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Iranian Journal of Pathology (2008)3 (3), 161 - 166 Original Article
Susceptibility to Vancomycin in Staphylococcus Aureus 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.
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, Staphylococcus aureus
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 protected]
162 Susceptibility to Vancomycin in Staphylococcus Aureus Isolated From Patients of .
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 tion (MIC)
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 Staphylococcus aureus 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
designed Sex Age Specimens Ward
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.
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 Discussion
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)) Conclusion
(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 IRANIAN JOURNAL OF PATHOLOGY
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