ANTIBIOTIC SUSCEPTIBILITY OF PYOGENIC
STREPTOCOCCI IN CATFISH AND TILAPIA FROM
FRESH WATER DEMONSTRATION FISH PONDS,
MANDO, KADUNA

U. G Chonoko,, E Sule, A Ado

► To cite this version:

U. G Chonoko,, E Sule, A Ado. ANTIBIOTIC SUSCEPTIBILITY OF PYOGENIC STREPTOCOCCI IN CATFISH AND TILAPIA FROM FRESH WATER DEMONSTRATION FISH PONDS, MANDO, KADUNA. Continental J. Pharmaceutical Sciences, 2014, 8 (1), pp.7-15. 10.5707/cjpharm-sci.2014.8.1.7.15. [hal-01326645](https://hal.science/hal-01326645)

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# 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

Corresponding Author:

Accepted for Publication: 26/10/14

## 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
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 β-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 manufacturer's instructions. Sterilization was done by autoclaving at 15lb/sq inch at 121°C for 15 minutes. All glass wares (pipettes and Petri dishes) were packed in canisters and

<figure><img src="image_2.png" /></figure>
sterilized in the hot air oven at 160°C 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 37°C for 24 hrs. β-Hemolytic single colonies on the plates were re-streaked on to fresh blood agar to obtain pure isolates (Al-harbi and Ahmed, 1994). The β-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 37°C 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).

$$ MARI = \frac{\text{Number of antibiotics to which resistant}}{\text{Total number of antibiotics tested}} $$

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, 27.6°C); pH (pond 1, 6.87); conductivity (pond 6, 113 µs /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. 27°C), pH (pond 6

<figure><img src="image_3.png" /></figure>
5.85); conductivity (pond 1; 40 µs /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, 27.6°C); pH pond 3; 7.26); conductivity, (pond 2, 227 µs /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, 27.4°C); pH, (pond 1, 6.98) conductivity, (pond 5, 61 µs /cm); B.O.D, (pond 3, 0.4 mg/l, pond 2, 0.5 mg/l); nitrate, (pond 1, 1.0 mg/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.
<table><caption>Table 1. Physicochemical Properties of Pond Water Samples in Wet and Dry Seasons</caption><thead><tr><th rowspan="3">Parameters<br>_Assessed</th><th colspan="12">Ponds Sampled</th></tr><tr><th colspan="2">1</th><th colspan="2">2</th><th colspan="2">3</th><th colspan="2">4</th><th colspan="2">5</th><th colspan="2">6</th><th rowspan="2">mean wet<br>season<br>X̄</th><th rowspan="2">mean dry<br>season<br>X̄</th></tr><tr><th>W</th><th>D</th><th>W</th><th>D</th><th>W</th><th>D</th><th>W</th><th>D</th><th>W</th><th>D</th><th>W</th><th>D</th></tr></thead><tbody><tr><td>Temperature<br>(°C)</td><td>27.5</td><td>27.9</td><td>27.3</td><td>27.5</td><td>27.0</td><td>27.4</td><td>27.2</td><td>27.6</td><td>27.6</td><td>27.8</td><td>17.0</td><td>27.6</td><td>25.60</td><td>27.63</td></tr><tr><td>pH</td><td>6.87</td><td>6.98</td><td>6.42</td><td>7.15</td><td>6.43</td><td>7.26</td><td>6.62</td><td>7.18</td><td>6.20</td><td>7.01</td><td>5.85</td><td>7.08</td><td>6.39</td><td>7.11</td></tr><tr><td>Conductivity<br>(μS/cm)</td><td>40</td><td>184</td><td>100</td><td>227</td><td>86</td><td>154</td><td>63</td><td>180</td><td>59</td><td>61</td><td>113</td><td>75</td><td>76.83</td><td>146.83</td></tr><tr><td>B.O.D<br>(mg/l)</td><td>1.1</td><td>0.9</td><td>0.5</td><td>0.5</td><td>0.6</td><td>0.4</td><td>1.7</td><td>1.4</td><td>1.0</td><td>0.8</td><td>0.9</td><td>0.7</td><td>0.97</td><td>0.78</td></tr><tr><td>Nitrates<br>(mg/l)</td><td>1.1</td><td>1.0</td><td>2.5</td><td>2.1</td><td>1.3</td><td>1.1</td><td>1.8</td><td>1.5</td><td>1.6</td><td>1.2</td><td>3.1</td><td>2.5</td><td>1.90</td><td>1.57</td></tr><tr><td>Turbidity<br>(NTU)</td><td>2.5</td><td>2.4</td><td>3.4</td><td>3.2</td><td>2.4</td><td>2.1</td><td>5.5</td><td>5.2</td><td>3.5</td><td>3.1</td><td>8.2</td><td>7.8</td><td>4.25</td><td>3.97</td></tr></tbody></table>

W = Wet Season

D = Dry Season

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<table><caption>Table 2. Incidence and % of Streptococci isolated from the fish ponds.</caption><thead><tr><th>Ponds</th><th>Number of Streptococci isolated</th><th>Number and % S. pyogenes</th><th>Number and % of non- Pyogenic cocci</th></tr></thead><tbody><tr><td colspan="4">Sampled</td></tr><tr><td>Pond 1</td><td>16</td><td>0 (00)</td><td>16 (100)</td></tr><tr><td>Pond 2</td><td>26</td><td>2 (7.7)</td><td>24 (92.3)</td></tr><tr><td>Pond 3</td><td>21</td><td>3 (14.3)</td><td>18 (85.7)</td></tr><tr><td>Pond 4</td><td>22</td><td>2 (9.1)</td><td>20 (90.9)</td></tr><tr><td>Pond 5</td><td>15</td><td>0 (00)</td><td>15 (100)</td></tr><tr><td>Pond 6</td><td>18</td><td>2 (11.1)</td><td>16 (88.9)</td></tr><tr><td colspan="2">ΣT = 118</td><td>ΣT 9 (42.2)</td><td>ΣT 109 (557.8)</td></tr></tbody></table>

<figure><img src="image_5.png" /></figure>
<table><caption>Table 3. Antibiotic susceptibility test of Streptococcus pyogenes isolated from the fish ponds.</caption><thead><tr><th colspan="11">Zones of Inhibition (mm) of antibiotics tested (µg)</th></tr><tr><th>Ponds</th><th>PEF</th><th>CN</th><th>APX</th><th>Z</th><th>AM</th><th>R</th><th>CPX</th><th>S</th><th>SXT</th><th>E</th></tr><tr><th>(P)</th><th>(10)</th><th>(10)</th><th>(30)</th><th>(20)</th><th>(30)</th><th>(35)</th><th>(10)</th><th>(30)</th><th>(30)</th><th>(10)</th><th>MARI</th></tr></thead><tbody><tr><td>P2</td><td>26</td><td>18</td><td>00</td><td>27</td><td>00</td><td>16</td><td>16</td><td>14</td><td>17</td><td>12</td><td>0.3</td></tr><tr><td>P3</td><td>25</td><td>12</td><td>00</td><td>27</td><td>00</td><td>18</td><td>18</td><td>13</td><td>20</td><td>10</td><td>0.5</td></tr><tr><td>P4</td><td>26</td><td>16</td><td>00</td><td>27</td><td>00</td><td>18</td><td>16</td><td>17</td><td>08</td><td>07</td><td>0.4</td></tr><tr><td>P6</td><td>18</td><td>16</td><td>00</td><td>19</td><td>00</td><td>17</td><td>18</td><td>15</td><td>15</td><td>06</td><td>0.3</td></tr></tbody></table>

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 Table 1 suggests 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 β-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 (10µg) 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, patient's 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.

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<figure><img src="image_7.png" /></figure>
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