Bacterial Isolates
220 clinical S. aureus isolates obtained from the blood of infected patients at the Buffalo Veterans Affairs Health System of Western New York from 2003 to 2005 were analyzed. Isolates were obtained from 220 different patients. All studies were conducted in accordance with the Institutional review board at the University at Buffalo and the VA Health System. Control MSSA strain S. aureus ATCC29213 and a standard hVISA strain Mu3 (ATCC700698) were utilized.
Antimicrobial agents, Media, and MIC Determination
Vancomycin powder (Sigma Chemical Co, St. Louis, MO) was obtained commercially. Stock solutions were made according to manufacturer's directions and stored at 4°C. Etest strips of vancomycin (AB Biodisk, Solna, Sweden) were utilized for the initial screening of hVISA. All susceptibility testing used Mueller-Hinton Broth (Difco Laboratories, Detroit, MI) supplemented with calcium (25 mg/L) and magnesium (12.5 mg/L). Brain-Heart Infusion agar (Difco Laboratories, Detroit, MI) was utilized for all analysis. Trypticase soy agar with 5% sheep blood agar (TSA II, Becton-Dickinson Diagnostics, Sparks, MD) was utilized for a bacteria growth medium and delta-hemolysin test. CLSI Broth microdilution MICs were determined for 220 clinical isolates and a control MSSA strain S. aureus ATCC29213 and a standard hVISA strain Mu3 in triplicate accordance with the Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards) guidelines [14].
Macro and Standard Etest
The detection of hVISA among 220 isolates was completed by using the Etest Macro Method as a preliminary screen as previously described [7, 15]. Overnight growth cultures of the 220 clinical isolates and a control MSSA strain S. aureus ATCC29213 and a standard hVISA strain Mu3 were suspended in saline solution. The suspensions were adjusted to 2.0 McFarland, swabbed on the BHI plates and dried at room temperature, and Etest strips were placed on the plates. After 48 h incubation at 35°C, the intersections of the elliptical inhibition zone and the subpopulation growth in the inhibition zone were recorded. The criterion for Macro Etest for detecting hVISA was the appearance of one or more subpopulation colonies in MIC ≥4 mg/L refers to the recent study results performed by Maor [16]. Additionally, Standard Etest was performed at a 0.5 McFarland using MHA that was also performed in the similar manner and interpreted according to CLSI breakpoints.
Modified PAP-AUC
Isolates which displayed hVISA profile by the Macro Etest Method were further analyzed using modified PAP-AUC method according to the standard PAP-AUC methodology as previously described [7]. Briefly, overnight growth culture of hVISA clinical isolates and a control isolates S. aureus ATCC29213 and a standard hVISA strain Mu3 were suspended in saline solution. The suspensions were adjusted to the modified starting inoculum 1010 CFU/mL. Cultures were serially diluted from 0 to 10-6, and 10 μL of each dilution was plated in quadruplicate on BHI containing vancomycin in the following concentrations: 0.5, 1, 2, 4, 6, 8 and 16 mg/L. Colonies were enumerated after 48 h incubation at 35°C. Bacterial colony counts (Log10 CFU/mL) were plotted against the vancomycin concentration (0 to 4 mg/L) using SigmaPlot 9.0. The area under the curve (AUC) was calculated for each isolate and divided by the AUC value of the reference strain Mu3. The criteria for identifying hVISA were either the modified PAP-AUC ratio of > 0.9 or > 0.8. Additionally, modified PAP-AUC ratios were plotted against Macro Etest MICs, which were evaluated for the relation between two variables using the linear regression equation and the correlation coefficient (r).
BHIAV4
To evaluate the inoculum effect of hVISA strains detected by the Macro Etest Method, a modified procedure involving Brain Heart Infusion agar contain vancomycin 4 mg/L BHIAV4 method was performed as described previously [6]. Briefly, overnight growth culture of hVISA clinical isolates and a control isolates S. aureus ATCC29213 and a standard hVISA strain Mu3 were suspended in saline solution. The suspensions were adjusted to the modified inoculum of 108 and an additional higher inoculum of 1010 CFU/mL. Each sample was plated in quadruplicate on BHI agar containing vancomycin 4 mg/mL. Colonies were enumerated after 48 h incubation at 35°C. Growth of 1 or more colonies indicate a positive result.
Delta-hemolysin
To evaluate the relationship between agr dysfunction and hVISA, delta-hemolysin expression was determined for hVISA isolates detected by Etest according to the study previously described [13]. Briefly, overnight growth culture of hVISA clinical isolates and S. aureus RN4220 were suspended in saline solution. The suspensions were adjusted to 0.5 McFarland standards and were streaked vertically near RN4220 on TSA II. After incubating plates at 35°C for 24 h, the expression of delta-hemolysin was evaluated.
Statistical analysis
The relation between MICs (mg/L) values and modified PAP-AUC ratios was evaluated using the linear regression analysis. The correlation coefficient (r) was calculated to display the correlation between the two variables. The relationships between the independent and dependent variables were analyzed using Mann-Whitney U test and chi-square test. P-value of < 0.05 was considered statistically significant. The number of false positive, false negative, the percentage of sensitivity and specificity were calculated for each method.