemolytic single colonies on the plates were re-streaked on to fresh blood agar to obtain pure isolates (Al-harbi and Ahmed, 1994). The -hemolytic colonies observed were gram stained. Representatives of pure isolates were preserved on nutrient agar slants. Biochemical Tests Characterization of the isolates was performed according to the methods described (Cheesbrough, 2000). This involved colonial morphology, catalase, bile solubility, Hippurate hydrolysis, Tolerance to 6.5% sodium chloride, Growth on 40% Bile agar, Motility test, Bacitracin susceptibility and Optochin susceptibility. Determination Of Zones Of Inhibition Using Disk Diffusion Test. Isolates of S. pyogenes were subjected to antibiotic susceptibility testing using the Kirby- Bauer method that has been standardized by Clinical Laboratory Institute Standards (CLIS) 2005, on Mueller-Hinton Agar plates using sensitivity disks containing the following antibiotics: (Pefloxacin, gentamicin, ampiclox, cefuroxime, amoxicillin, ceftriaxone, ciprofloxacin, streptomycin, cotrimoxazole and erythromycin). Isolates grown overnight on nutrient agar were suspended in sterile normal saline (0.9% w/v NaCl) using a sterile wire loop until the turbidity was equivalent to McFarland standard. Sterile nontoxic cotton swabs dipped into the standardized inocula were used to streak the entire surface of Mueller Hinton Agar plates. Antibiotic disks were aseptically placed using a sterile forceps, and all plates incubated (GallenKamp England model IH-150) at 37oC for 24hrs. The results were interpreted using CLIS guide lines (2005). Determination Of Multiple Antibiotic Resistance Index (MARI) The Multiple Antibiotic Resistance Index (MARI) of S pyogenes isolates was determined for each test pond by dividing the number of antibiotics to which the organism was resistant by the total number of antibiotics tested (Olonitola et al., 2006). MARI = Number of antibiotics to which resistant Total number of antibiotics tested The multiple antibiotic resistance index gives an indication of the probable source(s) of an organism. MARI index greater than 0.2 indicates that an organism must have originated from an environment where antibiotics are often used (Olonitola, et al., 2006). RESULTS Physicochemical parameters The physicochemical parameters of the ponds sampled in wet and dry seasons (Table 1) showed that higher values in wet season, which include, temperature, (pond 5, 27.6oC); pH (pond 1, 6.87); conductivity (pond 6, 113 s /cm); BOD (pond 4, 1.7 mg/l); nitrate (pond 2, 2.5 mg/l; pond 6, 3.1 mg/l) and turbidity (pond 4, 5.5 NTU; pond 6, 8.2 NTU). Lower values recorded in some ponds in wet season were; temperature (ponds 6 and 3 i.e. 270C), pH (pond 6


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Chonoko et al.,: Continental J. Pharmaceutical Sciences 8 (1): 7 - 15, 2014 5.85); conductivity (pond 1; 40 s /cm); B.O.D, (pond 2, 0.5 mg/l); nitrate (pond 1, 1.1 mg/l) and turbidity (pond 3, 2.4 NTU). However during the dry season, the parameters with higher values were; temperature (pond 5, 27.60C); pH pond 3; 7.26); conductivity, (pond 2, 227 s /cm); B.O.D (pond 4, 1.4 mg/l); nitrate, (pond 6, 2.5 mg/l) and turbidity, (pond 6, 7.8 NTU). Lower values were observed in the ponds thus: temperature, (pond 3, 27.40C); pH,(pond 1, 6.98) conductivity, (pond 5, 61 s /cm); B.O.D, (pond 3, 0.4 mg/l, pond 2, 0.5 mg/l); nitrate, (pond 1, 1.0mg/l, pond 3, 1.1 mg/l ); and turbidity, (pond 3, 2.1 NTU, and pond 1, 2.4 NTU) (Table 1). Incidence and percentage of Streptocci isolated from the ponds. A total of 9 (42.2%) Streptococcus pyogenes were isolated from the 300 samples taken from all the ponds. Out of this number, two (2) were isolated from ponds 2 (7.7%), 4 (9.1%) and 6 and only three (14.3%) from pond 3. However, non- pyogenic streptococci were also isolated from the ponds. Ponds 1 and 5 were negative for pyogenic and non pyogenic Streptococci (Table 2). Antibiotic Susceptibility Test. Antibiotic susceptibility test of Streptococcus pyogenes isolated from the fish ponds. The result of antibiotic susceptibility test is presented in Table 3. Ten antibiotics with varied disc potencies were used. No zone of inhibition was observed for ampiclox and amoxicillin, i.e they were resistant to these antibiotics. The isolates were highly sensitive to Pefloxacin, Cefuroxime, and Ceftriaxone because of the wide zones of inhibition. Strains of Streptococcus pyogenes isolated from four ponds were resistant to Erythromycin. Only one strain isolated from pond 3 was resistant to streptomycin.


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Table 1. Physicochemical Properties of Pond Water Samples in Wet and Dry Seasons Ponds Sampled mean wet mean dry Parameters 1 2 3 4 5 6 season season Assessed W D W D W D W D W D W D X X Temperature 27.5 27.9 27.3 27.5 27.0 27.4 27.2 27.6 27.6 27.8 17.0 27.6 25.60 27.63 ( 0C) pH 6.87 6.98 6.42 7.15 6.43 7.26 6.62 7.18 6.20 7.01 5.85 7.08 6.39 7.11 Conductivity 40 184 100 227 86 154 63 180 59 61 113 75 76.83 146.83 (s /cm) B.O.D 1.1 0.9 0.5 0.5 0.6 0.4 1.7 1.4 1.0 0.8 0.9 0.7 0.97 0.78 (mg/l) Nitrates 1.1 1.0 2.5 2.1 1.3 1.1 1.8 1.5 1.6 1.2 3.1 2.5 1.90 1.57 (mg/l) Turbidity 2.5 2.4 3.4 3.2 2.4 2.1 5.5 5.2 3.5 3.1 8.2 7.8 4.25 3.97 (NTU) W = Wet Season D = Dry Season


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Table 2. Incidence and % of Streptococci isolated from the fish ponds.

Ponds Number of Number and Number and % of Streptococci % S. pyogenes non- Pyogenic cocci isolated

Sampled Pond 1 16 0 (00) 16 (100) Pond 2 26 2 (7.7) 24 (92.3) Pond 3 21 3 (14.3) 18 (85.7) Pond 4 22 2 (9.1) 20 (90.9) Pond 5 15 0 (00) 15 (100) Pond 6 18 2 (11.1) 16 (88.9) = 118 9 (42.2) 109 (557.8)


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Table 3. Antibiotic susceptibility test of Streptococcus pyogenes isolated from the fish ponds.

Zones of Inhibition (mm) of antibiotics tested (g) Ponds PEF CN APX Z AM R CPX S SXT E (P) (10) (10) (30) (20) (30) (35) (10) (30) (30) (10) MARI P2 26 18 00 27 00 16 16 14 17 12 0.3 P3 25 12 00 27 00 18 18 13 20 10 0.5 P4 26 16 00 27 00 18 16 17 08 07 0.4 P6 18 16 00 19 00 17 18 15 15 06 0.3

Interpretation of zones of inhibition: 13mm= Resistant; 14mm = Susceptible. Source of Interpretation: Jaime et al., (1996). KEY: PEF= Pefloxacin; CN= Gentamycin; APX= Ampiclox; Z= Cefuroxime; AM= Amoxicillin; R= Ceftriaxone; CPX= Ciprofloxacin; S= Streptomycin; SXT= Cotrimoxaxole and E= Erythromycin.


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DISCUSSION The absence of pyogenic streptococci from the two of six test ponds (Pond 1 and Pond 5) in Table1suggests that the ponds are devoid of these organisms at the time of sampling. Environmental conditions in these ponds may be unfavourable for the organisms to survive. The presence of the pathogenic organisms in fish is clearly intolerable under any circumstances because they may pose health hazards to the fish handlers and consumers (Inglis et al., 1993). The response of S pyogenes from the four test ponds analyzed indicated significant susceptibility to pefloxacin, cefuroxime and ceftriaxone. These antibiotics belong to the -lactam class of antibiotics known for their broad spectrum of activity against a wide range of bacteria; besides, the relative cost of these antibiotics makes its indiscriminate use almost impossible, thus minimizing the problem of antibiotic resistance. Similarly, the remarkably low resistance of the isolates to erythromycin could be as a result of the low concentration/potency (10g) of erythromycin used. It could also be as a result of indiscriminate use by people. The resistance of isolates to ampiclox and amoxicillin and erythromycin could be as a result of indiscriminate use of these antibiotics by people without prescription which is mostly responsible for the development of plasmid mediated resistance to antibiotics. The high MAR index (< 0.3) shows that the antibiotics are used indiscriminately. This is in conformity with the work of Olonitola, et al., (2006), who reported that MAR index greater than 0.2 is an indication that an isolate originates from a place where antibiotics are commonly used. Olonitola, et al., (2006) also noted that availability of antibiotics without prescription, open sale of antibiotics by untrained people and in some instances, patients failure to comply with prescription instructions abound in the Nigerian environment result in the emergence of resistant microbial strains. This is also coupled with the fact that it is not compulsory to purchase prescribed drug within the hospitals but they can be purchased outside from hawkers or in shops where these drugs were kept in conditions not suitable for storage of drugs.

CONCLUSION Pyogenic streptococci were isolated in ponds 2, 3, 4 and 6 absent in ponds 1 and 5. This study has also showed that Streptococcus pyogenes isolates were resistant to ampiclox and amoxicillin but sensitive to pefloxacin, cefuroxime and ceftriaxone. The higher MAR indices mean that antibiotics are used indiscriminately.

REFERENCES Alharbi, A. H. (1994). First isolation of Streptococcus sp. from hybrid tilapia (Oreochromis niloticus and S. aureus) in Saudi Arabia. Journal of Aquaculture; 128; 195-201. Cheesbrough, M. (2000). Distric laboratory manual for tropical countries. Cambridge University Press United Kingdom. Pp203-254. Clinical Laboratory Institute Standards (2005). Performance standards for antimicrobial susceptibility, 15th informational supplement (CLIS document M-S 15), 25, Wayne, P. A. Eyo, A. A. (2001). Fish processing technology in the tropics. Nigerian institute for fresh water fisheries Research, University of Ilorin Press, Ilorin. P403. Facklam, R. R. and Carey R. B., (1985). Streptococci and Aerococci. Manual for Clinical Microbiology, 4th edition. American Society for Microbiology, Washington, D. C.. Pp. 154-175.


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Chonoko et al.,: Continental J. Pharmaceutical Sciences 8 (1): 7 - 15, 2014 Finegold, S. M., and Martin, W. J. (1982). Facultative streptococci and Aerococci Bailey and Scotts diagnostic Microbiology, 6th edition. The C. V. Mosby Company, St Louis. Pp. 169-174. Inglis, V., Roberts, R. J. and Bromage, N. R. (1993). Bacterial diseases of fish. Blackwell Scientific Publications, Oxford, London P312. Jaime, S. C., Mark, A. K., David, V. G and Raul, J. C. (1996). Laboratory exercises in microbiology West Publishing Company Los Angles. San Francisco P 254. Kenneth, T. (2002). Streptococcus pyogenes. Text book of bacteriology. University of Wisconsin-Madison Department of Bacteriology. Pp. 1-2. Miller, M. J. (1984). Isolation and identification of Streptococci. Techniques manual, United state Department of Health and Human Services. Centers for Disease Control, Atlanta. Pp 24-29. NCCLS (1999). Performance standard for the antimicrobial susceptibility testing. National Committee for Clinical Laboratory approved Standard M100-59. Ogbulie, N. N., Uwaezoke, J. C. and Ogiehor, S. I. (1998). Introductory microbiology practical Springfield Publishers, Owerri, Nigeria p162. Olonitola, O. S., Oniye, S. J., Abdullahi, I. O., Olayinka, B. O., Yakubu, S. C. and Unogwu, J. U. (2006). Community reservoirs of antibiotic resistant bacteria in Nigeria. Studies on prawns and shrimps. Biological and Environmental Sciences Journal for the Tropics 3 (1): 100-104.

antibiotic susceptibility of pyogenic streptococci in catfish and tilapia from fresh water...

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