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The Importance of Clindamycin Induction Test in Treating Patients Infected with
Methicillin Resistance Staphylococcus aureus
Iyad Kaddora
Bioinformatics
Introduction
In recent years, strains of Staphylococcus aureus (S. aureus) resistance to
erythromycin have been clinically isolated with increasing frequencies. S. aureus is a
Gram positive cocci bacterium that belongs to the Micrococcaceae family(4). S. aureus
can exist in air, dust, water, and can be normal flora of skin and respiratory tract of
human However, the most common mode of transmission is by skin-to-skin contact from
an infected host(4). This common bacterium is the number one cause of nosocomial
infections, which are infections that are acquired while clients are receiving care in the
hospital setting (8). S. aureus can live harmlessly on the skin surface, but when the
surface is punctured or broken, it can cause serious infections that may ultimately lead to
serious disease even death if untreated.
Antibiotics have been successful in killing bacteria, but due to over use of
medications and the incomplete drug course taken by infected individuals, the bacteria
have started to develop different, mutated strains that raise the degrees of virulence these
bacteria express to fight the effects of various antibiotics. In the past few years,
Staphylococcus aureus has begun to show more resistant to commonly used antibiotics,
which is expressed by developing mutated strains called Methicillin Resistant
staphylococcus aureus (MRSA). This methicillin resistance now refers to multiantibiotic resistant groups (8). This evidence represents the growing resistant to β-lactam
antibiotics such as penicillin and its offspring, as well as the cephalosporins drug. MRSA
can cause a serious infection if it is not treated quickly and spreads very easily in the
health care setting.
Macrolides is a class of antibiotic that inhibits growth of bacteria by several
different mechanisms (7). It is used clinically as a broad spectrum antibiotic against
gram positive bacteria. It inhibits the bacterial growth by binding to specific sites in the
bacteria’s ribosome to prevent the production of proteins. Resistance of the organisms to
macrolides can occur in many ways; however, in mechanism of resistant to erythromycin
occurs by one of these two mechanisms: efflux or ribosome alteration. Efflux
mechanism is mediated by the macrolide streptogramin (type B) gene (mrsA), where the
ribosome alteration is achieved by methylation of specific residues 23S rRNA and
mediated by erythromycin ribosome methylase (erm) gene (2, 6, 7).
Following exposure to macrolides the organism’s resistance can be either constantly
presents (appear) where the rRNA methylase is always produced, or it can be inducible
where methylase is produced only in the presence of inducing agent which is
erythromycin in this case (1,6). These two types of resistance are called respectively
constitutive and inducible resistance.
Determinant (gene)
Erythromycin
Clindamycin
Efflux
mrsA
R
S
Ribosome alteration
erm
R
S*
erm
R
R (constitutive)
mrsA: macrolide streptogramin (type B) resistant.
erm: erythromycin ribosome methylase; is a gene that encodes enzymes which confer inducible or
constitutive resistance to macrolide lincosamide (clindamycin) streptogramin type B agents via methylation
of the 23S rRNA.
Clindamycin is a lincosamide class of antibiotics that has activity against different
kinds of bacteria and possesses the ability to inhibit the synthesis of protein in bacterial
organism. This activity prevents the bacteria from replicating by binding exclusively to
the 50S ribosomal subunit of susceptible organisms (5). Clindamycin works very well in
treating MRSA However, some strains have recently begun showing resistance to this
drug, making treatment more difficult (2). The failure of Clindamycin therapy in the
treatment of MRSA infected patients, those expressed sensitivity to the drug at the
beginning of the course treatment, raises a big concern to health care professionals and
attending physicians.
In order to successfully treat individuals with MRSA, a new test has been
developed to test the future resistance of bacteria to medicines that are currently being
used to treat the infections. This test, clindamycin disk inductions test (D-Test) will aid
in the future treatment for clients with MRSA (2). Many studies have been preformed
regarding MRSA and the growing resistant strains that have begun to deter the normal
treatment of this infectious disease process. The aim of this study is to determine
whether Methicillin Resistant Staphylococcus aureus (MRSA) is showing more
resistance to the clindamycin antibiotic drug during 2000/2001 year compared to
2004/2005 year. By using this new test, treatment for MRSA in the future will be able to
be provided accurately and hopefully aid in the prolongation of the human race.
Materials and Methodology
The specimens were collected from patients presenting to clinical facilities in
Huntington, WV. A loopful of the sample was streaked on a new Trypticase soy agar
plate with 5% sheep’s blood (BAP) and incubated overnight at 37°C, with 5 % CO2
Another loopful of the sample was then combined with 5 ml of Trypticase soy broth
(TSB) in a test tube and incubated overnight at 37°C with 5% CO2. Two test tubes were
prepared from each sample. The test tubes were centrifuged at 1500 rpm (Beckman CS6R centrifuge with a GH-3.7 rotor) for 5 minutes to precipitate a pellet. After
centrifugation 0.5 ml of TSB/ bacterial pellet was mixed with 0.5 ml of a sterile 24%
glycerol solution and placed in a sterile 4 ml glass Wheaton vial. The vial was labeled
with the sample number and frozen at -70 to -80°C.
Two hundred and forty frozen samples of Methicillin Resistant Staphylococcus
aureus (MRSA) were collected in year 2000/ 2001 in order to be evaluated for
clindamycin resistance by performing the D-test. The frozen specimens were sub
cultured on blood agar plates and the D test was performed on all specimens. Positive Dtest indicates the ability of MRSA strains to demonstrate resistance to the clindamycin
during the antibiotic therapy. Negative D-test indicates the effectiveness of the
clindamycin drug in treating those patients with MRSA (6).
This test was performed on Mathecillin-resistant Staphylococcus aureus from
sources other than urine, such as sputum, blood culture, wound, or body fluid. When the
MRSA strains are resistant to erythromycin and sensitive to clindamycin, after using
routine antimicrobial susceptibility test method, the drugs sensitivity is determined.
Urine specimen is unacceptable because clindamycin is not used for treating urinary tract
infection caused by MRSA strains.
The principle of the D test is to make erythromycin induce production of the
methylase, which will allow clindamycin resistant to be expressed and detected by
forming a D shape letter on the blood agar plate (BAP). The first step in performing the
D test is to make 0.5 McFarland suspension of the organism in 3ml sterile inoculum’s
water. After the specimen is mixed in sterile water, a sterile swab is used to inoculate the
organism to the blood agar plate, making sure the organism is covering the entire surface
of the plate to ensure growth in the whole plate. An erythromycin disk is placed on the
plate. This disk is used as an inducing agent when placed 15mm to 26 mm away from
the clindamycin disk, on a (BAP). After the specimen is incubated for 18-24 hours at
35°C with 5% CO2, a flattening of the zone in the area between the two disks (letter D
shape) will indicate the organism’s ability to induce clindamycin resistance in the future
(6).
References
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specifies constitutive macrolide-lincosamide-streptogramin B resistance contains a novel
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Clindamycin treatment of Staphylococcus aureus expressing inducible clindamycin
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7. Weisblum, Bernard. 1995. Insights into Erythromycin Action from Studies of Its
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8. http://www.link.med.ed.ac.uk/RIDU/Mrsa.htm