Clinical Implications of Varying Degrees of Vancomycin Susceptibility in Methicillin-Resistant Staphylococcus aureus Bacteremia

Mitchell J. Schwaber, Sharon B. Wright, Yehuda Carmeli, Lata Venkataraman, Paola C. DeGirolami, Aneta Gramatikova, Trish M. Perl, George Sakoulas, Howard S. Gold


Emerging Infectious Diseases. 2003;9(6) 

In This Article


Bacterial isolates. MRSA bloodstream isolates that had been stored nonselectively at Beth Israel Deaconess Medical Center from September 1998 through November 2001 and nosocomial bloodstream isolates from patients in intensive care units at Johns Hopkins Hospital from July 1997 through April 2000 were used. In addition, the following strains were used as controls: ATCC 29213 (methicillin-susceptible S. aureus), ATCC 33591 (vancomycin-susceptible MRSA), ATCC 51299 (vancomycin-resistant Enterococcus faecium) and PC3 (VISA strain contributed by A. Tomasz).[19]

Screening for heterogeneously reduced susceptibility to vancomycin. Suspensions of 0.5 McFarland turbidity standard in brain heart infusion (BHI) broth were prepared from isolates after overnight incubation. Ten microliters of each suspension was injected onto BHI agar plates containing 4 mg/L vancomycin. Because of the reported inducibility of vancomycin heteroresistance in some strains of S. aureus by NaCl,[13] each isolate was screened on agar with and without 4% NaCl supplementation.

Plates were incubated at 35°C. Results were recorded after 24 h and 48 h of incubation. For any growth in excess of a single pinpoint colony on screening media, either with or without 4% NaCl supplementation, a positive result was recorded.

Susceptibility testing. Susceptibility to vancomycin was determined by agar dilution, according to NCCLS guidelines,[17] with Mueller-Hinton agar. Testing was performed on all MRSA bloodstream isolates (parent isolates), as well as on those colonies that grew on screening media (subclones). MIC testing was performed on colonies taken from the screening agar on which they exhibited optimal growth (i.e., BHI agar containing 4 mg/L vancomycin, with or without 4% NaCl).

Identity confirmation and strain typing. The identity of parent isolates and subclones was confirmed by Gram stain, catalase testing,[20] and latex agglutination testing (Staphaurex, Murex Biotech, Ltd., Dartford, UK). Strains were grouped by type and subtype by using pulsed-field gel electrophoresis (PFGE).[21] Plugs were made by using standard techniques. Macrorestriction was performed with SmaI.[22]

Population analysis. Suspensions of 3.0 McFarland turbidity standard in BHI broth were made from overnight cultures of selected parent isolates and subclones. Subclones were grown in 4 mg/L vancomycin before suspension in broth. Seven serial 10-fold dilutions of each suspension were prepared. Twenty-five microliters of each suspension and dilution was injected twice onto BHI agar containing 4% NaCl, at vancomycin concentrations of 0 mg/L, 1 mg/L, 2 mg/L, 4 mg/L, and 8 mg/L. NaCl was added due to the enhanced growth we observed on NaCl-containing media during screening. Colonies were counted after 48 h of incubation at 35°C, and sums of each inoculum pair were averaged.

Clinical data collection and inclusion criteria. For the epidemiologic analysis, the unit of observation shifted from the bacterial isolate to the patient with bacteremia. Demographic and clinical data for the patients with saved MRSA bloodstream isolates were collected from electronic medical records and from hospital databases. A patient could be included in the cohort only once, regardless of the number of isolates generated during the study period. Patients with multiple isolates were included at the time MRSA was first isolated from the bloodstream and the isolate grew on screening media. Those patients whose isolates never grew on screening media were included at the time of their first MRSA bloodstream isolation. Inpatients only were considered in the analysis. The study was approved by the ethics review boards of Beth Israel Deaconess Medical Center and of Johns Hopkins Hospital and performed at both institutions.

Study design. We conducted a two-part retrospective analysis on patients with MRSA bacteremia: a case-control analysis, which considered the clinical features at the time of culture, and a cohort analysis, which evaluated outcomes after culturing.

For the case-control study, patients whose MRSA bloodstream isolates exhibited growth on screening media (case-patients) were compared with patients whose isolates exhibited no growth (controls), in terms of the following features: age, sex, coexisting chronic conditions, hospital events, and antibiotic exposures before culture. Particular attention was given to exposure to vancomycin, examined both as a dichotomous variable and for cumulative days of exposure.

In the cohort study, we compared the above two groups of participants for the following postculture outcomes: deaths, discharge disposition, and duration of hospital stay after culture. Possible confounding variables were evaluated through multivariate modeling.

Statistical analysis. Statistical analyses were performed by using SAS statistical software (version 8e, SAS Institute, Inc., Cary, NC). Continuous variables were compared by using the Student t test or the Wilcoxon rank sum test, depending on the normality of the distribution. Binary variables were compared by using the Fisher exact test. Nonbinary categorical variables were compared by using the chi-square test. Multivariate logistic regression models were used in the death and disposition analyses to control for confounding. A Cox proportional hazards model was used in the time-to-discharge analysis; observations were censored at patient's death. For all statistical tests, a p value of ≤0.05 was considered significant.


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