Antibiotics Sensitivity Against Staphylococcus aureus and Escherichia coli using Industrially Manufactured Sensitivity Disc

Staphylococcus aureus a gram-positive bacteria cause diseases through direct destruction and invasion of tissue by the production of toxins and Escherichia coli a gram-negative bacteria causes a variety of diseases. Testing of antibacterial susceptibility plays a major role in antibacterial treatment. Their are some factors which affects the results are quality, type and structure of a disks. This study was carried out with the aim to determine the Zone of inhibition of antibiotics against Staphylococcus aureus and Escherichia coli using industrially manufactured sensitivity disc. In positive test against Staphylococcus aureus the zone of inhibition was highest in antibiotic streptomycin (10mm/dm) and lowest in Amoxil (0mm/dm). In negative test against Staphylococcus aureus the zone of inhibition was highest in antibiotic Reflacine (10mm/dm) and lowest in Amplicin (2mm/dm). In positive test against Escherichia coli the zone of inhibition was highest in antibiotic Ciproflox (7mm/dm) and lowest in Amoxil (2mm/dm). There is no effect on antibiotic Norfloxacin, Erythromycin, Levofloxacin and Ampiclox. In negative test against Escherichia coli the zone of inhibition was highest in antibiotic Streptomycin (12mm/dm) and lowest in Tarivid (2mm/dm).

Infectious diseases account for high proportion of health problems and are the leading cause of death worldwide [1]. Staphylococcus aureus a gram-positive bacteria cause diseases through direct destruction and invasion of tissue by the production of toxins which leads to mortality and morbidity. Escherichia coli a gram-negative bacteria causes a variety of diseases and infections which may leads to mortality at all ages. In some tropical countries infections caused by Staphylococcus aureus leads to mortality [2].

Resistance to antibiotics has become serious health problem all over the globe. The resistance of drug are determined by antibiotic sensitivity test . So many researchers studied correlation between zones of inhibition and diffusion tests [3-5].

Study area

Damaturu metropolis was considered as the study area. It has an area of 737km and population of 302, 145 according to 2006 population census, it's located in North-Eastern province of Nigeria.Damaturu is located between the latitude of 11°57'58.29''E or 11,7746996 respectively.The elevation of Damaturu is 376.23 meters above sea level and hence the population density of 48,014 person ( census 2006). The region is a Sahel Savannah which has some sustainable of the indigenous plant species.

Equipment used

1. Mueller Hinton agar
2. Autoclave
3. Hot air oven
4. Mortar and pestle
5. Conical flask
6. Cotton wool
7. Wire loop
8. Test tube
9. Weighing balance
10. Measuring cylinder
11. Aluminum foil
12. Petri-dishes
13. Incubator
14. Pipette
15. Soxhlet extractor (Apparatus)
16. Separating funnel
17. Cork  borrer
18. Spirit lamp
19. Beaker
20. Sensitivity disc
21. Filter paper

Reagents used

1. Distilled water
2. Petroleum ether
3. Dimethyl sulfoxide

Preparation of culture media

1. 9.5g of Mueller Hinton agar was weighed and was dissolved in 250g of distilled water in a conical flask.
2. The cornical flask was closed with a cotton and foil paper then sealed with a masking tape.
3. The conical flask was placed into an autoclave and sterilised at 121°c for 15mins.
4. The autoclave was turned off and the conical flask was removed and allowed to cool for sometimes.
5. An amount of the prepared media was aseptically poured into four petridishes   covered and allowed to solidify.

Innoculation of bacteria and plating

1. The two petri dishes containing the solidified Mueller Hinton agar were arranged and labelled according to the species of bacterias used i.e Escherichia coli and Staphylococcus aureus.
2. One Petri dish labelled as E.coli and  one as Staphylococcus aureus

Using strict plating method

1. Using a sterilised wire loop small amount of E.coli was inoculated in an aseptic condition into the two Petri dishes and was strictly rubbed on the surface of the media and the petridishes were closed.
2. After sterilising the wire loop again, calories of staphylococcus aureus was also inoculated through out the surface of the 2 plate and was closed.

Sensitivity test

Using 4 prepared plates of Mueller Hinton agar:-

• Two plates were used in testing the sensitivity of e.coli against some antibiotics using an industrial made sensitivity disc , which has positive and negative disc. After strict plating the 2 petridishes the positive and negative sensitivity disc was placed in each petridishes ,and incubated. The zone of inhibition was recorded using a transparent ruler and was recorded accordingly.
• Using the other two petridishes same was done to staphylococcus aureus too.  And the results were recorded.

In addition. The bacterias used were also tested using an industrially manufactured sensitivity disc that have both positive and negative effects and the results were obtained where some have higher zone of inhibition (this means higher effect on the bacteria) and some lower. This findings indicates that the bacterias where viable and active ,the antibiotics in the positive sensitivity disc include :-Ciproflox (CPX), Norfloxacin (NB), Gentamycin (CN),Amoxil (AML), Streptomycin (S), Rifampicin (RD), Erythromycin (E), Chloramphenicol (CH), Ampiclox (APX) and Levofloxacin (LEV), while the drugs in the negative sensitivity disc include:- Tarivid (OFX), Reflacine (PEF), Ciproflox (AU), Augmentin (AU), Gentamycin (CN), Streptomycin (S), Ceporex (CEP), Nacidixic acid (NA), Septin (SXT), Septin (SXT) and Amplicin (PN) respectively (Tables 1-4).

In positive test against Staphylococcus aureus the zone of inhibition was highest in antibiotic streptomycin (10mm/dm) and lowest in Amoxil (0mm/dm). There is no effect on antibiotic Erythromycin and Ampiclox (Table 1).

In negative test against Staphylococcus aureus the zone of inhibition was highest in antibiotic Reflacine (10mm/dm) and lowest in Amplicin (2mm/dm). There is no effect on antibiotic Ceporex and Nacidixic acid (Table 2).

I- Zone of inhibition of some antibiotics against Staphylococcus aureus using industrially manufactured sensitivity disc

In positive test against Escherichia coli the zone of inhibition was highest in antibiotic Ciproflox (7mm/dm) and lowest in Amoxil (2mm/dm). There is no effect on antibiotic Norfloxacin, Erythromycin, Levofloxacin and Ampiclox (Table3).

In negative test against Escherichia coli the zone of inhibition was highest in antibiotic Streptomycin (12mm/dm) and lowest in Tarivid (2mm/dm). There is no effect on antibiotic Ceporex and Nacidixic acid (Table4).

II- Zone of inhibition of some antibiotics against Escherichia coli using industrially manufactured sensitivity disc: Some researchers found that Staphylococcus aureus was the cause of wound sepsis and burns. A study from Ilorin , Nigeria found that 38% of wound infections are isolates of Staphylococcus aureus and 78% sensitivity was found in another study from Owerri, Nigeria [6-9].

1. Parekh J, Chanda S In vitro screening of antibacterial activity of aqueous and alcoholic extracts of various Indian plant species against selected pathogens from Enterobacteriaceae. Afr J Microbiol Res. 2007;1:92-99.
2. Onile BA, Odugbemi TO, Nwofor C. Antibiotic susceptibility of Bacterial agents of Septicemia in Ilorin. Big Med Pract. 1985;9(4):16-18.
3. Greenwood D. Detection of antibiotic resistance in vitro. Int J Antimicrob Agents. 2000 May;14(4):303-6. doi: 10.1016/s0924-8579(00)00141-2. PMID: 10794951.
4. Jones RN. Disk diffusion susceptibility test development for the new glycylcycline, GAR-936. Diagn Microbiol Infect Dis. 1999 Nov;35(3):249-52. doi: 10.1016/s0732-8893(99)00090-5. PMID: 10626138.
5. de Castillo MC, de Saab OA, de Nader OM, de Ruiz Holgado AP. In vitro comparison of disk diffusion and agar dilution antibiotic susceptibility test methods for Neisseria gonorrhoeae. Mem Inst Oswaldo Cruz. 1998 Jul-Aug;93(4):517-22. doi: 10.1590/s0074-02761998000400019. PMID: 9711344.
6. Emmerson M. Nosocomial staphylococcal outbreaks. Scand J Infect Dis Suppl. 1994;93:47-54. PMID: 8047857.
7. Nwankwo EO, Nasiru MS. Antibiotic sensitivity pattern of Staphylococcus aureus from clinical isolates in a tertiary health institution in Kano, Northwestern Nigeria. Pan Afr Med J. 2011;8:4. doi: 10.4314/pamj.v8i1.71050. Epub 2011 Jan 26. PMID: 22121413; PMCID: PMC3201603.
8. Uwazuoke JC, Aririatu LE. A survey of antibiotic resistant staphylococcus aureus strains from clinical sources in Owerri. J Appl Sci Environ Managt. 2004;8(1):67-68.
9. Frnest B, Hawkins MS. Health Education Resource; and Steven D Ehriich, N. M. D., private practice specialization in complementary and alternative medium, Rhoenix, AZ. Review provided by verimed Health care Network. 2007.