Methicillin-resistant Staphylococcus aureus heterogeneously resistant to vancomycin in a Turkish university hospital

Banu Sancak*, Serpil Ercis, Dilek Menemenlioglu, Sule Çolakoglu and Gülsen Hasçelik

Department of Clinical Microbiology and Microbiology, Hacettepe University Medical School, Ankara, 06100, Turkey


* Corresponding author. Tel: +90-312-3051562; Fax: +90-312-3115250; E-mail: banusancak{at}yahoo.com

Received 17 March 2005; returned 4 May 2005; revised 22 June 2005; accepted 2 July 2005


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Objectives: We investigated vancomycin-intermediate Staphylococcus aureus (VISA) and heterogeneously vancomycin-intermediate S. aureus (hetero-VISA) isolates from clinical specimens of hospitalized patients at Hacettepe University over a 4 year period.

Methods: Strains were screened for VISA and hetero-VISA by using brain heart infusion agar containing 4 mg/L vancomycin (BHI-V4) and macro Etest. Confirmation of the isolates that were found to have intermediate susceptibility to vancomycin with either of the methods was done by population analysis of subpopulations with reduced susceptibility to vancomycin. The MIC of vancomycin for the isolates grown on BHI-V4 was determined by the microdilution method.

Results: Among 256 methicillin-resistant S. aureus (MRSA) isolates, 145 grew on BHI-V4. Forty-six of these were also found to be heterogeneously vancomycin-intermediate strains when screened with the macro Etest. There were no VISA among 256 MRSA tested but 46 (17.97%) S. aureus strains with reduced susceptibility to vancomycin were identified by population analysis. Vancomycin MIC values for all isolates with reduced susceptibility were between ≤0.125 and 4 mg/L. Twelve of the 46 patients with hetero-VISA had a history of previously being treated with vancomycin or teicoplanin.

Conclusions: To our knowledge, this study is the first publication showing the presence of hetero-VISA in Turkey. In order to understand the epidemiological relationship between our isolates, molecular typing methods are needed.

Keywords: MRSA , hetero-VISA , VISA , S. aureus , Turkey


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The emergence of Staphylococcus aureus with reduced susceptibility to vancomycin (MIC 8–16 mg/L) was first reported from Japan1 and shortly afterwards from the United States,2 Europe3 and Korea,4 mainly from patients on vancomycin treatment. Until now, about 20 cases with reduced susceptibility have been reported from different countries.5 Thereafter, vancomycin resistance was documented in three isolates of S. aureus in the United States.68

After the first report of vancomycin-intermediate S. aureus (VISA) in 1997,1 a second type of resistant strain called heterogeneously vancomycin-intermediate S. aureus (hetero-VISA) was described.9 Following this first report by Hiramatsu et al.,9 there have been increasing reports of hetero-VISA from different countries.916 Until now, there has been no publication about the prevalence of VISA and/or hetero-VISA in Turkey.

S. aureus strains with reduced susceptibility to vancomycin are susceptible to vancomycin according to NCCLS MIC criteria (MIC ≤ 4 mg/L) but contain subpopulations at a frequency of 10–6 or higher with MICs of vancomycin of more than 4 mg/L.9 Because of the low frequency of these subpopulations, hetero-VISA strains could not be detected either by disc diffusion or by standard MIC methods. As a result, the differentiation of these isolates from the susceptible strains is a real problem. Therefore, alternative methods have been proposed such as population analysis, an aztreonam disc method based on the antagonism of vancomycin and ß-lactam12,17,18 and an Etest with a dense inoculum called the macro Etest.19

In this study, we investigated VISA and hetero-VISA in methicillin-resistant S. aureus (MRSA) strains by two methods (agar screening method and the macro Etest). The strains were isolated from clinical specimens of hospitalized patients.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Isolates

All MRSA (n = 256) isolated between January 1998 and January 2002, from clinical specimens (105 from blood, 102 from pus, 47 from tracheal aspiration/bronchoalveolar fluid and two from cerebrospinal fluid) of hospitalized patients at Hacettepe University were included in the study. All S. aureus isolates were identified by colony morphology, Gram staining, catalase and coagulase tests and the Sceptor automated system (Becton Dickinson, MD, USA). Oxacillin susceptibility testing was performed by the Kirby–Bauer disc diffusion method using NCCLS guidelines.20

Both agar screening and macro Etest methods were applied to all isolates for determination of susceptibility to vancomycin.

Agar screening method

For agar screening, we used in-house prepared brain heart infusion agar (Merck, Darmstadt, Germany) containing 4 mg/L vancomycin (BHI-V4). Bacterial suspensions were prepared from overnight cultures on blood agar and their turbidity was adjusted to be equivalent to that of a 0.5 McFarland standard (108 cfu/mL) in tryptone soy broth. Then, 10 µL of this suspension was used for inoculation. The inoculum was spotted onto BHI-V4. The plates were incubated at 35°C for 48 h and examined both at 24 and 48 h. If there was confluent growth after 24 h, the isolate was considered as potential VISA. If a countable growth (1–30 cfu) was seen within 48 h, the isolate was considered as possible hetero-VISA. If the growth was not apparent after 48 h, the isolate was considered as susceptible to vancomycin.9 All the isolates that showed growth on BHI-V4 were selected for vancomycin MIC determination by the NCCLS microdilution method21 and population analysis profiling (PAP).9

Macro Etest

MICs were determined according to the manufacturer's instructions (AB Biodisk, Solna, Sweden). BHI agar was inoculated with 200 µL of a McFarland 2 suspension in BHI broth (Merck, Darmstadt, Germany) and incubated for 48 h at 35°C.

The interpretative criteria for hetero-VISA categorization were vancomycin and teicoplanin MICs of ≥8 mg/L or only a teicoplanin MIC of ≥12 mg/L.22

Broth microdilution

The MIC of vancomycin was determined by a broth microdilution method according to NCCLS guidelines.21 S. aureus ATCC 29213 was used as a quality control strain.

Population analysis

For confirmation of the susceptibility level of hetero-VISA isolates, any isolate that was found as screen positive with either the BHI-V4 plate or macro Etest was subjected to population analysis. The population analysis was carried out as described previously with minor changes.9 A 50 µL aliquot was taken from a suspension with a turbidity equivalent to that of a 0.5 McFarland standard and serial 10-fold dilutions were spread onto BHI agar plates containing vancomycin at concentrations ranging from 1 to 10 mg/L with 1 mg/L increments. After incubation at 35°C for 48 h, the number of colonies on different concentrations of vancomycin was counted.

S. aureus ATCC 29213, Mu50 (VISA Japan) and Mu3 (hetero-VISA Japan) were used as controls.

Definitions

VISA was defined as an S. aureus isolate with the following characteristics: (i) reproducible growth on BHI-V4; (ii) a vancomycin MIC of 8–16 mg/L by the broth microdilution method; (iii) a population analysis profile similar to homogeneous VISA control strain Mu50.1,23

Hetero-VISA was defined as an S. aureus isolate with the following characteristics: (i) reproducible growth on BHI-V4; (ii) a vancomycin MIC ≤ 4 mg/L by the broth microdilution method; (iii) a population analysis profile similar to hetero-VISA control strain Mu39 (isolate that contained subpopulations with intermediate susceptibility to vancomycin at a frequency of 1 in 10–6 or higher.)


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Among 256 clinical MRSA isolates (from 256 patients), 145 grew on BHI-V4 screening plates within 48 h. Forty-six (17.97%) isolates were found as screen positive with both BHI-V4 and the macro Etest (Table 1). No VISA was detected with the microdilution test (MICs were between ≤0.125 and 4 mg/L). Population analysis confirmed 46 isolates as hetero-VISA since they produced subclones with MICs of vancomycin of more than 4 mg/L at a frequency of 10–6 or higher.


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Table 1. Results for 46 MRSA isolates found to have reduced susceptibilities to vancomycin

 

    Discussion
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 Abstract
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 Materials and methods
 Results
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There is a lot of confusion regarding the laboratory and clinical approach to patients with infections caused by S. aureus with reduced susceptibility to vancomycin. The low frequency (≤10–6) of the subpopulations decreases the sensitivity of the testing methods. Therefore, it is important to use effective methods that can detect heteroresistance of S. aureus isolates. Detection of VISA is possible with standard laboratory methods but the detection of hetero-VISA remains difficult.

Currently, no standardized method exists for identifying hetero-VISA. Population analysis profiling (PAP) has been proposed as the most precise method of determining heteroresistance, but this method is laborious, time-consuming and impractical for using in routine laboratories. It requires at least 106 cfu whereas 104 cfu is sufficient for the standard MIC methods. Hetero-VISA isolates are susceptible to vancomycin by NCCLS MIC criteria (MIC ≤ 4 mg/L) but contain subpopulations that can grow at a vancomycin concentration of ≥4 mg/L.9 Consequently, the conventional susceptibility tests may fail to detect the resistant cell subpopulation of hetero-VISA isolates, which occurs at a frequency of 1/106 or higher, and cannot differentiate these strains from susceptible ones. Similarly, in our study, MIC values for 46 hetero-VISA isolates ranged from ≤0.125 to 4 mg/L and all were categorized as susceptible, according to NCCLS criteria.

Although the reported cases of VISA infection are rare, the proportion of hetero-VISA ranges widely between surveys worldwide: Spain, 65%; France, 20%; Germany, 2–14%; Italy, 1%; Japan, 5–26%; Brazil, 4%; England, 0–16%; and Belgium, 0.4%.10,11,13,14,2426 Ariza et al.10 reported a high prevalence of 65% among MRSA isolates but most of these were from patients with orthopaedic implants. Even in the same country, there have been conflicting results concerning the prevalence of hetero-VISA. It is very difficult to compare the results of these studies because the study designs and the screening methods used differed. Although we found no VISA in our study, the prevalence of hetero-VISA was found to be 17.97% (46/256) in MRSA isolates. This prevalence is much higher than the results of several other studies. There may be several reasons for that.

First, in most of the studies, BHI-V4 has been used for screening although it is not sensitive and specific enough to detect either VISA or hetero-VISA.27,28 In addition, the method appeared to lack reproducibility.29 In our study, we used two different methods (BHI-V4 and macro Etest) for screening. Some of the researchers have applied standard breakpoint criteria to macro Etest results, which is not appropriate.12

Second, for the confirmation of hetero-VISA isolates, we used PAP, which has been proposed as the most precise method of determining hetero-resistance.22 However, the most common screening method in the literature was simplified population analysis described by Hiramatsu et al.9 It is a practical method for detecting hetero-VISA strains in a clinical laboratory. However, this method has some limitations as it cannot detect a strain that has a lower degree of heterogeneity. Also, Howe et al.30 found that this method lacks both specificity and reproducibility. This may be the reason for the variable rates of hetero-VISA detected up to the present time.

Finally, in most of the studies, both methicillin-susceptible and -resistant isolates were examined but there are some studies that included multiple isolates from the same patient. In our study, we only included MRSA strains, which were isolated from hospitalized patients.

Interestingly, there was an increase in the prevalence of hetero-VISA from 1.6% in 1998 to 36% in 2001 in our hospital. As these patients were hospitalized, there was a possibility of dissemination of epidemic MRSA clones with decreased susceptibility to vancomycin from patients who were under vancomycin or teicoplanin treatment to other patients. In order to understand the epidemiological association between our isolates, molecular typing methods are needed.

The clinical significance of the hetero-VISA phenotype and the role of hetero-VISA in treatment failures have not been defined yet because of lack of sufficient clinical data. Since it is not clear whether or not hetero-VISA isolates are precursors of VISA isolates in patients receiving a prolonged course of vancomycin, the differentiation of hetero-VISA isolates from susceptible ones is ascertained. In our study, only 12 of 46 patients were treated with vancomycin and/or teicoplanin. Unfortunately, it is hard to say that hetero-VISA isolates in our study originated from patients treated with vancomycin or teicoplanin.

Until now, the criteria for identifying hetero-VISA strains have not been standardized. Our results indicated that, for screening hetero-VISA, the macro Etest is more specific than the BHI-V4 agar screening method. It is obvious that the Etest is an expensive test to use for screening. Therefore, in microbiology laboratories, applying the macro Etest for isolates that grow on BHI-V4 agar may be the most suitable algorithm for screening hetero-VISA. As all S. aureus with reduced susceptibility to vancomycin, except one,31 were previously reported among MRSA isolates, it may be better to screen only MRSA rather than screening all S. aureus isolates until the clinical significance of hetero-VISA is better understood.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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18. Kim MN, Hwang SH, Pyo YJ et al. Clonal spread of Staphylococcus aureus heterogeneously resistant to vancomycin in a university hospital in Korea. J Clin Microbiol 2002; 40: 1376–80.[Abstract/Free Full Text]

19. Bolmström A, Karlsson A, Wong P. Macro-method conditions are optimal for detection of low level glycopeptide resistance in staphylococci. Clin Microbiol Infect 1999; 5 Suppl 3: 113.

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21. National Committee for Clinical Laboratory Standards. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Sixth Edition: Approved Standard M7-A6. NCCLS, Wayne, PA, USA, 2003.

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