The Prevalence of Vancomycin-Intermediate Staphylococcus aureus and Heterogeneous VISA Among Methicillin-Resistant Strains Isolated from Pediatric Population in a Turkish University Hospital

Abstract

There are limited data regarding the prevalence of vancomycin-intermediate Staphylococcus aureus (VISA)/heterogeneous VISA (hVISA) among pediatric population. Our objective was to determine the distribution of vancomycin and daptomycin minimum inhibitory concentrations (MICs) and explore the phenomenon of vancomycin MIC creep and the VISA/hVISA prevalence among the methicillin-resistant Staphylococcus aureus (MRSA) strains belonging to pediatric population by population analysis profile-area under the curve (PAP-AUC) and Etest macromethod. Vancomycin and daptomycin susceptibilities of 94 pediatric isolates of MRSA were tested by broth microdilution (BMD) and Etest methods. To determine the prevalence of VISA/hVISA, Etest macromethod and PAP-AUC was performed on all isolates. All isolates were susceptible to vancomycin and daptomycin by both BMD and Etest methods. Twenty-eight (29.8%) isolates had vancomycin MICs of 2 μg/ml by BMD. No increase in vancomycin MICs was observed over time. There were no VISA among 94 MRSA tested but 20 (21.3%) hVISA isolates were identified by PAP-AUC. Results of Etest macromethod were compared to PAP-AUC. Etest macromethod was 60.0% sensitive and 90.5% specific. The hVISA isolates represented 53.6% of isolates with vancomycin MICs of 2 μg/ml. Also, 75% of hVISA isolates had vancomycin MICs of 2 μg/ml. To our knowledge, this is the first study investigating the prevalence of VISA/hVISA among MRSA isolated from pediatric patients by PAP-AUC method. Based on our findings, MRSA isolates, which have vancomycin MIC of 2 μg/ml can be investigated for the presence of hVISA. In this study, daptomycin showed potent activity against all isolates and may represent a therapeutic option for MRSA infections.

Introduction

The glycopeptide vancomycin was first released in 1958. For decades, it has been the mainstay of treatment for methicillin-resistant Staphylococcus aureus (MRSA) infections.¹⁷ Rising incidences of MRSA has led to the extensive use of this antibiotic. The increased use of vancomycin in MRSA infections appears to be the cause of emergence of vancomycin-intermediate Staphylococcus aureus (VISA) and heterogeneous VISA (hVISA) strains.⁴⁷ The current definition for VISA is an S. aureus isolate with a vancomycin broth minimum inhibitory concentration (MIC) of 4–8 μg/ml.¹⁷ The vancomycin MIC among hVISA isolates is within the susceptible range, but subpopulations of VISA are present at proportions ranging from 1 per 10⁵ to 1 per 10⁶ organisms.²⁸

The first strains of VISA and hVISA were reported in 1997.^12,13 Although VISA strains are rare, there have been increasing reports of hVISA from different countries. The prevalence of hVISA significantly varies between studies.^{4,6,16,24,29,42} Some of these differences may be explained by the lack of standardized criteria for the definition of hVISA and the use of different methods to detect them in different laboratories. Currently, the population analysis profile-area under the curve (PAP-AUC) method is considered the gold standard and the most reliable method.^45,49,52

To our knowledge, until now there has been no publication about the investigation of VISA/hVISA prevalence using PAP-AUC method among the MRSA isolated from pediatric population.

The aim of this study was to determine the distribution of vancomycin and daptomycin MICs among MRSA isolates, to evaluate the possible vancomycin MIC creep of pediatric MRSA isolates, to investigate the VISA/hVISA prevalence among pediatric MRSA strains by PAP-AUC and to evaluate Etest macromethod for the detection of hVISA by using PAP-AUC as the reference method.

Materials and Methods

Isolates

All stored (−70°C) MRSA strains (n=94) isolated from pediatric patients between 2001 and 2011 at Hacettepe University hospital were included in this study. Only one isolate from each patient was analyzed. The isolates were recovered from various clinical sources, including blood (n=49), catheter (n=4), bone marrow (n=1), cerebrospinal fluid (n=4), pus (n=15), wound (n=2), bronchoalveolar lavage (n=2), tracheal aspirate (n=8), sputum (n=1), urine (n=1), and other sources (n=7). Isolates were subcultured twice before testing on Columbia agar supplemented with 5% sheep blood.

All S. aureus isolates were identified by colony morphology, Gram staining, catalase, and coagulase tests. Oxacillin and cefoxitin susceptibility test was performed by Kirby-Bauer disk diffusion method using Clinical and Laboratory Standards Institute (CLSI) guidelines.⁹

Antimicrobial agents and susceptibility testing

All isolates were tested for susceptibility to vancomycin and daptomycin by reference broth microdilution (BMD) method and by standard Etest. Daptomycin was supplied by Novartis and vancomycin powder was commercially purchased (Sigma Chemical Company). BMD was performed according to CLSI guidelines using cation-adjusted Mueller-Hinton broth (BBL; Becton Dickinson).⁸ Mueller-Hinton broth adjusted to contain physiological levels of calcium (50 μg/ml) was used when testing daptomycin. Etest was performed according to the manufacturer's manual using vancomycin and daptomycin Etest strips (bioMérieux).

MIC₅₀, MIC₉₀ values, and MIC ranges were calculated for BMD method and Etest. Vancomycin and daptomycin MIC breakpoints proposed by CLSI for BMD method were applied to and used for Etest to determine the categorical agreement between the two methods.⁹ For determining the percent agreement between BMD and the standard Etest, discrepancies between MIC values of not more than one dilution (±1 dilution) were calculated.

Detection of hVISA

Etest macromethod

Etest macromethod was performed on all MRSA strains according to the manufacturer's instructions (EAS 003; AB Biodisk). Bacterial suspensions were prepared in normal saline to obtain a 2.0 McFarland standard. One hundred microliters of this suspension was evenly spread on to a 90-mm brain heart infusion (BHI) agar plate and allowed to dry. Vancomycin and teicoplanin Etest strips were applied to the surface of the BHI agar. The plates were incubated at 35°C for 48 hr. Heteroresistance by the Etest macromethod was defined as MICs for vancomycin and teicoplanin of ≥8 μg/ml or a teicoplanin MIC of ≥12 μg/ml.

Population analysis profile-area under the curve

The PAP-AUC was calculated for all isolates by inoculating serial 10-fold dilutions of the test organism on to BHI agar, which contains increasing concentrations of vancomycin. The vancomycin concentrations used were 0, 0.5, 1, 2, 2.5, 4, and 8 μg/ml. Colony growth at 48 hr was calculated and graphed as log₁₀ CFU/ml versus the vancomycin concentration.^50,52

The AUC was calculated for each strain and compared to the AUC of Mu3 (700698; ATCC) for each group of PAP. The test isolate AUC/mean Mu3 ratios were calculated. Ratios of <0.9 and 0.9–1.3 were considered positive for vancomycin-susceptible Staphylococcus aureus (VSSA) and hVISA respectively. Ratio of >1.3 was considered positive for VISA.³⁶

Statistical analysis

For the analysis of vancomycin MIC trends over time, Spearman correlation test was used. For the detection of association between hVISA isolates and vancomycin MICs, Chi-Square Test (Linear by linear association) was used. All analyses were performed by the Statistical Program for Social Sciences (SPSS, Inc.) version 15.0 for Windows. Statistical significance was defined as a p of <0.05.

Results

Distribution of vancomycin and daptomycin MICs of 94 MRSA isolates based on testing methodology (BMD and Etest) were shown in Figs. 1 and 2, respectively. All of the isolates were susceptible to vancomycin by BMD and Etest. However, 28 (29.8%) and 49 (52.1%) isolates had vancomycin MICs of 2 μg/ml by BMD and Etest, respectively. All isolates were susceptible to daptomycin by both BMD and Etest methods.

FIG. 1.

Distribution of vancomycin minimum inhibitory concentration (MIC) based on testing methodology. Numbers above columns reflect the number of isolates, whereas numbers inside columns reflect the percentage of isolates.

FIG. 2.

Distribution of daptomycin MIC based on testing methodology. Numbers above columns reflect the number of isolates, whereas numbers inside columns reflect the percentage of isolates.

The vancomycin MIC₅₀, MIC₉₀, and MIC ranges were 1, 2, and 0.5–2 μg/ml by BMD and 2, 2, and 0.5–2 μg/ml by Etest, respectively. When yearly geometric mean results were analyzed, the geometric mean vancomycin MICs varied from 0.71 to 1.47 by BMD and 0.58 to 1.60 by Etest (Table 1). We could not detect vancomycin MIC creep by either method. The daptomycin MIC₅₀, MIC₉₀, and MIC ranges were 0.5, 1, and 0.25–1 μg/ml by BMD and 0.5, 1, and 0.125–1 μg/ml by Etest, respectively.

Table 1.

Known Infection Sources, Isolation Dates, and Geometric Mean Vancomycin MICs of MRSA Isolates

Year	Blood	Catheter	Bone marrow	CSF	Pus	Wound	BAL	TA	Sputum	Urine	Other	Total	GM BMD	GM Etest
2001	12		1									13	1.11	1.24
2002	8											8	1.00	1.02
2003	4											4	1.19	1.11
2004	4				1	1		1				7	1.10	0.99
2005	4	1			2	1		1				9	1.47	1.31
2006	4	2			2		2	3	1		2	16	1.24	1.21
2007	5			1	1			2				9	1.47	1.60
2008		1		2	4			1			2	10	1.15	1.11
2009	7			1	3						2	13	1.38	1.13
2010	1				1						1	3	1.00	0.58
2011					1					1		2	0.71	0.87

BAL, bronchoalveolar lavage; BMD, broth microdilution; CSF, cerebrospinal fluid; GM BMD, geometric mean vancomycin MICs by BMD; GM Etest, geometric mean vancomycin MICs by Etest; MIC, minimum inhibitory concentration; MRSA, methicillin-resistant Staphylococcus aureus; TA, tracheal aspirate.

The vancomycin and daptomycin MICs determined by Etest were compared with the BMD MICs for all isolates. Results of BMD and Etest are considered equivalent if they are identical or vary by±1 dilution. The overall agreement was 100% between standard Etest and BMD for vancomycin. For vancomycin MICs, the results were concordant for 53 isolates (56.4%). For the remaining isolates, the Etest MICs were one dilution higher than the BMD results for 27 isolates (28.7%) and one dilution lower for 14 isolates (14.9%).

The overall agreement was 88.3% between standard Etest and BMD for daptomycin. For daptomycin MICs, the results were concordant for 26 isolates (27.6%). The Etest MICs were one dilution higher than the BMD results for 12 isolates (12.8%), one dilution lower for 45 isolates (47.9%), and two dilutions lower for 11 isolates (11.7%).

No VRSA or VISA isolates were detected among the isolates tested. Among 94 MRSA isolates, PAP-AUC identified 74 isolates as VSSA and 20 isolates as hVISA. Thus, the prevalence of hVISA among MRSA isolates was obtained as 21.3% (20/94). Table 2 shows the characteristics of 20 hVISA isolates.

Table 2.

Sources, Isolation Dates, Vancomycin MICs, Daptomycin MICs, and Etest Macromethod Results of hVISA Isolates

			Vancomycin MIC (mg/L)		Daptomycin MIC (mg/L)
Isolate	Isolate source	Date of isolation	BMD	Etest	BMD	Etest	Etest macromethod result
1	Blood	2001	2	1.5	0.5	0.19	N
2	Blood	2003	2	1.5	1	0.5	P
3	Blood	2005	2	1.5	1	0.75	P
4	Blood	2006	2	2	0.5	0.75	P
5	Catheter	2006	2	1	1	0.5	N
6	TA	2006	2	1.5	1	0.75	P
7	Pus	2006	1	1.5	0.5	0.75	N
8	Other	2006	2	1.5	1	0.75	P
9	Pus	2006	1	1.5	1	1	N
10	Catheter	2006	1	1.5	0.25	0.38	N
11	Blood	2007	2	1.5	0.5	0.75	P
12	CSF	2007	2	1.5	1	0.38	N
13	Pus	2007	1	1.5	0.25	0.125	N
14	TA	2007	2	2	1	1	N
15	Other	2008	1	1.5	0.5	0.5	P
16	Pus	2008	2	1.5	0.5	0.25	P
17	Blood	2009	2	2	0.5	0.19	P
18	Blood	2009	2	1.5	1	0.75	P
19	CSF	2009	2	1	0.5	0.38	P
20	Pus	2009	2	1.5	1	0.38	P

hVISA, heterogeneous vancomycin-intermediate Staphylococcus aureus; N, negative; P, positive.

Using the Etest macromethod 19/94 (20.2%) isolates met the criteria for hVISA. The Etest macromethod identified 12 of the hVISA strains (sensitivity, 60.0%) and there were 7 strains identified as hVISA unconfirmed by PAP-AUC (specificity, 90.5%) (Table 3).

Table 3.

Comparison of Etest Macromethod to PAP-AUC Method for Detection of hVISA Isolates

	PAP-AUC
	+		−		Total
Etest macromethod	n	%	n	%	n	%
+	12	12.8	7	7.4	19	20.2
−	8	8.5	67	71.3	75	79.8
Total	20	21.3	74	78.7	94	100

PAP-AUC, population analysis profile-area under the curve.

The frequency of hVISA was MIC dependent irrespective of the method used. The percentage of hVISA strains increased in conjunction with increasing MICs of vancomycin (p<0.001). Of the 28 isolates with a vancomycin MIC of 2 μg/ml, 15 (53.6%) had the hVISA phenotype, whereas only 5 (7.8%) of the 64 MRSA isolates with a vancomycin MIC of 1 μg/ml obtained by BMD method were found as hVISA strains (Fig. 3). Similarly by Etest, 36.7% (18/49) of the isolates with vancomycin MIC>1 μg/ml showed heteroresistance, which was higher than that of the vancomycin MIC≤1 μg/ml (4.4% [2/45]).

FIG. 3.

Correlation between vancomycin broth microdilution MIC and heteroresistance (defined by population analysis profile-area under the curve) for methicillin-resistant Staphylococcus aureus isolates. hVISA, heterogeneous vancomycin-intermediate Staphylococcus aureus; VSSA, vancomycin-susceptible Staphylococcus aureus.

Discussion

The recommended therapeutic agents for treating invasive MRSA infections are the glycopeptides, in particular vancomycin. However, the increasing use of this agent contributes to the growing burden of nonsusceptible strains. Vancomycin treatment failure is not uncommon, despite MRSA strains are fully susceptible to vancomycin (MIC≤2 μg/ml).^18,30 In our study, although all isolates were susceptible to vancomycin, approximately one-third (29.8%) of the isolates by BMD and half (52.1%) of the isolates by Etest had vancomycin MIC of 2 μg/ml. The high percentage of MRSA isolates that have vancomycin MIC of 2 μg/ml is an important issue as the previous reports suggest a significant risk for vancomycin treatment failure in MRSA infections with increasing vancomycin MICs despite the organism being fully susceptible (vancomycin MIC≤2 μg/ml).^26,41

The gold standard method to determine vancomycin MIC is BMD. However, the Etest method is an option for alternative vancomycin testing since BMD is labor intensive but Etest is easy to perform. Also most of the studies reported that MIC results provided by the Etest method would reflect clinical efficacy more accurately than those provided by BMD method.¹⁹ Several studies comparing method-specific vancomycin MIC values reported that the Etest MIC results were consistently higher than BMD.^{19,27,31,33,39} On the other hand, in two studies it was reported that Etest results were higher than those provided by BMD at low MICs (≤1 μg/ml) but were either concordant or one dilution lower than BMD at high MIC (2 μg/ml).^22,48 In our study, the vancomycin MIC results were concordant for 53 isolates (56.4%) by both methods. Among the remainder of the isolates, the Etest MIC was one dilution higher than the vancomycin BMD MIC result for 27 isolates (28.7%) and one dilution lower for 14 isolates (14.9%). Therefore, Etest displayed same or higher MIC values than BMD for most of the isolates (85.1%). Our findings were similar to the findings of the study of Musta et al. and Sancak et al.^32,44 Musta et al.³² reported that BMD and Etest MIC results were concordant for 61% of the isolates. Among the remainder of the isolates, the Etest MIC was higher than the BMD result for 24% and lower for 15% of the isolates. Sancak et al.⁴⁴ reported that BMD and Etest MIC results were concordant for 57.5% of the isolates. The Etest MIC was 0.5–1 μg/ml higher than the BMD result for 27.1% and 0.5–1 μg/ml lower for 15.4% of the isolates.

In some studies, a gradual increase in vancomycin MICs among MRSA strains over time (MIC creep) has been noted.^14,23,46,54 This phenomenon is of clinical concern because poorer outcomes for vancomycin treatment of MRSA infections with higher vancomycin MICs has been demonsrated. However, vancomycin MIC creep has not been observed universally.^2,15,34 The question of why vancomycin MIC creep has been detected at some institutions but not all has not yet been sufficiently answered. The studies performed to date differ in the periods analyzed, the methods used for antimicrobial susceptibility testing (broth dilution vs. Etest), and the type of isolates analyzed (methicillin-susceptible Staphylococcus aureus [MSSA] vs. MRSA and blood isolates vs. all clinical isolates).²³ Also, the intensity of vancomycin usage and the clonal dissemination of more resistant strains can be the reasons of inconsistencies between the results of studies about vancomycin MIC creep. In our study, we did not find an increase in vancomycin MICs among MRSA isolates during the study period.

In this study, we also determined the prevalence of VISA/hVISA by performing PAP-AUC on all isolates. As far as we know, only three studies investigated the prevalence of VISA/hVISA among the MRSA isolates of the pediatric group.^11,20,51 In two studies Etest macromethod and in one study glycopeptide resistance detection (GRD) Etest method were used to detect hVISA. PAP-AUC, which is the gold standard for defining hVISA was not used. Jimenez-Truque et al.²⁰ evaluated vancomycin susceptibility of 71 invasive pediatric community acquired MRSA isolates and detected 2 (2.8%) hVISA isolates by Etest macromethod. Welsh et al.⁵¹ tested 22 MRSA isolates of pediatric patients with MRSA bacteremia using Etest macromethod and reported 1 (4.5%) hVISA isolate. Goldman et al.¹¹ used GRD Etest method to perform vancomycin heteroresistance testing on 71 MRSA blood isolates and couldn't detect hVISA.

To our knowledge this is the first study investigating the prevalence of VISA/hVISA isolates by PAP-AUC method among the MRSA isolates belong to pediatric group. By this method 20 (21.3%) hVISA isolates were detected. In many studies, it is reported that hVISA infections are associated with longer duration of bacteremia,^5,6,10,28 fever,⁶ treatment failure,^6,10 greater prevalence of complications such as endocarditis and osteomyelitis,³ and longer hospital stay.^6,10 Thus, the high percentage of hVISA is another important finding of our study.

Also, it appears that the incidence of hVISA correlates with increase of vancomycin MIC. In our study the proportion of hVISA isolates, similar to the case in other studies, increased with higher MICs.^7,35,49 Chen et al.⁷ found that as the vancomycin MIC increased from 0.5 to 2 μg/ml, the incidence of hVISA would rise from 2.1% to 38.9%. Richter et al.³⁵ reported that the hVISA isolates represented 0.1% of isolates with vancomycin MICs of 1 μg/ml and 10.5% of isolates with vancomycin MICs of 2 μg/ml. In our study, hVISA was detected among the isolates with vancomycin MICs of 1 μg/ml (7.8%) and 2 μg/ml (53.6%) (Fig. 3). Furthermore, 75% of hVISA isolates had vancomycin MIC of 2 μg/ml.

In this study, Etest macromethod was also used for detection of hVISA isolates. Results of Etest macromethod were compared to PAP-AUC method. Etest macromethod yielded sensitivity of 60.0% and specificity of 90.5%. Previous studies suggested that the Etest macromethod was a potentially effective test for detection of hVISA.^{3,16,42,43,50} However, the accuracy of this method shows significant variation between different studies. The usage of different bacterial suspension volumes (50, 100, and 200 μl) used in different studies^29,49,53 can be the reason for this variability.¹⁷ Currently, the manufacturer recommends a bacterial suspension of 100 μl and in our study we followed this recommendation. Sensitivity of this method may be increased by using larger volumes of bacterial suspension or changing the breakpoint criteria for defining hVISA.

Although the true prevalence of hVISA is unknown, estimates from several studies range from 0.2% to as high as 65% of all MRSA isolates.^{4,6,16,24,29,42} Some of these differences may be explained by the lack of standardized criteria for the definition of hVISA, the use of different methods to detect hVISA, and patient populations tested. The high prevalence of hVISA in our study can be explained by several reasons. First, in many studies, only the suspected isolates due to screening tests have been analyzed by the gold standard method PAP. Some hVISA isolates could not have been detected by screening tests.^1,24,25,37 Second, there was a possibility of transmission of epidemic MRSA clones with decreased susceptibility to vancomycin between patients. As given in Table 2, 7/20 hVISA isolates were detected in 2006, which leads us to think the possibility of transmission of a particular clone between the pediatric patients. This possibility can be eradicated by molecular typing methods.

Increasing reports of isolates with reduced susceptibility to vancomycin appears to revive the need for novel antibiotics such as daptomycin for the treatment of serious MRSA infections. Daptomycin has been reported to have a good antimicrobial activity against MRSA isolates.^21,38,40 Sader et al.⁴⁰ evaluated the in vitro activity of daptomycin against clinical strains of staphylococci (including 800 MRSA) and enterococci collected in European medical centers. All MRSA isolates were susceptible to daptomycin. Kao et al.²¹ determined the susceptibilities of 470 MRSA isolates to various antibiotics including daptomycin. The susceptibility rate to daptomycin was 99.6%. In another study, 22,858 S. aureus (including 12,181 MRSA) isolates were tested for daptomycin susceptibility. Among MRSA, only 13 (0.11%) daptomycin-nonsusceptible isolates were detected.³⁸ Our results were in agreement with previous studies. All isolates were susceptible to daptomycin by both BMD and Etest methods. It seems to be a promising alternative to vancomycin in the treatment of MRSA infections.

In conclusion, we did not observe an increase in vancomycin MICs during the study period. All isolates were susceptible to vancomycin by both BMD and Etest methods. However, 21.3% of the isolates were hVISA. It is important for the laboratories to accurately identify hVISA because in several studies it is reported that hVISA infections are associated with vancomycin treatment failure. Since PAP-AUC is labour intensive, alternative methods as Etest macromethod have been proposed. In our study Etest macromethod exhibited high specificity (90.5%) but low sensitivity (60.0%) for detecting hVISA isolates. Using larger volumes of bacterial suspension, more dense suspension or changing the breakpoint criteria for defining hVISA may increase the sensitivity of this method. In this study, 75% of hVISA isolates had vancomycin MICs of 2 μg/ml. Therefore, we recommend the investigation of MRSA isolates with vancomycin MIC of 2 μg/ml for the presence of hVISA by PAP-AUC method as early detection of vancomycin-nonsusceptible isolates could allow the change of treatment, which might lead to better patient outcomes. In our study, daptomycin showed potent activity against all isolates. Based on our results and those of previous publications, it appears to be a therapeutic option for MRSA infections.

Footnotes

Acknowledgments

This work was supported by Hacettepe University Scientific Research Unit (No. 010D03101003). We thank Dr. Mutlu Hayran and Dr. Jale Karakaya for statistical analysis.

Disclosure Statement

No competing financial interests exist.

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