Research Article
Screening of the Efficacy of Some Commonly Used Antibiotics in Ghana

G.K. Helegbe, L.Y. Anyidoho and F.N. Gyang

Research Journal of Microbiology, 2009, 4(6), 214-221.

Abstract

The objective of this study was to screen some commonly used antibiotics in Ghana for their efficacy in treating diseases so as to select sensitive organisms that can be used to design an assay in assessing their biological activity. The disc susceptibility test was used to screen stock antibiotics such as ampicilline, chloramphenicol, kanamycin and penicillin based antibiotics from different manufacturers (both local and foreign) which were obtained from different pharmacy shops against some bacteria species such as Salmonella typhi, Staphyloccus auresus and six strains of Escherichia coli. It was observed that both stock and field antibiotics (Antibiotics obtained from pharmacy shops for study) zone of inhibition were similar and compared with literature values. J916 (an E. coli isolate) and Salmonella typhi were found to be less sensitive to the penicillin-based antibiotics similar to literature values for both stock and pharmacy shop samples. This study revealed that the antibiotics produced by local and foreign pharmaceutical companies appear to be effective. In as much as this study demonstrate that, local and foreign pharmaceutical industries appear to be producing quality drugs, further studies are needed to substantiate this claim observed by this study, which was on a small scale.

ASCI-ID: 83-335

• The discs diameters were uniform, 6.0 mm in diameter

Volume of agar poured was about 15 mL for 90.0 mm diameter Petri dishes
Equal volume of peptone-water (10 mL) was measured into the culturing tube
Bacteria inoculum of 0.5 MacFarland was prepared by picking a colony of the organism into the peptone-water using sterile streaking loop
The prepared Mueller Hinton agar plates were dried before they were inoculated
The volume of culture used to inoculate the agar in the plates was 500.0 μL
Sub culturing of pure culture was done at least once a week to avoid any contamination
For long storage the microorganisms were transferred unto agar slopes and then incubated overnight of 37°C. Glycerol was added unto the surface of agar slope with microbial growth

In performing the sensitivity test 20.0 μL of the inoculum was poured gently onto a dried prepared Mueller Hinton Agar in an agar plate using a sterile micropipette. The plate was gently rotated for uniform distribution on the inoculums on the medium. After the inoculation the impregnated disc was picked by a sterile forceps and placed gently unto the inoculated agar. Susceptibilities to various antibiotics were determined by modified Kirby-Bauer disk diffusion methods according to the clinical Laboratory Standards Institute as described elsewhere (Ceri et al., 1999).

Zone of Inhibition Test for Stock and Field Antibiotics
Solution of ampicillin, chloramphenicol and kanamycin were prepared from the stock solution as described earlier to obtain the following concentration per disc 10.0, 30.0 and 30.0 μg, respectively.

After inoculation for about 15 min, each of the impregnated discs was placed gently unto the inoculated agar. The distance between each impregnated disc on agar plate was about 5cm and approximately 15 mm from edge of plate as described by Clutterbuck et al. (2007). The agar plates were incubated overnight at a temperature of 37°C. Reasonable zones of inhibition were measured to establish which microorganisms were sensitive to the stock antibiotics. The procedure was repeated once to obtain reliable and valid results. The Penicillin based antibiotics that were used were ampicillin, amoxicillin, cloxacillin and penicillin. They were prepared with the same method to have concentration of 10 μg each and the susceptibility testing performed with the same bacteria species.

Statistical Analysis
Data were entered into Excel programme and statistical analysis done using the one-way ANOVA. Values were considered significant when p value was less than 0.05.

RESULTS AND DISCUSSION

Table 1 gives a summary of zone of inhibition against 10 μg AM, 30 μg KA and 30 μg CH. S. typhi and S. aureus did not respond to 30 μg CH concentration. Also strains of E. coli were relatively more sensitive to 30 μg CH whereas, J1060LT was resistant to10 μg AM. S. typhi and S. aureus did not show any zone of inhibition for cloxacillin (data not shown).

Evaluating Potency of Field Antibiotics
In order to evaluate the potency of the field antibiotics against Gram negative bacteria, S. typhi and J916 were used as representative clinical microorganism for the screening of the field antibiotics. The sensitivity test and zone of inhibition to ampicillin and amoxicillin antibiotics from pharmacy shops are shown in Table 3. Both show similar show zone of inhibition to the selected microorganisms except zone of inhibition by ampicillin (CD 0201 by a local company), which was higher compared to amoxicillin (1000D01 also from local company), in relation to J916. Again for J916, zone of inhibition for ampicillin (D 594001, from a foreign company) was less than that of amoxicillin (AX 33A, from a local company), (Table 3). The zone of inhibition for the different forms of penicillin preparations of the field antibiotic are summarized in (Table 4). Except for benzyl procaine penicillin injection (5-501 from a foreign company) whose zone of inhibition could not be measured against J916, the others showed some zone of inhibition but were below literature values (Clutterbuck et al., 2007; Table 2).

Table 1: Mean (±SD) zones of inhibition around all the Stock Antibiotics against microorganisms provided
AM: Ampicillin; CH: Chloramphenicol; KA: Kanamycin; 0 implies no inhibition; SD: Standard deviation; ND: Not done; S: Sensitive; R: Resistant. NB: Each value was the mean of 4 readings and the expiry dates of the stock antibiotics were between May-June 2001

Table 2: Range of zone of sensitivity of some antibiotics against gram negative and positive bacteria species
CH, KA, AM and AMX have their usual meaning

Table 3: Mean (±SD) mm zones of inhibition around different states of Penicillin Injection Preparations antibiotics from different manufactures against selected microorganisms
BNo.: Batch number; ZI: Zone of Inhibition; S: sensitive; R: resistant; NM: Not measured; FPC: Foreign Pharmaceutical Company. Exp. Date: Expiry date. NB: Each value was the mean of 4 readings

Table 4: Mean (±SD) mm zones of inhibition around different states of Penicillin Injection Preparations antibiotics from different manufactures against selected microorganisms
BNo.: Batch number; ZI: Zone of Inhibition; S: Sensitive; R: Resistant; NM: Not measured; FPC: Foreign Pharmaceutical Company. Exp. Date: Expiry date. NB: Each value was the mean of 4 readings

This may probably be due to the gram negative nature of these microorganisms. The other observation made was that cloxacillin did not show any zone of inhibition whatsoever (data not shown). However, 5-501 and 1537-03 also from a foreign company were sensitive to S. typhi.

Clinical laboratories have used antibiotic diagnostic devices as guides to selection of therapy nearly as long as such drugs have been commercially produced. And in this study, the disc diffusion testing, which was used to screen for the biological activity of the field antibiotics, gave results which are reproducible, thus indicating the quality of the field antibiotics. And that the significance of this study is such that the biological activity of the antibiotics can be carried out to assess the potency of field antibiotics to complement the chemical activity. In addition, the results obtained are of a high standard which has a reasonable degree of accuracy and it is the common laboratory test for antibiotic susceptibility (Livermore, 2003; Bauer et al., 1966; Victor, 1981), compared with agar dilution.

However, one major disadvantage of the agar dilution compared with disc diffusions is the fact that test plates cannot be sub cultured easily in order to determine the bactericidal activity of the antimicrobial agent. In view of the above reasons the disc diffusion has been the most widely used procedure. Furthermore, the suitability of Mueller Hinton agar for antimicrobial sensitivity testing, stemmed from the fact that it is found not to contain inhibitory substances to antimicrobial compounds and its pH for routine sensitivity work is 7.2-7.4.

In the sensitivity test of the stock antibiotics, the ampicillin stock preparation did show a relative wide spectrum of biological activity. Most of the microorganisms were sensitive to it with the exception of J1060 LT and 101695 ST. Ampicillin is generally active against gram-negative and gram-positive bacteria (Clutterbuck et al., 2007). The resistance of J1060 LT and 101695 ST to ampicillin could be that these strains of E. coli may have produced beta-lactamase or acid that hydrolyses the ampicillin thereby losing its activity.

It is surprising to note that S. aureus was resistant to chloramphenicol. This is because 1 μg mL-1 chloramphenicol inhibits most gram positive bacteria and many gram-negative bacteria are inhibited by concentrations of 0.2-5 μg mL-1 (Katzung, 1992). In this study, however, when compared to the other two antibiotic stocks (ampicillin and kanamycin) it is found that the strains of E. coli were more sensitive to chloramphenicol (Table 1). The fact that J1060 was sensitive to both chloramphenicol and kanamycin but not ampicillin emphasizes the point mentioned earlier that J1060 may have produced beta-lactamase or acid that hydrolyzed the ampicillin structure.

In the sensitivity test of some of the antibiotics from the pharmacy shops, the zone of inhibition for J916 (a strain of E. coli) and S. typhi were comparable and similar to the stock as well, which were comparable to literature. It has also been shown that, there are instances when such organisms become resistant to lower concentrations, which we observe in preliminary findings of this study. Other possibility is that the growth of the bacteria might be in a biofilm state, which has been found to be recalcitrant to antibiotic treatment (Ceri et al., 1999), as a result the choice and concentration of antibiotic are often unsuccessful and must be increased.

The fact that the zone of inhibition of the stock and the field antibiotic were similar could imply that the potency of the antibiotic by the pharmaceuticals was of good quality. However, the difference in the zone of inhibition of amoxicillin made by one local Pharmaceutical company in Accra on J916 was 14.0±0.5 mm as against the stock of 17.1±0.5 mm, were statistically significant (p<0.05). Since, studies has shown that a 10 μg disc concentration of antibiotic giving a zone of inhibition of more than 14mm is sensitive, we can conclude that the stock is more sensitive compared to the one by the pharmaceutical company, which is also effective. Furthermore the zone of inhibition by the stock of 23.4±0.7 mm by ampicillin (Table 1) and that of amoxicillin by the same local pharmaceutical company in Accra was 20.5±0.5 mm (Table 3) to S. typhi, suggesting that the antibiotics will be effective in the clearance of microorganisms during an infection.

One other clear observation made was the lack of sensitivity of S. typhi and J916 to cloxacillin. This was not unusual due to the fact that isoxazolyl penicillins such as cloxacillin have no activity against such gram-negative bacteria. However, these isoxazolyl penicillins are used in combination with either ampicillin or one of the cephalosporins for the treatment of urinary tract infections caused by gram-negative bacilli (Astal et al., 2002). The function of the isoxazyl penicillin is to protect the action by binding bacteria beta-lactamases which destroy these drugs (Bennet and Kuccers, 1979). It is possible to obtain high value of zones of inhibition with diameters of 30-35 mm, which are arbitrarily indicative of high susceptibility and zones of up to 15 mm and are related to organisms resistant to the drug concentration employed (Jorgensen and Ferraro, 2000). Exceptions to this generation are those organisms whose zones of inhibition are approximately 20 mm when tested with penicillin and which must be checked for penicillinase production.

Within experimental error, zone of inhibition by the penicillin antibiotics (namely benzyl penicillin injection, crystalline penicillin injection and procaine penicillin) were similar. However, benzyl penicillin injection made in India (Table 4) did not inhibit the growth of J916 and not indicative of the potency of benzyl penicillin injection being lost or of low quality, as it has been shown to have inhibited growth of S. typhi. There could be possible implication of penicillinase production as its zone of inhibition on J916 was below 20 mm. Date of manufacture and expiratory date of almost all the field antibiotic were the same and had no effect on the potency of the antibiotic.

Taken together, the results in this study is significant in that the inhibition of growth by both stock and field antibiotics enabled the selection of microorganisms that can be used to design an assay system to examine the biological activity of antibiotics.

CONCLUSION

The strains of E. coli were sensitive to ampicillin stock with the exception of J1060 LT. The zone of inhibition of the field antibiotic and that of the stock did not vary much, when compared to that in literature, affirming that this method of screening antibiotics from the pharmacy shops is reliable, thus the biological activity assay can be designed to assess the potency of the antibiotics. In as much, this study demonstrate that, local and foreign pharmaceutical industries appear to be producing quality drugs, further studies are needed to substantiate this claim observed by this study, which was on a small scale.

ACKNOWLEDGMENTS

We wish to appreciate the help offered by Mrs. Rose Nyarko of the Biochemistry Department, Legon and to all the technical staff of the Department. We also want to thank Dr. Yaa Difie Osei for providing us with the antibiotics stock. We are also grateful to Bacteriology Department of Noguchi Memorial Institute for Medical Research (NMIMR) for their assistance in providing us with bacteria species and the owners of Pharmacy shops that participated in the study.

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