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African Journal of Biomedical Research
Ibadan Biomedical Communications Group
ISSN: 1119-5096
Vol. 6, Num. 2, 2003, pp. 91-94
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African Journal of Biomedical Research, Vol. 6, No. 2, May, 2003, pp. 91-94
THE EFFECT OF A FIXED NON-MINERAL OIL ON THE ANTIBACTERIAL
ACTIVITY OF AMPICILLIN TRIHYDRATE AGAINST RESISTANT CLINICAL STRAINS OF STAPHYLOCOCCUS
AUREUS .
O.E ADELEKE* and J.O. OLAITAN
Microbiology Department, University of Agriculture , Abeokuta , Nigeria
*Author for correspondence
Received: May 2001
Accepted in final form: February
2003
Code Number: md03017
ABSTRACT
The effect of King's Vegetable oil, a fixed non-mineral oil, on the antibacterial
activity of ampicillin trihydrate, a water- insoluble form of ampicillin,
was investigated against resistant clinical strains of Staphylococcus aureus.
In the agar-diffusion method employed, 40% of the resistant clinical strains
tested showed sensitivity to different oil-dispersed concentrations of ampicillin
trihydrate, which ranged from 0.06µg/ml to 1.25µg/ml. The resistant
strains were among the clinical strains detected with Beta-lactamase. This
finding is presented as a preliminary report on the potentiality of employing
an oil medium to effect a "cure" of antibiotic resistance in staphylococci
besides the use of acridine dyes, ethidium bromide, ultraviolet radiation
and other measures.
Keywords: King's vegetable oil, ampicillin trihydrate, staphylococcus aureus,
INTRODUCTION
Ampicillin trihydrate is a water-insoluble form of ampicillin for its activity
against sensitive strains of S. aureus (Hugbo and Ruczaj, 1983). The
poor water solubility profile of ampicillin trihydrate (Clarke,1986) implicates
a poor antibacterial activity due to lack of effective concentration of drug
molecules across the bacterial cell membrane. In a previous study (Adeleke
and Agunbiade, 1991), ampicillin trihydrate dispersed in an oil medium was
associated with a greater antibacterial activity against sensitive strains
of S.aureus , than water-dispersed ampicillin trihydrate and the water-soluble
form of ampicillin (ampicillin sodium) both in tube broth-dilution and agar-diffusion
methods. These results were predicated upon reports of preferential distribution
of some microorganisms and water-insoluble antimicrobial compounds into oily
organic phase relative to aqueous phase, in partioning coeeficient based distribution
experiments (Adeleke, 1991). The minimal water content of fixed non-mineral
oils (Diem and Letner, 1974) was among the reasons suggested for the better
antibacterial activity.
Resistance of S. aureus is atttributed largely to transducible R- plasmids
which mediate production of Beta-lactamase in the resistant strains (Davis et
al ., 1973). The inactivation process of Beta-lactamase on susceptible
Beta-lactam antibiotics such as ampicillin, is hydrolysis (Franklin and Snow,
1975). Application of oil in the dispersion of a water-insoluble antibiotic
would be expected to avoid the hydrolytic reaction, thereby, facilitating the
anti-bacterial activity of the compound. This property could therefore, find
application in the elimination or "curring" of bacterial resistance
to antibiotics that are susceptible to hydrolysis by Beta-lactamase, as an
alternative to elimination with acridine dyes and ethidium bromide, some of
which have mutagenic effects on bacterial cells (Bouanchaud et al .
1969).
In the present study, the effect of King's Vegetable oil, a fixed non-mineral
oil, on the antibacterial activity of ampicillin trihydrate, a water- insoluble
form of ampicillin, was investigated against resistant clinical strains of Staphylococcus
aureus . This is with a view to finding out the potentiality of employing
an oil medium to effect a "cure" of antibiotic resistance in staphylococci
besides the use of acridine dyes, ethidium bromide, ultraviolet radiation and
other measures
MATERIALS AND METHODS
Microorganisms: Fifty (50) clinical strains of S. aureus and
a standard strain,NCTC 6571,were used in this study. The clinical strains were
isolates from specimens of different clinical manifestations obtained from
the university College Hospital , Ibadan . All the strains were confirmed coagulase
and deoxyribonuclease positive as well as fermentative in mannitol, both under
aerobic and anaerobic incubations. They were maintained as purified cultures
on tryptone soya agar (OXOID) slants and subcultured periodically.
Fixed non-mineral oil: King's vegetable oil (bleached palm olein),
Devon Industries SDN RHD RS, was obtained locally. It was sterilised by dry
heat in an oven sterilizer at 160 o C for 1hr.
Multodisk R Agar-diffussion test: As specified for staphylococci
(Brown and Blowers, 1978), a 4mm loop-full of an overnight nutrient broth (OXOID)
culture of each strain of S. aureus was mixed with 3ml. of sterile
nutrient broth, for a 1:1000 dilution of the culture. The mixture was used
to flood a plate of dried Seed Agar antibiotic medium I (BBL) to form an overlay
and excess of the mixture was siphoned out. A strip of Multodisk R (OXOID)
was placed on the medium followed by incubation at 37 o C for 24hrs. The multodisk
R heads carried penicillin G (1.6 i.u or 1µg), ampicillin (2µg),
cloxacillin (5µg) and erythromycin (10µg).
Screening for Beta-lactamase production: All the strains of S.
aureus were screened for the production of Beta-lactamase enzyme by the
iodometric cell-suspension method (Sykes, 1978). The cell population in every
cell-suspension prepared in phosphate bufferd penicillinG was estimated at10
9 cells per ml.on Mcfarland turbidity standards (Bauer et al ., 1966).
MIC of ampicillin trihydrate in an oil medium: Sterile distilled
water served as a medium of transference of ampicillin trihydrate into sterile
oil by removing 5ml. of aqueous 1,000µg/ml.concentration into 45g of
sterile oil to give 100µg/ml.from which graded decreasing concentrations
were prepared in nutrient broth. Each mixture was inoculated with 0.1ml.of
1:100 dilution of overnight broth culture of S. aureus NCTC 6571 and
shaken on a vortex Mixer (Griffin) followed by incubation with appropriate
controls at 37 0 C for 24hrs.
Similar determination was carried out using sterile distilled water entirely
as the dispersion medium for ampicillin trihydrate.
Agar-diffusion (well-in-seeded plate) test: Selected resistant
clinical strains were exposed to three successively high concentrations of
ampicillin trihydarte in oil medium including the MIC obtained for the standard
strain, NCTC 6571. Wells were cut in duplicates in the seeded plate of each
strain. Each well was filled with 0.1ml. of x10 of a particular concentration
so as to obtain the required concentration in well. For instance, 0.1ml. of
0.6µg/ml. of oil-dispersed ampicillin trihydrate was filled into each
of two wells to give a final concentration of 0.06µg/ml. The other two
concentrations used were 0.125µg/ml and 1.25µg/ml. The plates were
incubated after allowing a pre-incubation diffusion period of 2hrs at 37 o
C for 48hrs.
RESULTS
Forty-seven (94%) of the 50 clinical strains of S..aureus were resistant
to penicillin G and ampicillin while 16 (32%) of the strains were sensitive
to cloxacillin. All the strains but one was sensitive to erythromycin. Most
of the resistant strains were from pyoderma which also represented 42% of the
specimen collection sources. Beta-lactamase was detected in 80% of the 47 resistant
strains, leaving only a strain without the enzyme.
The minimum inhibitory concentration (MIC) obtained for ampicillin trihydrate
dispersed in oil medium against S. aureus NCTC6571, was 0.06µg/ml.
against 0.25µg/ml. for aqueous ampicillin trihydrate (Table 1). In the
subsequent Agar-diffusion (well-in-seeded plate) sensitivity test using 0.06,
0.125 and 1.25µg/ml.in oil medium against selected resistant clinical
strains, sensitivity of 40% of the strains test was recorded to one or more
of the three concentrations (Table 2).
DISCUSSION
The preponderance of Beta-lactamase producers among the resistant clinical
strains of S. aureus underscores the menace associated with the implication
of B-lactamase in bacterial resistance to susceptible antibiotics (Oyelese
and Oyewo, 1995). This observation further paints an apprehensive picture with
only 16 of the 47 resistant strains showing sensitivity to cloxacillin, a Beta-lactamase
stable penicillin antibiotic. However, while virtually all the strains were
sensitive to erythromycin, a macrolide antibiotic, it is interesting to note
that 40% of the resistant clinical strains tested for elimination of antibiotic
resistance, to different concentrations of ampicillin trihydarte in oil medium,
reverted to sensitive strains. This result suggests the potentials of using
fixed non-mineral oils, especially King's Vegetable oil, in the elimination
of staphylococcal resistance to Beta-lactamase sensitive antibiotics such as
the parent penicillins and some semi-synthetic Beta-lactam antibiotics. This
would be a radical departure from the use of "traditional" curring
agents such as acriflavine, acridine orange, mepacrine and ethidium bromide
(Bounchaud et al ., 1969; Wantanabe and
Table1 MIC of ampicilin trihydrate B. P. in aqueous medium and oil
medium against S.
aureus NCTC 6571
Type of medium
|
Antibiotic concentration (µg/ml.)
|
MIC (µg/ml.)
|
1 |
0.5 |
0.25 |
0.125 |
0.0625 |
0.031 |
Aqueous medium |
- |
- |
- |
+ |
+ |
+ |
0.25 |
Oil medium |
- |
- |
- |
- |
- |
+ |
0.065 |
KEY: MIC = minimum inhibitory concentration; NCTC = National
collection of typed cultures
+ = growth; - = no growth
Table 2 Agar-diffusion test on resistant clinical strains of S.
aureus with
oil-dispersed ampicillin trihydrate
*Strain of S. aureus |
Antibiotic concentration (µg/ml.) |
Oil (5g/ml.) |
0.06 |
0.125 |
1.0 |
4611 |
- a |
12mm** |
15mm |
- b |
6370 |
15mm |
15.5mm |
16.6mm |
- |
1096 |
- |
- |
- |
- |
4747 |
- |
- |
- |
- |
5843 |
13mm |
14mm |
- |
- |
UR-0F |
- |
- |
- |
- |
2018 |
- |
- |
- |
- |
700 |
- |
- |
- |
- |
1566 |
- |
- |
- |
- |
14.5mm |
14.5mm |
16.5mm |
18mm |
- |
KEY: ** = Zone of inhibition in millimetres; a = No zone of inhibition; b
= No growth in oil
*= Strain of S. aureus with resistance to penicillinG,
ampicillin, cloxacillin and erythromycin.
Fukasawa, 1960d; Watanabe and Fukasawa, 1961b; Levy and Watanabe, 1966). Some
of these agents such as ethidium bromide are mutagenic (Bouachaud et al., 1969;
Naomi, 1978), as such, their curring effect only constitutes an evidence but
not a proof that a resistance is plasmid mediated (Naomi, 1978). Moreover,
the curring effect of these agents on resistant bacteria is yet to be associated
with application in clinical situations. Interestingly, metabolisable fixed
non-mineral oils are commonly used in the formulation of small-volume oily
injections (Allwood, 1983).
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