Control of Staphylococcus aureus Sensitivity to Sweet Pepper (Capsicumannum) by a Chemical Mutation
Chemical mutation was carried out on Staphylococcus aureus
using ethylmethyl sulphonate. The bacterium and its mutants were tested
for sensitivity to the extract of Capsicum annum (sweet pepper).
Mutants with varying degrees of sensitivity to the extract were obtained.
On the basis of zone of inhibition, mutants were classified as Non-sensitive
(NS), Slightly-sensitive (SS), Fairly-sensitive (FS), Normal-sensitive
(NMS) and Super-sensitive (SUS) mutants with zones of growth inhibition
on Mannitol Salt Agar (MSA) in the range of 0.00-0.09, 0.10-1.09, 1.10-2.09,
2.10-3.09 and 3.10-4.09 mm respectively. About 26, 7 and 23.5% mutants
were screened as NS, SS and FS respectively. Other mutants, NS and SS
constituted 27 and 16.5% of the total mutants population. Some of the
mutants appeared bacteriostatic, bactericidal and bacteriolytic in actions.
Also, mutation caused a change in colour of MSA from red to yellow. Some
mutants completely changed the colour (complete colour change, CCC mutants)
with zone of colour change of 9.00 mm. Size of colour change exhibited
by other mutants ranged from 2.40 to 3.20 mm (slight colour change, SCC),
3.50 to 4.90 mm (strong colour change, STCC) and 0.00 mm (no colour change,
NCC) relative to the wild-type which showed 4.70 mm zone of colour change.
Capsicum annum (sweet pepper) belongs to the night shade family
of Solanaceae introduced into India by the Portuguese in the 17th century.
It is a native of tropical America and West Indies; cultivated in many
tropical countries including Nigeria. It is used for flavouring and giving
taste to curries, chutneys, salads etc. (Dutta, 1981). Traditionally,
it is used to treat various diseases including dropsy, colic, diarrhoea,
asthma, arthritis, muscle cramps, tooth aches, high and low blood pressures,
apoplexy, gangrene or mortification and asphyxia. It is a powerful stimulant,
carminative, anti-oxidant, anti-inflammatory and anti-cancer agent (Demmig-Adams
et al., 1996; Kelloff et al., 2000). Al-Qarawi and Adams
(2003) reported that C. annum helped to lower human blood pressure;
a property, which has been applied locally.
Capsicum annum possesses antibacterial, antifungal and antiviral
activities (Cereaga et al., 2003). It is active against both gram-positive
and gram-negative bacteria such as Staphylococcus aureus, Listeria
monocytogenes, Bacillus subtilis, Escherichia coli
and Pseudomonas aureginosa (Barber et al., 2000; Dorantes
et al., 2000). Its antifungal action is directed towards many fungi
like Candida tropicalis and Saccharomyces cerevisiae ATCC
9763 (Kivanc and Akgul, 1998). Farag et al. (2003) showed that
C. annum enhanced the production of neutralizing antibodies to
combat viral infections including Herpes zooster virus.
In a previous research done by Boboye et al. (2007), it was noted
that Capsicum annum and C. frutescens inhibited the growth
of Klebsiella pneumoniae, Streptococcus faecalis, Corynebacterium
diphtheriae, Pseudomonas aeruginosa and Escherichia coli.
It was also demonstrated that the minimum inhibitory concentrations of
the pepper on gram-positive, Staph. aureus and gram-negative, Ps.
aureginosa were 402 and 335 mg mL-1 respectively (Boboye,
2004). Recently, we carried out a chemical mutation with ethylmethyl sulphonate
(EMS) on Staph. aureus to study its sensitivity to sweet pepper.
This forms a genetical basis for the reaction of Staph. aureus
to antibacterial agent like sweet pepper and also gives a clue to the
mode of action of the Capsicum on the bacterium.
MATERIALS AND METHODS
Capsicum annum and Staphylococcus aureus were obtained
from `Oba` market and State Specialist Hospital, Akure, Nigeria respectively.
The bacterium was stored on nutrient agar slant.
Experiment on Mutation
Fresh culture of Staph. aureus was inoculated into nutrient
broth and grown at 37°C for 18 h. It was pour plated on Mannitol Salt
Agar (MSA) and incubated for 24 h. Chemical mutation by EMS was performed
using method of Parkinson (1976) with slight modification as described
by Boboye and Alao (2008). Mutational rate was calculated and mutants
were used for further studies.
Preparation of Pepper Extract
The extract of pepper was prepared according to the method of Boboye
and Dayo-Owoyemi (2004) with little change. Sweet pepper was ground with
sterile water using sterile mortar/pestle and blender to obtain 667 mg
mL-1. The ground material was filtered to constitute the extract.
Test for Growth Inhibition
Agar diffusion method was employed. An 18 h grown culture containing
26X107 cells was pour plated in MSA. A well was bored into
the agar with a 17 mm diameter cork borer and filled with 1 mL of the
extract. Incubation was carried out and distance of growth restriction
formed around the well bored was measured at 24 and 48 h. This was repeated
for every mutant and the wild-type strain. Isolate`s ability to change
colour of the agar from red to yellow was recorded in distance (mm) covered
by the colour from the well.
RESULTS AND DISCUSSION
Growth Effect of Capsicum annum on the Staphylococcus aureus
Mutational rate of the mutagen was 1.44% survival of the Staph
aureus. Two hundred mutants screened exhibited various levels of growth
inhibition to the Capsicum annum. The mutants were grouped into
five classes (Table 1). Some mutants were resistant
without any appreciable clear distance (0.00 to 0.09 mm) created between
the holed extract and the grown cells. These are the Non-sensitive mutants
(class 1) with 52 members (25% of the total mutants screened). In contrast,
many mutants were restricted in growth towards the extract with a clear
gap ranging from 0.10 to 4.09 mm between the agar well and the grown cells.
These mutants were rated as Slightly- sensitive (SS) class 2 (0.10-1.09
mm), Fairly-sensitive (FS) class 3 (1.10-2.09 mm), Normal-sensitive (NMS)
class 4 (2.10-3.09 mm) and Super-sensitive (SS) class 5 (3.10-4.09 mm)
with 14, 47, 54 and 33 members respectively. The wild-type grew 3.00 mm
away from the extract.
||Growth resumption of the mutants of Staphylococcus
aureus after 24 h treatment with the extract of Capsicum annum
Growth Resumption of the Mutants of Staphylococcus aureus
At 48 h of incubation, growth inhibition zone of the wild-type
strain was observed to increase from 3.00 to 3.10 mm while many of the
mutants (above 85%) retained their zones of inhibition. Other mutants
in classes 2, 3, 4 and 5 resumed growth (Table 2). These
constituted 7.14, 8.51, 11.11 and 3.03% mutants of the classes respectively.
This action implies that the pepper was bacteriostatic to the mutants;
they are thus termed pepper-bacteriostatic-respondents. On the other hand,
all the Non-sensitive mutants in class 1 with the exception of MU 24,
26, 32, 64 and 81, remained resistant to the inhibitory power of the pepper
extract. Clear zones of growth inhibition measuring 0.30, 0.20, 0.80,
0.20 and 0.40 mm were observed around wells of pepper extract on agar
seeded with the MU 24, 26, 32, 64 and 81 mutants, respectively after 48
h of incubation. This indicates exhibition of delayed sensitivity to the
extract. These mutants are pepper-bactericidal-respondents.
||Colour change of growth medium by the mutants of Staphylococcus
The pepper appeared to inhibit growth of wild-type Staph. aureus by
killing the cells; the cells were intact microscopically although dead.
This indicates that the pepper is naturally bactericidal in action to
the Staph. aureus. This mechanism was altered to bacteriostatic
and bacteriolytic by mutation as depicted in many mutants. Microscopic
examination showed that the cells of some mutants have ruptured appearing
as particles making the mutants pepper-bacteriolytic-respondents. Bacteriostatic
agent binds loosely to ribosomes during which there is growth inhibition;
later when the agent becomes free from the ribosomes; growth is resumed.
This pepper acting as bacteriostatic agent must have- inhibited protein
synthesis but did not kill the organism. Bactericidal and bacteriolytic
agents bind tightly to cellular target of the relevant organism. The former
agent leave cells unbroken while the latter induced killing by lysis which
was observed in this experiment as turbidity in the MSA after growth.
They inhibit cell membrane and cell wall synthesis (Brock et al.,
1984; Madigan et al., 2001).
Medium Colour Change Effect of Mutation on Staphylococcus aureus
In this experiment, it was also observed that the wild-type strain
changed the colour of the MSA growth medium to yellow from red with a
zone of 4.70 mm yellow colour. The mutants exhibited colour change at
various levels. This variation range from red colour change to complete
yellow (CCC mutants) through Normal colour change (NMCC mutants), Slight
colour change (SLC mutants) to No colour change (NCC mutants) (Table
3). This change in colour implies that many of the mutants fermented
the mannitol to release acid as the wild-type. Variation in the growth
inhibition of the cells, mode of action of the pepper and medium colour
change was caused by random hits of the mutagen (EMS) on the genes encoding
these properties. The genes in some cells of class 1 mutants were hit
to inactivate the genes causing varying sensitivity of the mutants to
inhibitory action of the pepper with concomitant differing levels in growth
inhibition and fermentation of mannitol by the mutant cells. The results
obtained in this work are similar to that of Vivjer et al. (2001)
who reported that Staph. aureus treated with EMS formed mutants
with various pathogenic virulence relative to the wild-type strain. Ethylmethane
sulphonate causes mutation by transforming DNA strand. It introduces a
methyl group to various points on G nucleotide. This causes guanine to
faulty pairs with thymine resulting in GC:AT transition (Suzuki et
Spontaneous mutation and transfer of resistant genes among microbes in
the same environment could cause resistance in microorganisms to antibiotic;
this however occurs rarely (106 or 109 times) (Brock
et al., 1984). Resistance to antimicrobial agent occurs in nature
after a long use of the agent. This research has revealed that Capsicum
annum can act differently on Staphylococcus aureus when subjected
to mutation. This suggests that sweet pepper can be used effectively to
cure diseases caused by Staph. aureus and the bacterium can be
made resistant and sensitive like any other microorganism pointing to
future possible occurrence after along use.
We thank the Department of Microbiology and the chief technologist (O.E.
Achor) of the department and G.F. Hassan in the Department of Crop, Soil
and Pest Management, Federal University of Technology, Akure, Ondo State,
Nigeria for instrumentation and chemicals used for this work.
1: Al-Qarawi, A.A. and S.E. Adams, 2003. Effect of combination of Capsicum annum and lutein on growth, haematological and pathophysiological parameter of rats. J. Qafaz Univ., 5: 34-36.
2: Barber, M.S., V.S. Mc Connell and B.S. De Caux, 2000. Antimicrobial intermediates of general phenylpropanoids and lignin specific pathway. Phytochemistry, 54: 53-65.
Direct Link |
3: Boboye, B., 2004. Antibacterial effect of Capsicum annum on Pseudomonas aeruginosa and Staphylococcus aureus. Biosci. Biotechnol. Res. Asia, 1: 49-50.
4: Boboye, B. and I.D.Owoyemi, 2004. Antibacterial effect and minimum inhibitory concentrations of garlic (Allium sativum) extract on some human pathogenic bacteria. Biosci. Biotechnol. Res. Asia, 1: 37-40.
5: Boboye, B., T. Babatunde and A. Onoriode, 2007. Antibacterial activities of some plants used as condiments and spices in Nigeria. World Curr. Environ., 2: 171-174.
Direct Link |
6: Boboye, B. and A. Alao, 2008. Effect of mutation on trehalose-catabolic-enzyme synthesized by a tropical Rhizobium species F1. Res. J. Microbiol., 3: 269-275.
CrossRef | Direct Link |
7: Brock, T.D., D.W. Smith and M.T. Madigan, 1984. Biology of Microorganisms. 4th Edn., Prentice-Hall International Inc., New Jersey, USA
8: Cereaga, M., E. Fernandezi, L. Dorantes, L. Mota, M.E. Jaramillo and H. Hernandez-Sanchez, 2003. Antibacterial activity of Capsicum extract against Salmonella typhimurium and Pseudomonas aeruginosa inoculated in raw beef meat. Int. J. Food Microbiol., 83: 331-335.
CrossRef | Direct Link |
9: Demmig-Adams, B., A. Gilmore and W.W. Adams, 1996. In vivo function of carotenoids in higher plants. FASEB. J., 10: 403-412.
10: Dorantes, L., R. Colmenero, H. Hernandez, L. Mora, M.E. Jaramillo, E. Fernandez and C. Solano, 2000. Inhibition of growth of some food borne pathogenic bacteria by Capsicum annum. Int. J. Food Microbiol., 57: 125-128.
Direct Link |
11: Dutta, A.C., 1981. Botany for Degree Students. 5th Edn., Oxford University Press, Calcutta, India, ISBN-13: 978-0195611021, Pages: 825.
12: Farag, R.S., Z.Y. Daw, F.M. Hewedi and G.S. El-Baroty, 1989. Antimicrobial activity of some Egyptian spice essential oils. J. Food Prot., 52: 665-667.
Direct Link |
13: Kelloff, G.J., J.A. Growell and V.A. Steele, 2000. Progression of cancer chemoprevention: Development of diet derived thermopreventive agents. J. Nutr., 130: 467S-471S.
14: Kivanc, M. and A. Akgul, 1986. Antibacterial activities of essential oils from Turkish spices and citrus. Flavour Fragrance J., 1: 175-179.
CrossRef | Direct Link |
15: Madigan, M., P. Martino and J. Parker, 2001. Brocks Biology of Microorganisms. 9th Edn., Prentice-Hall International Inc., London, pp: 204-208
16: Parkinson, J.S., 1976. Che A, Che B and Che C genes of Escherichia coli and their role in chemotaxis. J. Bacteriol., 126: 758-770.
Direct Link |
17: Suzuki, D.T., J.E. Griffin, H.J. Miller and C.R. Lewontin, 2001. An Introduction to Genetic Analysis. 7th Edn., Freeman Publishers, USA., pp: 101-105
18: Vivjer, J.C., M.M. Es-Boon and M.F. Micheal, 2001. Introduction to mutation in Staphylococcus aureus by ethylmethane sulphonate. Acta Microbiological Polonica, 3: 151-162.