ABSTRACT
Doxycycline and Vibramycin are newer members of
tetracycline which appears to offer enhanced antimicrobial effectiveness and
more efficient absorption in the body. The comparism between the activities of
Doxycycline (Malvin Medics) a local brand and Vibramycin (Pfizer) an imported
brand in this study showed that doxycycline is more effective and cheaper than
vibramycin. The minimum inhibitory concentration (MIC) of both antibiotics were
experimentally determine using fifteen (15) clinical isolates of Staphylococcus aureus, Pseudomonas
aeruginosa, Klebsiella pnuemoniae, Proteus species and Escherichia coli isolated
from Otitis media. Although, there was a co-relationship in the activities of
both antibiotics but the isolates appeared more sensitive to Doxycycline at
lower MIC (0.05µg/ml) than Vibramycin (0.8µg/ml). These findings clearly
suggest that medical practitioners should encourage the use of doxycyline over
vibramycin.
TABLE
OF CONTENTS
Title
page i
Certification iii
Dedication iv
Acknowledgement v
Abstract vii
Table
of content viii
CHAPTER ONE
1.0 History and development of
chemotheraphy 1
1.1 Review
of the antibiotic in study 5
1.2 Discovery
and development of the tetracycline 8
1.3 Mode
of action 13
1.4 Resistance
15
1.5 Ribosomal
protection protein 18
1.6 Interactions
with the absorption of tetracycline 20
1.7 Pharmaco-therapeutics
of the newer tetracycline 21
CHAPTER TWO
Material and methods
2.0 Materials/equipment 23
2.1 Media 23
2.2 Sample
source and storage of isolate 23
2.3 Cleaning
and sterilization and glasswares 24
2.4 Preparation
and sterilization of media 24
2.5 Storage
and maintenance of isolates 24
2.6 Identification
of test isolates 25
2.6.1
Biochemical test 25
2.7 The
dilution method adapted to agar cell diffusion method 31
CHAPTER THREE
3.0 Results 33
CHAPTER FOUR
4.0 Discussion
and Conclusion 37
4.1 Recommendation 39
References 41
Appendix 51
CHAPTER ONE
1. 0 HISTORY AND DEVELOPMENT OF CHEMOTHERAPHY
One
of the greatest triumphs of modern medicine has been the introduction of a
rational system of antimicrobial chemotherapy to combat infection diseases. The
first recorded use was an extract of cinchona bark (quinine) for treating
malaria in 1619. In 1631 Quinine was used to treat malaria in Rome by the Jesuit brother Agostino
Salumbrino (1561-1642). He used the bark of the cinchona tree to treat the
Romans.
In
the 1860’s, a French scientist, Louis Pasteur proved that many diseases were
caused by bacteria and concluded that man could learn to fight these bacteria
using other bacteria.
Two
German doctors, Ruddy Emmerich and Oskar Low, were the first to make an
effective medicine from microbes. Among other things, they successfully proved
that the germ which causes other diseases can successfully cure other diseases.
The
history of antibiotics officially begins in the late 1920s when the Scottish
bacteriologist, Alexander Fleming (1881-1955) identified penicillin as an
antibacterial agent; however, the use of antibiotics dates back to ancient
times. The Chinese were know to use molds to fight infections as early as 400BC
while medical folklore in Europe treated
infections with plants materials and extracts some of which were later shown to
have antimicrobials properties. Chinese traditional medicine, as well as
European medical folklore achieved some successes in the treatment of infection
with the selected molds and plants extracts but it was not until the 19th
century when the scientist identified bacteria as a disease-causing agent
(Atkinson et al., 1997)
As
a result, the scientist began to devote time to searching for drugs that would
kill the disease-causing bacteria. The goal was to find the magic bullet which
would destroy the bacteria without being toxic to the host taking the drug
(Andres et al., 1998).
The
first chemotherapeutically effective antibiotic was discovered in 1929 by
Alexander Fleming (1881-1955); a British bacteriologist who had long being
interested in the treatment of wound infections. On returning from a vacation
on Sept 3rd 1929 noticed among a pile of petri-dishes on his bench
one that had being streaked with a culture of staphylococcus aureus
which was also contaminated by a single colony of mold. As Fleming observed the
plate, he noted that the colonies immediately surrounding the mold were
transparent and appeared to be undergoing lysis. He reasoned that the mold was
excreting into the medium a chemical that caused the surrounding colonies to
lyse. Sensing the possible chemotherapeutic significance of his observation,
Fleming isolated the mold which proved to be a species of penicillium and established that the culture filtrates contained an
antibacterial substance which he called pencillin (Freeman et al.,1994). He
investigated its positive antibacterial effect on many organisms and noticed
that it affected bacteria such as staphylococci and many more gram positive
pathogens that causes scarlet fever, pneumonia, meningitis, diphtheria and neisseria gonorrhoeae which causes gonorrhoea.
In 1932, Gerhard Johannes Paul (1895-1964) a German
pathologist and bacteriologist turned his attention away from natural
antibiotics and towards synthetic ones. The first sulfonamide, sulfanilamudo
(prototype of which was synthesized in 1908) and used widely as dye before in
1935. Gerhard Johannes reported its effectiveness against streptococci. Later
it proved effective against several other bacterial including those causing
scarlet fever, certain venereal disease and meningitis (Grave et al., 1999).
In
1939 Rene Jules Dubos (1901-1982) a French Born American Microbiologist
isolated the antibacterial agents tyrothricin and gramicidine from bacteria (Bacillus brevis). This organism was found in the soil and it inhibited or
killed gram positive bacteria but proved highly toxic to human (Gales and
Jonnes, 2000)
In
1943 Albert Schatz (1922-2005) a graduate student in the laboratory of Selman
Abraham Waksman (1888-1973) isolated another antibacterial agent from the soil Streptomyces griseus. Streptomycin
proved effective against several common infections most note-worthy was its
ability to kill the bacterium mycobacterium tuberculosis, the microbe causing
tuberculosis which had up till to that point resisted numerous methods of
treatment (Kingston, 2004).
In
1945, Benjmanin Minge Duggar (1872 to 1956) an American plant physiologist
working at Lederle laboratories under the supervision of Drs Sebba Rao
identified chlorotetracycline (the first tetracyline to be identified) as the
product of an actinomycete he cultured from a soil sample collected from
Sanborn field. The organism was named streptomyces
aurofaciens and the isolated drug
Aureomycin because of their golden colour (Roberts, 1997).
1.1 REVIEW OF THE ANTIBIOTIC IN
STUDY: TETRACYCLINE
Tetracycline
is a broad spectrum polyketide produced by the streptomyces genes of actinobacteria, indicated for use against
many bacteria infections. It is protein synthesis inhibitor, which is commonly
used to treat acne today, and more recently rosacea, and also it is
historically important in reducing the number of deaths from cholera.
Tetracycline is antibiotic produced, marketed under the brand names declomycin,
vibramycin, doxycycline, panmycin among others. Actisile is a thread like fiber
formulation used to produce several semisynthetic derivatives, which together
are known as tetracycline antibiotics. The term tetracycline is used to denote
the 4-ring system of this compound. Tetracyclines are related substances that
contain the same 4- ring system with their different individual side chains (De
Rossi et al.,1999).
STRUCTURE OF TETRACYCLINE
 |
Chlorotetracycline differs from
tetracycline having a chlorine atom; doxycycline consists of tetracycline and
an extra hydroxyl oxytetracycline and chlorotetracycline which are produced
naturally by streptomycin species. These antibiotics are similar to
the aminogylycosides and acts on 30s subunit of the ribosome. This inhibits the
binding of aminocyl-tRNA molecules to the acceptor (A) site of the ribosome
because their action is only bacteriostatic, the effectiveness of treatment
depends on active host resistance of pathogen (Barden et al; 1994).
Tetracycline
are broad spectrum antibiotic that are active against gram negative as well as
gram positive bacteria, staphylococcus and some protozoans (Bertram et al.,
1991) the favourable antimicrobial properties of these agents and the extensive
use in the therapy of human and animal infections. They are also used
prophylactic drugs for the prevention of malaria caused by mefloquine resistant
Plasmodium falciparum (Bunnag et al., 1996) Although the tetracycline
retain important role in both human and veterinary medicine, the emergence of
microbial resistance has limited their effectiveness.
The
use of tetracycline, in clinical practice has being responsible for the
selection of resistance organism. Never the less, the use of tetracycline and
other antibiotics as animal growth promoters is becoming increasingly controversial
because of concerns that these practices may be contributing to the emergence
of resistance in human pathogens. The increasing incidence of bacterial
resistance to tetracycline has in turn resulted in efforts to establish the
mechanism by which genetic bacteria and the molecular basis of the resistance
mechanism themselves (Atkinson et al., 1997).
High doses of
tetracycline may result in nausea, diarrhea, skin photo-sensitivity, breathing
complication as well as anaphylactic shock, discoloration of the teeth in
children and damage to skin and liver. Although their use has decline in recent
years, they are skill sometimes used to treat acne.
1.2 DISCOVERY
AND DEVELOPMENT OF THE TETRACYCE
Chlortetracycline
and oxytetracycline both discovered in the late 1940s were the first members of
the tetracycline group to be described. These molecules were products of streptomyces aureofaciens and streptomyces rimosus respectively. Other
tetracyclines were identified later either as naturally occurring molecules
e.g. tetracycline from s. aureofaciens, streptomyces
rimosus and streptomyces viridafacines
and demethylchloretetracycline from streptomyces
aurofacines or as products of
semisynthetic approaches e.g. methacycline, doxycyline, minocycline. Despite the
success of the early tetracyclines, analogs were sought with improved water solubility
either to allow parental administration or to enhance and absorption ( George et al.,). These approaches resulted in
the development of the semosynthetic compound rolitetracycline and lymecycline.
The most recently discovered tetracyclines are the semisynthetic group referred
to as glycyclines e..g 9-(N,N–dimethylglcylamido)-6-demethly-6-
doxytetracycline, 9- (XI,XI dimethyl glycyclamid)-minocycline and 9-t-
(butylglycychmido) – monocycline these compound posses a 9-glyclamido
substituent.
Antibiotic between (1948 to 1963) can be
referred to as first generation, second generation (1965-1922) and third
generation (glycylcycline) tetracycline. The 9-t-butylglycylamindo derivative
of minocycline (yigilyclcine; formetly know as GAR-936) commenced phase I study
in October 1999 and is currently under going clinical trials (Johnson, 2000)
some of the earlier compound e.g. chlortetracycline are not available in all
countries (Finch, 1997).
By
studying the literature of tetracyclines it becomes clearly evident that
tetracycline are very dynamic molecules. In some cases their structure-activity
relationship are know especially against bacteria while against other targets
they are virtually unknown (Guay et al., 1994).
Tetracycline
molecules comprises a linear fused tetracycline nucleus (ring, designated A,B,C
and D) to which a variety of functional groups are attached. The simplest
tetracycline to display detectable antibacterial activity is 6-deoxy -6demethyltetracycline
and so this structure may be regarded as minimum pharmacophore (Chopra et al.,
1992). Features important for antibacterial activities among the tetracycline
are maintenance of the linear fused tetracycline, naturally occurring α- stereochemical
configurations at the 4a 12a (AB ring junction) and 4 (dimethylamino group)
positions and conservation of the keto system (position 11, 12 and 12a) in
proximity of the phenolic, D-ring.
The
tetracycline are strong chelating agents (Balckwood, 1985; Chopra et al.,
1992) and both their antimicrobial and pharmacokinetic properties are influenced
by chelation of metal ions. Chelation sites includes the β-diketone system
(position 11 and 12) and the need (position 1 and 3) and carboxamide (position
2) groups of the α ring (Blackwood, 1985; Chopra et al., 1992). The newly
discovered glycycline, like other tetracycline derivatives also form chelation
complexes with divalent cations (Somani et
al., 2000). Replacement of the c-2
carboxamide moiety with other groups has generally resulted in analogs with
inferior antibacterial activities (Oethinger et al., 2000) probably
because bacteria accumulate these molecules poorly (Chopra,1996) however, the
addition of subsistent to the amide nitrogen can impact significant water
solubility as in the case of rolitetracycline and lymecycline.
Dissociation
of the pro-drugs in vivo liberates free tetracycline (Mitscher, 1999)
consistent with the above observations, substitution at positions 1,3,4a,10,11
or 12 are invariably detrimental to antibacterial activity (Rogalski, 2000)
nevertheless, a number of other substitutions of different positions on the B,C
and D rings are tolerated and molecules possessing these substituents have
given rise to the tetracyclines in clinical use today as well as new glycycline
molecules that are currently under going clinical trials.
 |
The structure-activity studies referred to above, revealed
that with an exception, each of the rings in the linear fused tetracycline
nucleus must be six-membered and purely carboxyclic for the molecules to retain
antibacterial activity. For instance the nortetracyclines derivatives in which
the β ring comprises a five-membered carbocyclic, are essentially devoid of
antibacterial activity (Rogalski, 2000). It is now been established that the
thiatetracycline and a number of other tetracyclines exhibit a different
structure activity relationship from the majority of tetracyclines (Konishi et al.,
1999). These molecules which also include the anhydrotetracyclines, 4-epi anhydrotetracyclien
and chelocardin, appear to directly perturb the bacterial cytoplasmic membrane
leading bacteriocidal response (Olivia, 1992) this contrasts with the
typical tetracycline, which interact
with the ribosome to inhibit bacterial protein synthesis and display a
reversible bacteriostatic effect. The membrane disrupting properties of the
atypical tetracycline are probably related to the relative planting of the B,C,
and D rings so that a lipophilic, non-ionized molecule predominates.
On
interaction with the cell, the atypical tetracycliens are likely to be
preferentially trapped in the hydrophobic environment of the cytoplasmic
membrane, disrupting its function. These molecules are of no interest as
therapeutic candidates because they
causes adverse side effects in human which are probably related to their
ability to interact non-specifically
with eukaryotic as prokaryotic all membrane (Chopra, 1996).
1.3 MODE OF ACTION
Tetracycline
antibiotics are protein synthesis inhibitors inhibiting the binding of
aminoacyl tRNA to the mRNA ribosome complex. They do so mainly by binding to
the 30s ribosomal unit in the mRNA translation complex (Tauch et al.,
2000).
Tetracyclines
transverse the outer membrane of gram negative enteric bacteria through the
Ompf and OmpC porin channels as positively charge cations (probably magnesium-tetracycline
coordination complexes) (Wexler et al., 1994).
The
cationic metal ion antibiotic complex is attracted by the Donnan potential
across the outer membrane leading to accumulation in the periplasm, where the
metal ion-tetracycline complex probably dissociates to liberates uncharged
tetracycline, a weakly lipophilic molecule able to diffuse through the lipid
di-layer regions of the inner membrane. Similarly, the electro neutral, lipophilic
form is assumed to be the species transferred across the cytoplasmic membrane
of gram positive bacteria. Uptake of tetracyclines across the cytoplasmic
membrane is energy dependent and driven by the DpH component of the proton
motive force (Zhao and Awoki, 1992).
Within
the cytoplasm, tetracycline molecules are likely to become chelated since the
internal pH and divalent metal ion concentrations are higher than those outside
the cell. (Tamayo et al., 1999).
Association
of tetracycline with the ribosome is irreversible providing an explanation of
the bacteriostatic effects of these antibiotics (Taylor et al., 2008) several
studies have indicated a single high affinity binding site for tetracyclines in
the ribosomal 30s submit, with indication through photoaffinity labeling and
chemical foot printing studies that protein S7 and 16S rRNA bases G693, A892,
U1052, C1054, G1300 and G1338 contribute to the binding pocket.Interpretation
of the probing studies above is complicated by the observation that binding of
tetracycline (which measure approximately 8 by 12 Ao) to the
ribosome appears to cause which ranging structural change in 16S rRNA.
Naturally
occurring tetracycline resistant propioni bacteria contain a cytosine to
guanine point mutation of position, 1058 in 16S rRNA which does at least
suggested that the neigbouring bases U1052 and U1054 identified by chemical
foot printing may have functional significance for the binding of tetracycline
to the 30S subunit.
The
absence of major antieukaryotic activity explans the selective antimicrobial
properties of the tetracycline. At the molecular level, this results from
relatively weak inhibition of protein synthesis supported by 80S ribosomes and
poor accumulation of the antibiotics by mammalian cells. However, tetracyclines
protein synthesis in mitochondria due to the presence of 70S ribosomes in these
organelle. It has being recognized for some time that the spectrum of activity
of tetracycline encompasses various protozoan parasites such as plasmodium falciparum, Entamoeba histolytica, trichomonas vaginalis and toxoplasma
gondinio.
1.4 RESISTANCE
Tetracycline
have a broad spectrum activity against pathogenic bacteria and the absence of
major adverse side effects. They have been used extensively on therapy for
human and animal infections and as growth promoters in agriculture.
Due
to this, its clinical usefulness has been declining because of the appearance
of an increasing number of tetracycline resistant isolates to clinically
important bacteria. Two type of resistance mechanism predominate tetracycline
efflux and ribosomal protection. A third and 4th mechanism of
resistance tetracycline modification has being identified. Chemical
modification of tetracycline and a mutation in the 16S rRNA affecting the
binding site.
In efflux genes found in gram negative enteric
bacteria, regulation is via a repressor that interacts with tetracycline. Gram
positive efflux genes appear to be regulated by an attenuation mechanism.
Recently it’s being reported that at least one of the ribosome protention genes
is regulated by attenuation. Efflux mediated resistance to tigecycline
(Charpentire et al., 1993) in 2012 used pseudomonas
aeruginosa as the test organism.
Pseudomonas aeruginosa strains are less susceptible to tigecycline (previously
GAR 936; MIC, 8 µg/ml than any other bacteria (Petersen et al., 1999) to
elucidate the mechanism of resistance to tetracycline P. aeruginosa PAOI strain, detective in the Mex AB-OPrM and Mex
XYCOPrM) efflux pumps were tested for susceptibility to tetracycline. Increase
susceptibility to tetracycline (MIC, 0.5 to 1 µg/ml) was specifically
associated with loss of Mex X Y. Transcription of Mex X and Mex Y was also
responsive to exposure of cells to tetracycline to test for the emergence of
compensatory efflux pumps in the absence of Mex X Y and OPrM, mutants lacking
Mex X Y-OPrM were plated on medium containing tetracycline of 4 or 6 µg/ml
resistant mutant were readily discovered and these also had decreased
susceptibility of several other antibiotic suggesting efflux pump recruitment.
One representative carbenicillin resistant strain over expressed OPrM, the
outer membrane channel component of the Mex AB-OPrM efflux pump. The Mex AB –
OprM repressor gene, MexR, from this strain contained a 15-bp in frame
deletion.
Two representative chloramphenicol
resistant strains showed expression of an outer membrane protein slightly
larger than OPrM. The Mex CD OPrJ repressor gene, nf x B from these mutants
contained a 327-bp in-frame deletion and is element insertion, respectively.
Together these data indicated drug efflux mediated by mexCD-OPrJ. The MIC of
the narrow spectrum semisynthetic tetracycline doxycline and minocycline
increased more substantially than those of tigecycline and other glycycline,
against the Mex AB-OPrM and mexCD-OPrJ over expressing mutant strains. This
suggests that glycyclines, although they are subject to efflux from P. aeruginosa
are generally inferior substrate of P.
aeruginosa efflux pumps that are
narrower spectrum tetracycline.
1.5 RIBOSOMAL PROTECTION PROTEIN
Ribosomal
protection represents an important tactic for promoting tetracycline resistance
in both gram positive and gram negative species.
Tet
(a) and tet (m) are the best studies of these determinants and were originally
isolated for campylobacter jejuni,
streptococcus spp respectively. Although both are widely distributed these
are the only the ribosomal protection proteins that have being studies in
detail others include tet (S), tet (T) tet (Q) tet (P), tet B (P) tet (W) and
Otr (A) function through similar mechanism. The distribution of these
determinants in the eubacteia have extensively being reviewed by Copra and
Roberts.
The tet (M) and tet (O) proteins are
the most extensively characterized of the ribosomal protection group they have
been shown to have ribosome dependent GTpase activity. The tet (0) protein
binds GDP and GTP site directed mutations in the test (0) protein which reduces
the binding of GTP, were correlated with reduction in the susceptibility to tetracycline
in isolates. This suggests that the GTP binding is important to the function of
the tet (0) protein.
Burdett
found that the tet (M) protein allows the aminoacyle tRNA to bind to the receptor
site of the ribosome in the presence of tetracycline concentrations that could
normally inhibit translation. In the presence of the tet (M) protein,
tetracycline is apparently released from the ribosome. In the presence of
either the tet (M) or the tet (O) protein, tetracycline binding to the
ribosomes to reduce with GTP but not when GDP is present. Burdett found that
energy from GTP hydrolysis released the tetracycline from the ribosome when a
non ionizable GTP analog was used.
It
has being assumed that the other proteins in the ribosomal protection group{
tet (S), tet (T), tet (Q) tet B (P), tet (W) and Otrs (A)}; have GTpase activity and interact with tetracycline
and the ribosomes in similar ways to those described for the tet (M) and tet
(O) proteins because of their similarities at the amino acid sequence level.
The ribosomal protection proteins can be divided into groups based an amino
acid sequence comparision. The first group includes tet (M) tet (0) tet (S) and
tet (W) the second group includes the otr (A) and the Tet B (P) while the third
group includes tet (Q) and tet (T) proteins.
1.6 INTERACTIONS WITH THE
ABSORPTION OF TETRACYCLINE
All
tetracycline derivatives are bacteriostatics and their concentration in serum
should not fall during the therapy below the originally accepted minimum
therapeutic concentration of 0.5 to 1.5µg/ml.
Tetracyclines
have a high affinity to form chelates with polyvalent metallic cations such as
Fe3+, Fe2+, Al3+, Mg2+ and Ca2+.
Many of these tetracycline metal complexes are either insoluble or otherwise
poorly absorbable from the gastro intestinal tract. Milk and other diary
products, antacids containing polyvalent cation, such as various ion salts
ingested simultaneously with tetracycline derivative, might interfere with
their absorption by 50 to 90% or even more. The severity of interaction depend
both on the nature of the tetracycline derivative and of the cation, on the
does used on pharmaceutical factors and on time schedules in dosing. An
interval of 3 hours between the ingestion of tetracycline and cations prevents
the interaction. The pharmacokinetic interaction on absorption of tetracycline
likely to be clinically significant in case which the infecting pathogens are
moderately resistant to tetracycline and relativity high serum concentrations
are needed for proper bacteriostasis (Nelson et al., 1993).
1.7 PHARMACO-THERAPEUTICS OF THE
NEWER TETRACYCLINE
The
newer tetracycline are defined as those tetracycline available in the United States
but not approved for veterinary use. These include democlocycline, metacycline,
doxycycline and minocycline appear to
offer advantages that would render them useful in certain situation in
veterinary medicine. Their major advantage lies in their greater lipid
solubility relative to other tetracycline.
These
characteristics probably accounts for their enhanced antimicrobial
effectiveness for some organisms, more efficient absorption after oral
administration, and enhanced distribution in the body. The principal excretory
organ for doxycycline is the intestine where the drug diffuses through the
intestinal mucosa into the intestinal tracts. These unique characteristics make
these drugs useful in cases of pre-existing renal dysfunction and may render
this drug superior to other tetracycline in the treatment of intestinal
infection. Doxycycline is used in other countries for respiratory tract and
intestinal tract diseases of poultry. The usefulness of doxycycline and
minocycline in food producing animal may be limited because of persistent drug
residue. The superiority of doxycylcine and minocycline relative to other
tetracyclines in their distribution to areas to other tetracycline in their
distribution to areas of the body such as the eye, brain, cerebrospinal fluid
and postrate gland suggest that trials of their efficacy in tetracycline-sensitive
infection of these areas are indicated.
CHAPTER TWO
MATERIALS AND METHODS
2.0 MATERIAL/EQUIPMENT
The
materials used in this research work include Weighing balance, Petri-dishes, Bunsen
burner, Inoculating Wire loop, Disinfectant, Bijoux bottle, Aluminum foil, Incubator,
Cotton wool, Autoclave, Test tubes, Conical flask, Spatula, Measuring Cylinder,
Test tube racks, Distilled water, Pasteur pipette and Hot air oven.
2.1 MEDIA
The
media used were Mac Conkey Agar, Nutrient Agar and Peptone water.
2.2 SAMPLE SOURCE AND STORAGE OF ISOLATES
Sample
received from ENT unit (ear, nose, throat), UBTH (University of Benin
Teaching Hospital) were used for the test. Test
isolates were kept on nutrient agar slope and stored at 40oC before
use. Provisional identification was done in UBTH and confirmatory test was done
in laboratory of Microbiology Department ,Ambrose Alli University Ekpoma.
2.3 CLEANING AND STERILIZATION OF GLASSWARES
The
glassware used were washed with detergent water and rinsed in distilled water.
All glasswares were sterilized using the hot air oven at 1600C for 1
hour.
2.4 PREPARATION AND STERILIZATION OF MEDIA
Media
were reconstituted with water according to manufacturer’s specification and
sterilized at 1210C for 15 minutes in the bucket autoclave. Media
were dispensed into sterilize Petri dishes and allowed to solidify at room
temperature before use.
2.5 STORAGE AND MAINTENANCE OF ISOLATES
Pure
culture of four (4) game-positive ad gram negative bacteria of Staphylococcus aureus and Escherichia coli received from the
Medical Microbiology Laboratory of University of Benin Teaching Hospital on the 12th of October 2010
were used for the test.
Test
isolates were kept on nutrient agar slope and stored at 40C before
use.
2.6 IDENTIFICATION OF TEST ISOLATES
Confirmatory
tests were done on two test isolates which had been identified in University of
Benin Teaching Hospital (UBTH) as follows:
Test
strains were inoculated into Peptone water allowed to stay for 2 hours on the
bench before inoculating onto MacConkey agar and nutrient agar.
The
colonial morphologies were noted, Gram stain, Catalase, Oxidase, Coagulase,
Indole, Motility, Urease, Citrate and Sugar utilization tests were done. See
identification test procedures below:
2.6.1 BIOCHEMICAL TESTS
GRAM STAINING
Difference
in gram reaction between bacteria is thought to be due to difference in the
permeability of the cell wall of gram- positive and gram negative organism
during the staining processes.
Procedure
Heat-fix
the dried smear with methanol for 2 minutes, cover the fixed smear with Crystal
violet stain for 30-60 seconds. Rapidly washed off the stain with clean water;
tip off all the water and cover the smears with Lugols iodine for 30-60
seconds, wash off the iodine water. Decolorize rapidly with acetone, wash
immediately with clean water. Cover the smear with neutral red stain for 2
minutes, wash off the stain with clean water, wipe the back of the slide and
place it in a draining rack for the smear to air dry. Examine the smear
microscopically first with 40 objective to check the staining and to see the
distribution of material and then with oil immersion objective to report the
bacteria and cells.
Catalase test
The test is used to differentiate
those bacteria that produce enzymes catalase, such as staphylococci from
non-catalase producing bacteria such as streptococci.
Procedure
Pour
2-3ml of hydrogen peroxide solution into a test tube. Using a sterile glass
rod, remove several colonies of test organism and immense in the hydrogen
peroxide solution. Look for immediate bubbling, active bubbling show a positive
catalase test.
Oxidase test
This
test is used to test for the bacteria which have the ability to produce the
enzyme cytochrome oxidase which is capable of catalyzing the transfer of
electron (e) from an e-donor in the bacteria and a redox-dye.
Procedure
Place
a piece of filter paper in a clean Petri-dish and add 2 or 3 drops of freshly
prepared oxidase reagent. Using a piece of stick or glass rod. Remove a colony
of the test organism and smear it on the filter paper. Look for the development
of a bubble purple colour within a few seconds.
Coagulase test
This
is used to identify staphylococcus
aureus, which produce the enzyme coagulase.
Procedure
Pour
1-2 drops of distilled water on each end of a slide or on two separate slides.
Emulsify a colony of the test organism in each of the drops to make two thick
suspensions. Add loopful plasma to one of the suspensions, and mix gently, look
for clumping of the organism within 10 seconds.
Motility test
Motility
test was used to differentiate motile from non-motile organism. Motility was
detected by using the handing drop method.
Hanging drop method
One
or two drops of peptone water culture of the test organism was placed on a
cover glass. A plastacin was then used to form a circle. This was then placed
on the slide. The slide now carrying the plasticin was then turned on the cover
slip that contains the test organism. This was subsequently inverted such that
the drop on the cover slop hangs. This was observed under the microscope for
dangling movement.
Indole test
Testing
for indole production is important in the identification of enterobacteriacae.
This demonstrates the ability of certain bacteria to decompose the amino acid;
typtophan to indole which accumulates in the medium.
Procedure
The test organism was inoculated in a
Bijoux bottles containing 3ml of sterile water and was incubated at 35-370C
for up to 48 hours. After incubation 0.5ml of Kovac’s reagent was added and
shaken gently and examined for a red colour in the surface layer after 5
minutes.
Urease test
Testing
for urease enzyme activity is important in differentiating enterobacteriacae.
Proteus strains are strong urease producers. Salmonella and Shigella do
not produce urease.
Procedure
Inoculate
the test organism in a bijoux bottle, containing 3ml sterile urea broth.
Incubate at 35-370C for 24 hours. After incubation, positive urease
test showed pinkish colour.
Citrate test
The
test is used for the identification of enterobacteriacae. This test is based on
the ability of an organism to use citrate as its sole source of carbon.
Procedure
Prepare
slopes of the medium in bijoux bottles and store at 2-80C with the
use of sterile straight wire, first streak the slope with the saline suspension
of the test organism and then stab the butt. Incubate at 350C for 48hours,
bright blue colour indicates a positive citrate test.
Sugar fermentation test
The
sugars used were Mannitol, Ducittol, Lactose, Malatose, sucrose and glucose
were prepared in a bijoux bottle containing 1% of each specific sugar and a pH
indicator usually Andrade’s indicator is employed.
Note
that peptone was first dissolved with distilled water before sugar preparation.
A Durham tube
was positioned in each bijoux bottle in order to detect if gas was produced
during the fermentation of sugars. It was then sterilized by autoclaving, then
using a wire loop, the organisms was inoculated from the MacConkey agar to each
bijoux bottle containing different sugars and incubated at 370C for
24 hours.
2.7 THE DILUTION METHOD ADAPTED
TO AGAR CELL DIFFUSION METHOD
stock dilution of each antibiotics
(Doxycyline and Vibramycin) were prepared. A row of 10 sterile test tubes were
set upon on test tube rack; with the aid of a sterile Pasteur pipette, 1ml of
sterile saline was dispensed into each test tube. 1ml of the antibiotic
solution was dispensed to the 1st test tube and the serial dilution
was completed until the 10th test tube was discarded. From each test
tube, 1ml was proved to each appropriately labeled Petri-dish i.e., one Petri
dish correspond to a test tube of a rack. 19ml of molten nutrient agar was
dispensed into each Petri-dish with the aid of a sterile pipette and rock round
to the drop to properly mix-up with the agar. The Petri-dish was allowed to set
at room temperature, in inverted position, the bottom of the Petri-dish was
segmented into fifteen (15) parts. The segments was labeled 1-15 parts. The
Petri-dish was allowed to dry at 450c for 30 minutes. With the aid
of a sterile wire loop, each isolate was inoculated from a broth into the
appropriate labeled segment on each Petri-dish. This was done for all the
isolate. The Petri-dish was incubated for 24-48 hours. The solution of
antibiotics were sub-cultured into freshly prepared Petri-dish of nutrient agar
and incubated at 370c for 24-48 hours to test for their purity.
Since each Petri-dish corresponded to each dilution tube, absence or presence
of growth on a segment represent sensitive or resistance to the corresponding
dilution in the Petri-dish.
CHAPTER THREE
3.0
RESULT
TABLE 1: Shows The Minimum Inhibitory Concentration
(MIC) Of Fifteen Bacterial Isolates From Cases Of Otitis Media
|
Isolates
|
Bacterial organism
|
Concentration of Doxycylycine
(µg/ml
|
Concentration of vibramycin
(µg/ml)
|
|
1
|
Klebsiella spp
|
0
|
0
|
|
2
|
Klebsiella spp
|
0
|
0
|
|
3
|
Pseudomonas aeruginosa
|
0
|
0
|
|
4
|
Pseudomonas aeruginosa
|
0
|
0
|
|
5
|
Staphylococcus aureus
|
0
|
0
|
|
6
|
Staphylococcus aureus
|
0.05
|
0.8
|
|
7
|
Pseudomonas aeruginosa
|
0
|
0
|
|
8
|
Klebsiella spp
|
0
|
0
|
|
9
|
Proteus spp
|
0
|
0
|
|
10
|
Proteus spp
|
0
|
0
|
|
11
|
Proteus spp
|
0
|
0
|
|
12
|
Escherichia coli
|
0
|
0
|
|
13
|
Klebsiella spp
|
0
|
0
|
|
14
|
Proteus spp
|
0
|
0
|
|
15
|
Klebsiella spp
|
0
|
0
|
From
the table above, only isolate Number 6 was sensitive to Doxycycline and
Vibramycin with Minimum Inhibitory Concentration (MIC) of 0.05µg/ml and 0.8:g/ml
respectively, other isolates were resistant to both antibiotics irrespective of
their concentration.
Table
2: Shows
the Morphological Characteristics and Biochemical Reactions of the Bacterial
Isolates
|
Bacterial
isolates
|
Cultural
Characteristics & colonial Appearance
|
Gram
Reaction Morphology
|
         BIOCHEMICAL REACTION |
|||||||||||||
|
|
|
 |
|
 |
|
|
|
|
 |
|
|
|
|
 |
 |
|
|
Staphylococcus aureus
|
Yellowish
clusters
|
+Cocci
|
+
|
-
|
+
|
-
|
-
|
-
|
NA
|
NA
|
NA
|
NA
|
NA
|
NA
|
-
|
-
|
|
Proteus spp
|
Creamy,
swarming colonies
|
-Rods
|
+
|
-
|
NA
|
+
|
V
|
+
|
+
|
+
|
-
|
+
|
+
|
+
|
-
|
-
|
|
Klebsiella spp
|
Large
mucoid, pinkish colonies
|
-Rods
|
+
|
-
|
NA
|
-
|
-
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
-
|
-
|
|
Escherichia coli
|
Round
pinkish colonies
|
-Rods
|
+
|
-
|
NA
|
+
|
+
|
-
|
-
|
+
|
+
|
+
|
+
|
+
|
-
|
-
|
|
Pseudomonas aeruginosa
|
Dark
greenish colonies
|
-Rods
|
+
|
+
|
NA
|
+
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
|
CHAPTER FOUR
4.0 DISCUSSION
The results of this experiment have
shown the comparative invitro activity of doxycycline and vibramycin though
both antibiotivcs were only effective against one (1) strain. The activity of
doxycycline was a little bit higher (0.5:g/ml) compared to that of
vibramycin (0.8:g/ml). This findings is similar to
that of (Agalar et al., 1999) who evaluated the resistance
of most gram negative organisms to tetracycline and disagrees with the findings
of (Gang et al., 1994) who reported that Pseudomonas
aeruginosa was sensitive to
vibramycin at various concentration.
The effectiveness and the MIC values of
vibramycin and doxycycline in this report does not agree with the findings of
(Ogbaber, 2010) who stated that vibramycin and doxycycline functions equally at
the same MIC, but conforms with the statement of (Scott et al., 2000) who reported
the efficacy of vibramycin and doxycycline against bacteria isolates at various
concentration.
However,
carefully analysis of this study revealed that vibramycin is not as effective
as doxycyline. Doxycyline, a locally manufactured drug in Nigeria was discovered
to more effective and inhibited the growth of Staphylococcus aureus (6)
having a Minimium Inhibitory Concentration (MIC) of 0.05 µg/ml while Vibramycin,
a foreign brand had an (MIC) of 0.8µg/ml. Although, other strains all
other strains of bacterial
isolates were resistant to doxycycline and vibramycin, this could be as a
result of drug resistance on the part of the individual from which the sample
was taken.
Interestingly, since the minimum
inhibitory concentration (MIC) of doxycyline is far lesser compared to that of
vibramycin, it is however irrational to assume that foreign made drugs are more
effective than those made in Nigeria
as shown by this study. The inequality in the cost between doxycyline and
vibramycin is totally aberrant, doxycycline which is made in Nigeria is more effective and
cheaper than vibramycin which is an imported brand, therefore it suggested that
medical practitioners should encourage the use of doxycyline over vibramycin.
4.1 CONCLUSION AND RECOMMENDATION.
Since the effectiveness of any drug
depends on the minimum inhibitory concentration (MIC) of such drug, it is
however necessary for the manufacturers of such drug to state its MIC to enable
the health provider have an idea of the dosage to be prescribed. Unfortunately,
since resistance of new tetracycline derivates may occur quickly, other
strategies must be considered to combat bacterial resistance such as the
development of new classes of drugs with unique antibacterial mechanisms. More studies are required to understand the
mechanisms of resistance of microbes to all antibiotics including tetracycline,
this could lead to the design of new intervention strategies. The experience
gained from this study highlights the need for a better understanding of
bacterial resistance at the global level.
Also, we need a variety of approaches to
reduce the amounts of antimicrobial being used worldwide in human and animal
medicine hence people should be educated on the effect of drug abuse. The
National Agency for Food and Drug Administration and Control (NAFDAC) should
intensify efforts in monitoring the Quality control of Drugs as this is the
only way we will be able to keep tetracycline and other antibiotics as
resources for the next generation. Massive health education and health
communication agenda should be propagated to keep the public updated on the
negative outcome of drug abuse and this could achieved through radio jingles,
community out-reach, seminars and workshops.
I hereby recommend that medical
practitioners should be well informed of the economic advantage of prescribing
doxycycline over vibramycin and the continuous importation of vibramycin should
be discouraged.
REFERENCES
Agalar, C., Usubutun, S. and Turkyilmaz, R.
(1999). Ciprofloxacin and Rifampcin versus Doxycycline and Rifampicin in the
treatment of brucellosis. Eur. J. Clin.
Microbiol. Infect. Dis. 18:535-538.
Atkinson, B.A., Abu-Al-Jaibat, A. and LeBlanc,
D.K. (1997). Antibiotic resistance among enterococci isolated from clinical
specimens between 1953 and 1954. Antimicrob.
Agents Chemother 41: 1598-1600.
Alekshum, M.N. and Levy, S.B., (1997). Regulation
of chromosomally medicated multiple antibiotic resistance. Antimicrob. Agents Chemother. 41: 2067-2075
Andres, MT., Chung, W.O. and Fierro, J.F. (1993).
Antimicrobial susceptibilities of Porphyromonas
gingivalas, Prevotella intermedia and Provotella nigrescens isolated in Spain.
Antimicrob. Agents Chemother. 42: 3022-3023.
Antonio, M.A.D., Hawes, S.E. and Hillier S.L.
(1999). The identification of vaginal Lactobacillus
species by these species. J. Infect. Dis. 180:1950-1956
Barboa, T.M., K. P. Scott, and H.J. Flint. (1999).
Evidence for recent intergeneric transfer of new tetracycline resistance gene,
tet (W), isolated from Butyrivibro
fibrisolens, and the occurrence of tet (O) in ruminal bacteria. Environ. Microbial. 1:53-64.
Barden, T.C., Buckwalter, B.L., Testa, R.T.,
Petersen, P.J. and Lee. V.J. (1994). “Glycylcydints”. 9-Aminodoxycycline
carboxamides J. Med. Clin. 37:3205-3211.
Bertram, J., Stratz, M., and Durre, P. 1991
Natural transfer of conjugative transposon In 916 between gram-positive and
gram-negative bacteria. J. Bacteriol 173: 443-448.
Biggs, C.E., and Fratamico, P.M. (1999). Molecular characterization of an
antibiotic resistance gene cluster of Salmonella typhimurium D.T 104. Antimicrob. Agents Chemother. 43:846-849.
Blackwood, R.K. (1985). Structure determination
and total synthesis of the tetracyclines.
Handbook of Experimental Pharmacology. 59-136.
Bremon, A.R., Ruiz-Tovar, M.,Gonicho, B., Torres,
P.D. and Rodgriguez, R.I. (2000). Non-hospital consumption of
antibiotics in Spain.
J. Antimicrob. Chemother. 45:395-400.
Brown, B.A., Wallace, R.J. and Onyi, G. (1996).
Activities of the glycylcyclines N, N-dimethylglylamido-minocydine and NN-dimethylgly
cylamido-6-demethyl-6-deoxytetracycline against Nocardia spp. And tetracycline
resistant isolates of rapidly growing mycobacteria. Antimicrob. Agents Chemother. 40: 874-878.
Brown, M.B. and Roberts, M.C. (1991). Tetracycline
resistance determinants in streptococcal species isolated from the bovine
mammary gland. Vet. Microb. 29:173-180.
Bunnag, D., Karbwang, J., Na-Bangchang, A.
Thanavibul, A., Cittamas, S. and Harinasuta. T. (1996). Quinine-tetracycline
for multidrug resistant falciparum malaria. Southeast Asia J. Trop. Med. Public Health. 27:15-18.
Burdett, V. (1992). TRNA modification activity is
necessary for Tet(m)-mediated tetracycline resistance. J. Bacteriol. 175: 7200-7215.
Burns J.L., Wadsworth,
C.D., Barry, J.J.and Goodall, C.P. (1996). Nucleotide sequence analysis of a
gene from Burkholderia (Pseudomonas) Capacia encoding an outer membrane
lipoprotein involved in multiple antibiotic resistance. Antimicrob. Agents Chemother. 40: 307-313.
Charpentier, E., Gerband, G.and Courvalin, P.
(1993). Characterization of a new class or tetracycline-resistance gene tet (B)
in Listeria monocytogenes BM4210. Gene
131:26-34.
Chermont, D., Chesmean, O., De Cespedes, G. and
Horaud, T. (1997). New tetracycline resistance determinants coding for ribosomal
protection in streptocci and nucleotide sequence of tet (T) isolated from
streptococcus pyogenes A498. Antimicrob.
Agents Chemother 41: 112-116.
Chopra, I.
(1986). Transport or tetracycline into Escherichia
Coli requires a carboxamide group at the C2 position of the
molecule. J. Microb. Chemother 18: 661-666.
Chopra, I.,
Hawkey, P.M. and Hinton, M. (1992). Tetracycline’s. molecular and chemical
aspects. J. Antimicrob. Chemother. 29:245-277
Clewell, D.B., Flannagan, S.E. and Jaworski, D.D.
(1995). Unconstrained bacterial promiscuity: The In 916-In1545 family or
conjugative transpons. Trends Microbiol 3:
229-236
Dantley, K.A., Dannelly, H,K. and Budett, V.
(1998). Binding interaction between Tet (M) and the ribosome: requirements for
binding. J. Bacteriol. 180:4089-4092.
Debets-Ossenkopp, Y.J., Herscheid, A.K., Pot,
R.G.K., Knipers, E.J., Kusters, J.G. and Dandenbrouk- Grenuls, J.E. (1999).
Prevalence of Helicobacter pylori assistance to metronidazole,
clanthromycine. Amoxyellin, tetracycline and troxafloxacin in the Netherlands.
J. Antimicrob. Chemother. 43:511-515.
Finch, R.G (1997). Tetracyclines. N. Engl.
J. Med. 511:617
Gales, D.C and Jones, R.N. (2000). Antimicrobial
activity and spectrum of the new glycyline, GAR-936 tested against 1,203 recent
clinical bacterial isolates. Diagn Microbiol. Infect. Dis. 36:19-36
George, A.M., Hall. R.M and Stokes, H.W. (1995).
Multi drug resistance in Kelbsiella pneumoniae: a novel gene, ram, confers a
multidrug resistance phenotype in Escherichia
coli. Microbiology 141:
1909-1920.
Grave, K., Lingas, E. and Running M. (1999).
Survelliance of the overall consumption of antibacterial drugs in humans,
domestic animals and farmed fish in Norway in 1992 and 1996. J. Antimicrob.
Chemother 43:
243-252.
Guay. G.G, Tuckman, M. and Rothstein, D.M. (1994).
Mutations in the tet A (B) gene that cause a change in substrate specifity of
the tetracycline efflux pump. Antimicrob.
Agents Chemother 38:857-860.
Johnson, A.P 2000. Gar-936. Curr. Opin. Anti-infect. Investig. Drugs 2: 164-170
Konishi, S., Iwaki,
S., Kimura-Someya, T. and Yamagudi, A.
(1999). Cysteine-Scanning mutagenesis around transmembrane segment vi of
Tn10-encoded metal tetracycline. FEBS
Lett 461: 315-318.
Oethinger, M., Kern, W.V., Jellen-Ritter, A.S.,
McMurry, L.M. and Levy, S.B. (2000). Ineffectiveness of toporsomerase mutations
in mediating clinically significant flouroquinolone resistance in Escherichia Coli in the absence of the Acr AB efflux pump. Antimicrob. Agents Chemother. 44:10-13
Petersen P.J., Jacobus, N.V., Weisis, W.K., Sum,
P.E. and Testa, R.T. (1999). In Nitro
and in vivo antibacterial activities of a novel glycylcyline, the 9-6- butyl
glycylamido derivative of minocycline (GAR-936). Antimicrob. Agents Chemother. 43: 738-744
Roberts, M.C. (1997). Oral Bacteria: reservoirs
for antibiotic resistance traits. AFUA
News. 15: 1-6.
Rogalski, W. (2000). Chemical modification of the
tetracyclines. Handbook of Experimental Pharmacology. 78:179-187
Somani, J., Bhullar, V.B., Workowski, K.A.,
Farshy, C.E. and Black, C.M (2000). Multiple drug resistant Chlamydia trachomatis associated with clinical treatment failure. J. Infect.
Dis. 181:1420-1427
Tamayo, M.R., Sa-Leao R., Sanches, I.S.,
Castaneda, E. and Lencastre, H. (1999). Dissemination of a chloramphenicol and
tetracycline resistant but penicillin-susceptible invasive clone of serotype S Streptococcus Pneumoniae in Colombia J. Clin.
Microbiol 7: 2337-2342.
Taylor,
D.E., Trieber, C.A, Trescher, G. and Bekkering, M. (1998). Host mutations
reduce tetracycline resistance mediated by Tet (0) and Tet (m). Antimicrob. Agents. Chemother. 42: 59-64.
Treeman, C.O., Nightingale, C.H and Quintilliani
R. (1994). Minocycline: old and new therapeutic uses. Int. J. Antimicrob. Agents. 4: 325-335.
Wexler H.M, Mobtoris, E. and Finegold S.M. (1994).
In vitro activities of the new glycylcylclines, N, N-dimethylglycylamido
derivatives of minocyline and 6-demethyl-6-deoxytetracycline against 339
strains of anaerobic bacteria. Antimicrob.
Agents Chemother. 38:
2513-2515.
Zhao, J. and Aoki, T. (1992). Nucleotide sequence
analysis of the class G tetracycline resistance determinant from Vibrio anguillarum. Microbiol. Immunol. 36: 1051-1060
APPENDIX
FORMULATION AND PREPARATION OF
MEDIA
NUTRIENT
AGAR
Base
formulation Grams/litre
Meat extract 1.0
Yeast Extract 2.0
Peptone water 5.0
Sodium chloride 5.0
Agar 15
pH 7.4
METHOD
28g of the medium is suspended in 1
litre of distilled water and dissolved by boiling and it was sterilized by
autoclaving at 1210c for 15 minutes.
MACKCONKEY AGAR
Base
formulation
Peptone
Sodium
chloride
Lactose
Bile
salts
Neutral
red
Agar
METHOD
5.20g of the medium is suspended in 1
litre of distilled water. It was boiled to dissolve completely and sterilized
by autoclaving at 1210c for 15 minutes.
No comments:
Post a Comment