Iranian Journal of Pathology (2008)3 (3), 161 - 166
Original Article Susceptibility to Vancomycin in StaphylococcusAureus Isolated From Patients of Four University-Afﬁliated Hospitals in Tehran Horieh Saderi1, Parviz Owlia1, Zohreh Maleki2, Mehri Habibi1, Nayere Rahmati1 1. Dept. of Microbiology, School of Medicine, Shahed University, Tehran, Iran. 2. Vali-ye-Asr Hospital, Tehran, Iran.ABSTRACT Background and Objective: Vancomycin is frequently the antibiotic of choice for the treatment of infections caused by methicillin-resistant Staphylococcus aureus (MRSA). For the last years, the incidence of vancomycin-intermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA) has been increased in various parts of the world. The present study was carried out to determine the presence of VISA and VRSA in Tehran. Materials and Methods: A total of 164 S. aureus strains were isolated from clinical specimens in four university-afﬁliated hospitals in Tehran from November 2006 to June 2007. Minimum inhibitory concentration (MIC) of vancomycin of isolates was determined by agar dilution method. Vancomycin (6 mg/l) screen agar plate method and E-test were used to conﬁrm presence of resistance to vancomycin. Disc diffusion agar test was also used to detect resistance to other antimicrobial Results: Only one VRSA (MIC 256 mg/l) was detected and three strains with MIC 4 mg/l considered VISA according to recent CLSI breakpoints for vancomycin. Only VRSA strain had shown growth on vancomycin screen agar plate and was also resistant to several antimicrobial agents but susceptible to quinupristin/dalfopristin, linezolid, chloramphenicol, mupirocin and cotrimoxazole. Isolated VISA were also multi-resistant but showed susceptibility to quinupristin/ dalfopristin, linezolid, chloramphenicol and mupirocin. Conclusion: Detection of vancomycin resistance in Iranian S. aureus isolates emphasizes the challenges confronted by the infection control specialists in hospitals in Iran as well as causing problems in the treatment of patients with S. aureus infections. Key words: Vancomycin, Staphylococcusaureus Received: 25 March 2008Accepted: 3 May 2008Address communications to: Dr. Horieh Saderi, Department of Microbiology, School of Medicine, Abdollahzade Ave., Keshavarz Blvd., Shahed University, Tehran, Iran. Email: email@example.comIRANIAN JOURNAL OF PATHOLOGY
162 Susceptibility to Vancomycin in Staphylococcus Aureus Isolated From Patients of . Introduction
sputum and CSF from the patients of different inpatient and outpatient departments of 4 university-afﬁliated
Staphylococcus aureus causes serious infections hospitals in Tehran. The identiﬁcation of isolates was
done according to standard methods (12).
The growing prevalence of methicillin-resistant
Determination of minimum inhibitory concentra- Staphylococcus aureus (MRSA) as a cause of these
infections has increased the use of the glycopeptide
MIC of vancomycin (LKT laboratories, USA)
antibiotic vancomycin over the past 3 decades (1).
was determined by agar dilution method according
As a consequence, selective pressure was established
to CLSI guidelines (16). Brieﬂy, gradient plates
that eventually lead to the emergence of strains of S.
of Mueller-Hinton agar (Merck, Germany) were
aureus with decreased susceptibility to vancomycin
prepared with vancomycin (0.5–256 mg/l). By direct
and other glycopeptides (2). In 1997, the ﬁrst strain
colony suspension method, 0.5 McFarland equivalent
of vancomycin-intermediate S. aureus (VISA) was
inoculum was prepared in normal saline from 18–
reported from Japan (3). Shortly after, two additional
24 h agar plate culture. The suspension was further
cases were reported from United States (4). However,
diluted to achieve desired inoculum concentration of
ﬁrst clinical isolate of vancomycin-resistant S. aureus
105 CFU/ml. All strains were spotted onto gradient
(VRSA) was reported from United States in 2002
plates. Plates were incubated overnight at 35 °C
(5). Subsequent isolation of VISA and VRSA isolates
for any visible growth. For strains showing MIC
from United States (6;7) and other countries including
≥4 mg/l to vancomycin in agar dilution method,
Brazil (8), France (9), United Kingdom (10), Germany
E-test (AB Biodisk, Sweden) was used according
(11), India (12;13), and Belgium (14) has conﬁrmed
to manufacture guidelines, to conﬁrm presence of
that emergence of these strains is a global issue.
vancomycin resistance. S. aureus ATCC 29213 was
The choice of method for susceptibility testing
used as vancomycin susceptible control. Readings
and test conditions are essential considerations in
were taken according to recent CLSI guideline:
accurately applying a deﬁnition of VISA or VRSA
MIC≤2 mg/l for vancomycin-susceptible, MIC of 4-8
for any given isolate. VISA and VRSA isolates are
mg/l for vancomycin-intermediate S. aureus (VISA)
not detected by disc diffusion, whilst vancomycin
and MIC≥16 mg/l for vancomycin-resistant S. aureus
screen agar plate are only suitable for isolates with
a minimum inhibitory concentration (MIC) >6 mg/l. Acceptable methods used to detect these strains are
Vancomycin screen agar plate method
non-automated and include broth or agar dilution and
In-house vancomycin screen agar plate was
E-test. Also, CLSI has recently lowered breakpoints
prepared by addition of 6 mg/l vancomycin (LKT
for vancomycin and strains with MIC of 4-8 mg/l are
laboratories, USA) to brain heart infusion (BHI)
considered VISA and with MIC ≥ 32 mg/l are VRSA
agar (Merck, Germany). Inoculum suspension was
prepared by transferring colonies from overnight
In our previous study in 2003, ﬁve out of the 139
growth on nutrient agar plate to sterile saline to
of S. aureus strains isolated in Tehran had shown a
produce a suspension that matches the turbidity
vancomycin MIC of ≥128 mg/l by agar dilution and
of a 0.5 McFarland standard. Then, 0.1 ml of this
E-test methods (19). Emaneini et al have recently
suspension was spread on vancomycin screen agar
reported isolation of VRSA from one Iranian patient
plate and was incubated for 24 h at 35 °C in ambient
(20). Keeping these in view we performed this study
air. Any visible growth indicated the vancomycin
on 164 S. aureus strains for the assessment of current
resistance. In addition, S. aureus ATCC 29213 was
situation of vancomycin resistance in Tehran.
used as a vancomycin-susceptible control strain. Materials and Methods Disc diffusion agar test Disc diffusion agar test was carried out using Kirby- S. aureus isolates
Bauer method by following discs: penicillin G (10
A total 164 S. aureus were investigated for the period
U), ampicillin (10 μg), ampicillin-sulbactam (20 μg),
of 8 months from November 2006 to June 2007. The
oxacillin (1 μg), gentamicin (10 μg), ciproﬂoxacin
strains were collected from various clinical specimens
(5 μg), chloramphenicol (30 μg), erythromycin (15
including pus, urine, wound swabs, catheters, blood,
μg), tetracycline (30 μg), cotrimoxazole (25 μg),
IRANIAN JOURNAL OF PATHOLOGY
vancomycin (30 μg), cefoxitin (30 μg), clindamycin
(MIC 256 mg/l). This VRSA strain was isolated
(2 μg), rifampicin (5 μg), mupirocin (5 μg), linezolid
from pus of wound of a 36 years old female patient
(30 μg) and quinupristin/dalfopristin (15 μg), all
admitted in operation ward. It was not distinguished
purchased from MAST (UK). Mueller-Hinton agar
from vancomycin susceptible S. aureus isolates in disc
plates were overlaid with the inoculum (turbidity
diffusion agar test because it has produced a 20 mm
equivalent to that of a 0.5 McFarland Standard) of
zone of growth inhibition around vancomycin disc.
the S. aureus strains. Zone diameters were measured
From 164 S. aureus strains, only this VRSA strain
at 24 h following CLSI criteria (16). S. aureus ATCC
showed growth on vancomycin screen agar and was
resistant to most of the commonly used antimicrobial agents including penicillin G, ampicillin, oxacillin,
cefoxitin, clindamycin, ampicillin-sulbactam, ciprof-loxacin, gentamicin, erythromycin, rifampicin
MIC for 164 S. aureus strains against vancomycin
and tetracycline but susceptible to quinupristin/
is shown in Table 1. MIC for 97.5% of isolates was ≤2
dalfopristin, linezolid, chloramphenicol, mupirocin
mg/l. Only one strain was found to be VRSA strains
Table 1: Distribution of vancomycin MICs for 164 isolates of Staphylococcusaureus as determined by agar dilution method MIC (mg/l) No. of strains Table 2: Detailed description of VISA and VRSA (detected by agar dilution method and E-test) including antibiotic susceptibility patterns as determined by disc diffusion method Characterization of patients Resistance to Susceptibility to vancomycin designed Sex Age Specimens Ward vancomycin
VA, vancomycin; CD, clindamycin; TS, cotrimoxazole; OX, oxacillin; C, chloramphenicol; RP , rifampicin;
PG, penicillin G; LZD, linezolid; SAM, ampicillin-sulbactam; AP, ampicillin; FOX, cefoxitin; SYN, quinupristin/dalfopristin; CIP, ciproﬂoxacin; G, gentamicin; T , tetracycline; E, erythromycin; MUP, mupirocin. IRANIAN JOURNAL OF PATHOLOGY
164 Susceptibility to Vancomycin in Staphylococcus Aureus Isolated From Patients of .
Three strains of S.aureus had vancomycin MIC of
identify these strains. Broth or agar dilution methods
4 mg/l and considered VISA according to recent CLSI
and E-test are standard methods for detection of VISA
breakpoints for vancomycin (16). However, these
and VRSA (17-18), although discrepancies between
strains were not distinguished from vancomycin-
their results have been reported (22;23). Vancomycin
susceptible S. aureus isolates in disc diffusion test
screen agar plate are only suitable for isolates with
because they had shown ≥20 mm zone of growth
MIC> 6 mg/l and is unsuitable in view of the lower
inhibition around vancomycin disc. All isolated VISA
CLSI susceptibility breakpoint of 2 mg/l (18). The
were resistant to penicillin G, ampicillin, oxacillin,
Center for Disease Control and Prevention (CDC)
cefoxitin, clindamycin, ampicillin sulbactam,
recommends that laboratories use MIC method plus
ciproﬂoxacin, gentamicin and erythromycin but
vancomycin screen agar for detection of VISA and
susceptible to quinupristin/dalfopristin, linezolid, chloramphenicol and mupirocin and susceptibility
VRSA (17). Therefore, we used different methods to
to other antimicrobial agents were different among
identify vancomycin resistance in S. aureus.
these strains. The isolated VISAs were isolated from
On the other hand, VISA and VRSA tend to be multi-
respiratory specimens of female patients admitted in
drug resistant against a large number of currently
different wards with various ages (Table 2).
available antimicrobial agents, compromising
treatment options and increasing the likelihood of
inadequate antimicrobial therapy and increase in
morbidity and mortality (18). In the present study,
Infections caused by methicillin-resistant S. aureus
isolated VRSA and VISA showed resistance to a wide
have been associated with high morbidity and mortality
range of different antimicrobial agents but retained
rates (18). Vancomycin is the main antimicrobial agent
susceptibility to linezolid, quinupristin/dalfopristin,
available to treat serious infections with MRSA but
chloramphenicol and mupirocin. Isolated VRSA was
unfortunately, decreases in vancomycin susceptibility
also sensitive to cotrimoxazole. Sensitivity of isolated
of S.aureus and isolation of vancomycin-intermediate
VRSA to linezolid and quinupristin/dalfopristin; two
and resistant S.aureus were recently reported from
recently approved antimicrobials by the Food and
many countries (3-14). Until now, two reports from
Drug Administration (FDA) has also been shown in
Iran were published about VISA and VRSA. In our
other studies (6;7;21;24;25) and linezolid was used
previous study in 2003 (19), ﬁve out of the 139 S.
for treatment of clinical infection of VRSA (6).
aureus strains isolated in Tehran were VRSA (MIC
Also, most VISA and VRSA strains isolated in other
≥128 mg/l). Emaneini et al. have recently reported
studies (12;24;26) and this study were susceptible
isolation of one VRSA strain in a teaching hospital
to cotrimoxazole, which for the time being, may
in Tehran (20). In this study, one of 164 S.aureus
represent adequate therapy for skin and soft tissue
strains was VRSA (MIC ≥256 mg/l). There has been
infections caused by these strains. However, VRSA
increasing evidence that strains with a vancomycin
isolated in our previous study (19) and some of
MIC of 4 mg/l behave similar in the clinical setting
isolated VRSA in other studies has shown in vitro
to VRSA strains as clinical failure generally results if
resistance to cotrimoxazole (21;26). Also, widespread
treatment with vancomycin is continued (18;21;22).
use of these drugs will surely lead to resistance and
So, the previous breakpoints for vancomycin (≤4 mg/
newer therapeutic modalities are urgently needed.
l (S); 8-16 mg/l (I); ≥32 mg/l (R) (15) have recently
been revised (≤2 mg/l (S); 4-8 mg/l (I); ≥16 mg/l (R))
(16). Therefore, three isolated S.aureus strains in this
study with MIC 4 mg/l have been considered VISA.
The importance of discovering vancomycin
Isolation of VRSA and VISA in Tehran calls for
resistance in S. aureus isolates underscores the
the implementation of a regional and nationwide
fact that physicians should include vancomycin
surveillance system to monitor presence of these
susceptibility tests in strategies for managing patients with S. aureus infections and use of an active infection
strains in other regions in Iran. Unfortunately, VISA
control policy to prevent the spread of VRSA in the
and VRSA isolates are not detected by disc diffusion
agar test and automated methods did not accurately
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