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ORIGINAL ARTICLE
Inducible Clindamycin Resistance in Staphylococcus Species
Faisal Iqbal Afridi1, Mubarak Zeb1, Arif Hussain2, Badar Jahan Farooqi1 and Ghulam Murtuza1
ABSTRACT
Objective: To determine the frequency of inducible clindamycin resistance in clinical isolates of Staphylococcus species
by phenotypic D-test.
Study Design: Observational study.
Place and Duration of Study: Ziauddin University Hospital, Karachi, from July to December 2011.
Methodology: Consecutive clinical isolates of Staphylococcus species were collected and identified by conventional
microbiological techniques. Antimicrobial susceptibility testing and inducible clindamycin resistance was carried out by
performing D-test using CLSI criteria. Methicillin resistance was detected by using Cefoxitin disk as a surrogate marker.
Statistical analysis was performed by SPSS version-17.
Results: A total of 667 clinical isolates of Staphylococcus species were obtained during the study period. In these isolates,
177 (26.5%) were Staphylococcus aureus, and 490 (73.5%) were coagulase negative Staphylococci. The total frequency
of inducible clindamycin resistance among isolates of Staphylococcus species was 120/667 (18%). Frequency of inducible
clindamycin resistance among coagulase negative Staphylococci group and Staphylococcus aureus group were 18.57%
and 16.38% respectively. Median age of patients in D-test positive group was 19.5 (1 - 54) years.
Conclusion: The frequency of inducible clindamycin resistance among Staphylococcus species may differ in different
hospital setup. Clinical microbiology laboratories should implement testing simple and effective D-test on all
Staphylococcus species. D-test positive isolates should be reported clindamycin resistant to decrease treatment failure.
Key Words: Inducible clindamycin resistance. Staphylococcus. Phenotype. D-test positive staphylococcus isolates.
INTRODUCTION
Staphylococci are Gram-positive bacteria, which form
grape-like clusters. Almost 30% of the normal healthy
population is affected by Staphylococcus aureus (S.
aureus) as it asymptomatically colonizes human hosts.1
S. aureus is the common cause of various diseases
including: mild skin infections (impetigo, folliculitis, etc.),
invasive diseases (wound infections, osteomyelitis,
bacteremia with metastatic complications, etc.), and
toxin mediated diseases (food poisoning, toxic shock
syndrome, scaled skin syndrome, etc.). Common
superficial infections include carbuncles, impetigo,
cellulitis, and folliculitis. Community-acquired infections
include bacteremia, endocarditis, osteomyelitis, and
pneumonia.2 Staphylococcus species are the major
cause of hospital-acquired infections. Foreign bodies,
such as sutures, indwelling catheters, and implanted
joints are extremely susceptible to Staphylococcus
epidermidis (S. epidermidis) colonization, and often
serve as the point of entry for infection.
By forming biofilms, S. epidermidis is resistant to
antibiotics, and can serve as a reservoir for antibiotic
resistant genes that can be transferred to other
Department of Microbiology1 / Pathology2, Dr. Ziauddin
University Hospital, North Nazimabad Campus, Karachi.
Correspondence: Dr. Faisal Iqbal Afridi, A-79/6 Block No. 14,
Gulistan-e-Jauhar, Karachi-75290.
E-mail: [email protected]
Received: January 01, 2013; Accepted: March 20, 2014.
bacteria.3 The first serious emergence of antibiotic
resistant Staphylococcus occurred with a specific strain
refers to as Methicillin-Resistant Staphylococcus aureus
(MRSA).4 This strain expressed a modified penicillinbinding protein encoded by mecA gene and is present in
4 forms of Staphylococcus cassette.4
Clindamycin is considered as a useful alternate agent in
penicillin-allergic patients in the treatment of skin and
soft tissue infections caused by S. aureus. It has an
excellent tissue penetration, accumulates in abscesses,
and no dosage adjustments are required in the presence
of renal disease. The good oral absorption of
clindamycin makes it an alternative option for use in
outpatients or as follow-up treatment after intravenous
therapy (de-escalation).5
Target site modification is the most common mechanism
of acquired resistance to macrolides, lincosamides, and
streptogramin B (MLSB) antibiotics in Staphylococci and
confers cross-resistance to the MLS antibiotics.6 MLSB
resistance can be either constitutive or inducible, if it is
inducible, bacteria often test resistant to Erythromycin
(E) but susceptible to Clindamycin (DA).7 When the disk
diffusion test is used to determine susceptibility, a
distorted “D-shaped” zone of inhibition is observed
around DA if an E disk is placed nearby.7 Although
isolates appear susceptible to DA in the absence of an
inducing agent, there is wide spread reluctance to
prescribe DA for treatment of patient with infections
caused by such organisms because of concerns that
resistance to DA may cause treatment failure in vivo.8
Journal of the College of Physicians and Surgeons Pakistan 2014, Vol. 24 (7): 481-484
481
Faisal Iqbal Afridi, Mubarak Zeb, Arif Hussain, Badar Jahan Farooqi and Ghulam Murtuza
This study was conducted to investigate the frequency of
inducible clindamycin resistance in Staphylococcus
species to assess the resistant pattern.
METHODOLOGY
This observational study was conducted over a period of
6 months from July to December, 2011 in the
Department of Clinical Microbiology of Ziauddin Medical
University Hospital, Karachi. Six hundred and sixty
seven consecutive clinical isolates of Staphylococcus
species were collected from different clinical samples by
convenient sampling and included in the study. Sources
were blood, respiratory secretions, wound swabs,
central venous pressure (CVP) lines tips, and pus. All
the duplicate isolates were excluded from the study.
Written approval from the institutional ethical committee
was obtained. Informed consent was taken from either
the patient or any other patient's relative.
Clinical samples were received in a sterile container or
in an Amies transport medium supplied from the
Microbiology laboratory. These samples were processed
and incubated at 37ºC in ambient air for 24 - 48 hours,
using standard microbiological techniques.9 Staphylococcus species including S. aureus were identified using
conventional techniques (colony morphology, gram
staining, catalase test, coagulase test, mannitol salt
agar, and DNase test).9
Antimicrobial susceptibility testing was performed on
Muellar Hinton agar (MHA) medium (Oxoid Ltd.,
England) using modified Kirby-Bauer disk diffusion
method according to Clinical and Laboratory Standards
Institute (CLSI) 2010 guidelines.10 Detection of inducible
clindamycin resistance was carried out by performing
D-test using CLSI 2010 guideline.10 A 0.5 McFarland
equivalent suspension of organism was inoculated onto
a MHA plate, the E (15 ug) disk was placed 15 - 26 mm
(edge-to-edge) apart from DA (2 ug) disk on this MHA
plate. The exact distance between the two disks (edgeto-edge) was 20 mm apart on most of the occasions.
The plates were incubated overnight at 37ºC in an
ambient air incubator. After incubation, isolates showing
rounded zones of inhibition with diameter of ≤ 13 mm for
E and ≥ 21 mm for DA were interpreted as negative for
inducible resistance (D-test negative). Isolates with
similar inhibitory zones of inhibition as above but a
D-shape zone around DA, were interpreted as positive
for inducible resistance (D-test positive) as shown in
Figure 1. S. aureus American Type Culture Collection
(ATCC®) 25923, S. aureus ATCC® BAA-977 (containing
inducible ermA-mediated resistance), and S. aureus
ATCC® BAA-976 (containing msrA-mediated macrolidesonly efflux) were used as controls. Methicillin resistance
was detected by using Cefoxitin (30 µg) disk as a
surrogate marker.10
A research proforma was used to document the
essential data including age and gender. Data analysis
482
was performed by using Statistical Package for Social
Sciences (SPSS) version-17. Frequencies and percentages were computed for presentation of all categorical
variables like microorganisms, gender and D-test
positivity. Median Inter-quartile range (IQR) is reported
for median age of patients.
RESULTS
A total of 667 clinical isolates of Staphylococcus species
were obtained during the study period. Distribution of
isolates of Staphylococcus species from different clinical
samples are shown in Figure 2. In these 667 isolates,
177 (26.5%) were S. aureus, and 490 (73.5%) were
coagulase negative Staphylococci (CoNS). Predominantly, the isolates were from male patients 361/667
(54.1%), while isolates from female patients were
306/667 (45.9%). Male to female ratio was 1.17:1. In
total, the frequency of inducible clindamycin resistance
among isolates of Staphylococcus species is 120/667
(18%). Out of these 120 D-test positive isolates, 91 were
Figure 1: D-Test. (A); positive test showing D-shaped zone of inhibition of
clindamycin (DA) adjacent to erythromycin disk (E). (B); negative test
showing circular zone of inhibition of clindamycin (DA) adjacent to
erythromycin disk (E).
Figure 2: Distribution of isolates of Staphylococcus species from different
clinical samples.
Journal of the College of Physicians and Surgeons Pakistan 2014, Vol. 24 (7): 481-484
Inducible clindamycin resistance in Staphylococcus species
CoNS and 29 were S. aureus. Frequency of inducible
clindamycin resistance among CoNS group was 91/490
= 18.57% and S. aureus group was 29/177 = 16.38%. In
total 363/667 (54.4%) isolates of Staphylococcus
species were resistant to methicillin. Among the isolates
of CoNS 239/490 (48.77%) were methicillin sensitive
and 251/490 (51.22%) were methicillin resistant.
Among the isolates of S. aureus 65/177 (36.72%)
were methicillin sensitive and 112/177 (63.27%) were
methicillin resistant.
Median age (IQR) of patients in D-test positive group
was 19.5 (53) years versus 30 (56) years for patients in
D-test negative group.
DISCUSSION
The determination of antimicrobial susceptibility of a
clinical isolate is often crucial for optimal antimicrobial
therapy of infected patients. This is particularly important
considering the increase of resistance and the
emergence of multi-drug resistant organisms. The
emergence of resistant to multiple antibiotics among
gram-positive cocci has left very little therapeutic options
for clinicians.
The increased frequency of Staphylococcal infections,
combined with resistant to antimicrobial agents which
are used to treat Staphylococcal infections, have caused
a great deal of problem in therapeutic treatment of skin,
soft tissues as well as other organs infected by
Staphylococci.11 Clindamycin which is a Lincosamide
has long been an attractive option because of its efficacy
against Methicillin Sensitive Staphylococcus aureus
(MSSA) and MRSA for its good bone marrow and tissue
penetration and potential antitoxin effects.12
In this study, a total of 667 Staphylococcus species were
isolated from different sources. Majority of isolates were
recovered from blood-culture samples that were 51%.
Ako-Nai et al. reported higher percentage of Staphylococcus species isolated from skin specimens which is
68% while blood isolates were 17%.13 Gunduz et al.
showed higher percentages of Staphylococcus species
in urinary isolates which is 34%.14 This indicate that
distribution of isolates may be different in different health
care setups. In this study, the predominance of isolates
were from male patients which is very similar to the
study of Patel et al. in which 54% of Staphylococcal
isolates were from male patients. Male to female ratio is
also almost similar that is 1.19:1.15
This study findings are different with studies published
by other authors. Gunduz et al. showed 6.5% inducible
clindamycin resistance in Staphylococcal isolates.14
Saderi et al. from Iran showed 6.4% inducible
clindamycin resistance in the isolates of S. aureus,16
while another study from Iran also reported 9.7%
inducible clindamycin resistance among the isolates of
S. aureus.17 Fasih et al. reported a high percentage
(72%) of inducible clindamycin resistance phenotype
among the discordant (Clindamycin-sensitive, Erythromycin-resistant) isolates of S. aureus.18 Fokas et al.
from Greece documented 35% inducible clindamycin
resistance among the isolates of S. aureus.19 These
results indicate that inducible clindamycin resistance
phenotype may vary in different hospital setups.
With respect to methicillin resistance in S. aureus
isolates, the present results are also different from other
authors. Vivek et al., Fasih et al., and Seifi et al. reported
32.5%, 36%, and 41.7% methicillin resistance in the
isolates of S. aureus respectively.20,18,21 Moreover, Cetin
et al. from Turkey reported a very high percentage of
methicillin resistance (91%) among the isolates of S.
aureus.22 This indicates the overall non-judicious use of
cloxacillin in these setups.
There is a possibility that Staphylococcal strains may be
inducible resistant to MLSB antibiotics, if erythromycin
resistance and clindamycin susceptibility is detected in
these strains by standard disc diffusion method. This
inducible resistance can easily be missed by clinical
laboratory, if erythromycin and clindamycin discs are not
placed at adjacent positions by using standard disc
diffusion test. One of the therapeutic options in the
treatment of MRSA is considered to be clindamycin.23 It
is noticed that clindamycin cannot be considered as
useful treatment option for hospital-associated MRSA
infections.
However, the lower positivity rate of inducible
clindamycin resistance in this study by D-test among
both S. aureus and CoNS isolates made us think that
clindamycin can be considered as an effective treatment
option in D-test negative MRSA and CoNS isolates
especially in lower respiratory tract isolates in the
situation of aspiration pneumonia where anaerobic
cover is also required. Clindamycin also has good
anaerobic activity. The proper use of clindamycin in
methicillin resistant Staphylococcal species will also
reduce the non-judicious use of glycopeptides. This can
be achieved by accurate results of antimicrobial
susceptibility testing including the application of D-test
on a routine basis.
CONCLUSION
The frequency of inducible clindamycin resistance
among Staphylococcus species may differ in different
hospital setups. Clinical microbiology laboratories
should implement testing simple and effective D-test on
all Staphylococcus species. D-test positive isolates
should be reported clindamycin resistant to decrease
treatment failure.
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