William Michael Dunne

BBL CHROMagar VanRE (CVRE) was compared with bile esculin azide agar plus vancomycin to screen for vancomycin-resistant enterococcus (VRE) colonization. CVRE distinguishes Enterococcus faecalis (green colonies) from Enterococcus faecium (mauve colonies) on the basis of chromogenic substrate use. CVRE sensitivity and specificity were 98.6% and 99.1%. Positive and negative predictive values were 95.9% and 99.7%.

In January 2006, the Clinical and Laboratory Standards Institute (CLSI) updated breakpoints for vancomycin susceptibility testing for Staphylococcus aureus such that an MIC greater than 2 mg/liter was considered to represent nonsusceptibility to vancomycin. Previously, an MIC of 4 mg/liter had been considered to represent susceptibility. Additionally, in 2009, the CLSI altered the guidelines for staphylococci such that disk diffusion was no longer an acceptable means for testing vancomycin susceptibility in these organisms. To accommodate the change in breakpoints and methodological updates, we designed a medium consisting of brain heart infusion agar with 3 mg/liter vancomycin (BHI-V3) to screen for isolates of S. aureus with reduced susceptibility to vancomycin. We challenged this medium using a previously characterized collection of 100 isolates of S. aureus , including 55 vancomycin-susceptible isolates and 45 isolates representing vancomycin-intermediate strains of S. aureus (VISA) (with vancomycin MICs of 4 mg/liter or greater). All of the VISA isolates grew on the agar, for 100% sensitivity. Nineteen vancomycin-susceptible isolates also grew on the agar, for 65% specificity. We then incorporated BHI-V3 into clinical practice. In the first 60 days postimplementation, we identified 17 potential VISA isolates out of 421 S. aureus isolates tested. Thirteen out of the 17 were confirmed to represent VISA isolates. In light of the excellent sensitivity of this medium, we recommend that clinical laboratories incorporate BHI-V3 to screen for vancomycin-nonsusceptible isolates of S. aureus .

The performance of the VersaTREK blood culture system for the detection of a bacterial contamination of platelet concentrates was assessed using samples spiked with serial dilutions of nine bacterial species associated with platelet contamination. The system detected growth for all organisms in <20 h, and the detection sensitivity was <20 CFU/ml.

An isolate of Proteus mirabilis recovered from blood cultures of a diabetic patient was shown to be resistant to imipenem, meropenem, and ertapenem by disk diffusion susceptibility testing. Amplification of whole-cell and/or plasmid DNA recovered from the isolate with primers specific for the bla _KPC carbapenemase gene produced an amplicon of the expected size which was confirmed to be bla _KPC-2 by sequence analysis. Transformation of a susceptible Escherichia coli host with plasmid preparations from the isolate generated a transformant for which the MICs of all of the carbapenems tested were increased three- to fourfold. We believe this to be the first report of carbapenem resistance in P . mirabilis caused by the acquisition of bla _KPC .

The development of reliable and rapid methods for the identification of patients colonized with vancomycin-resistant enterococci (VRE) is central to the containment of this agent within a hospital environment. To this end, we evaluated a prototype chromogenic agar medium (VRE-BMX; bioMérieux, Marcy l'Etoile, France) used to recover VRE from clinical specimens. This medium can also identify isolated colonies as either vancomycin-resistant Enterococcus faecium or Enterococcus faecalis , based on distinct colony colors. We compared the performance of VRE-BMX with bile esculin azide agar supplemented with vancomycin (BEAV). For this study, 147 stool samples were plated on each test medium and examined after 24 and 48 h of incubation. At 24 h, the sensitivity and specificity of each medium were as follows: BEAV, 90.9% and 89.9%, respectively; VRE-BMX, 96.4% and 96.6%, respectively. The positive predictive values (PPV) of VRE-BMX and BEAV at 24 h were 89.8% and 80.7%, respectively. VRE-BMX provided the identification of 10 isolates of vancomycin-resistant E. faecalis and 4 isolates of vancomycin-resistant E. faecium that were not recovered by BEAV. Further, VRE-BMX was capable of identifying patients colonized with both E. faecium and E. faecalis , a feature useful for infection control purposes that is not a function of BEAV. In terms of the recovery of vancomycin-resistant E. faecium and E. faecalis , the sensitivity and PPV were as follows: BEAV, 75.7% and 74.6%, respectively; VRE-BMX, 95.5% and 91.3%, respectively. In this initial evaluation, we found that VRE-BMX provided improved recovery of VRE from stool specimens, with the added advantage of being able to differentiate between vancomycin-resistant E. faecalis and E. faecium . Extending the incubation period beyond 24 h did not significantly improve the recovery of VRE and resulted in decreased specificity.

We performed a blinded study to compare repetitive-sequence PCR and multilocus sequence typing for genotyping hospital- and community-acquired methicillin-resistant Staphylococcus aureus (MRSA). The MRSA strains that were sequence type 8 (ST8), staphylococcal cassette chromosome mec (SCC mec ) type IV, and Panton-Valentine leukocidin-positive clustered separately from those that were ST5 and SCC mec type II.

Two-hundred consecutive, single patient isolates of Enterobacter spp., Serratia spp., Citrobacter spp., and Pseudomonas aeruginosa were evaluated for AmpC production using a variety of inducer-substrate antibiotic combinations in a disk approximation format. The combinations examined included cefoxitin-piperacillin, imipenem-cefotaxime, imipenem-ceftazidime, imipenem-piperacillin-tazobactam, and imipenem-cefoxitin. All isolates were also screened for the presence of extended-spectrum β-lactamase (ESBL) activity. In total, 85.5% of isolates were shown to be inducible for the production of AmpC by one or more inducer/substrate combinations and 11% of all isolates were stably derepressed for the expression of AmpC. Of all of the combinations, imipenem/piperacillin-tazobactam provided the greatest sensitivity (97.1%). All combinations were 100% specific when a positive test was observed. Given this background among these organisms in our institution, it is reasonable to develop an antibiotic reporting strategy that favors the selection of agents for therapy of these organisms that do not serve as labile substrates of AmpC.

At present, only two commercially available automated culture systems are cleared by the FDA for the purpose of quality control (QC) testing for bacterial contamination of platelet (PLT) concentrates: the BacT/ALERT blood culture system (bioMérieux) and the Pall eBDS (Pall Corporation), both of which allow testing of leukoreduced apheresis as well as whole blood-derived PLTs. After the decision of the AABB to institute universal QC testing of PLT concentrates for evidence of bacterial contamination, in-house validation of the performance of our current blood culture system, the BACTEC 9240, was carried out for this purpose. Serial dilutions of nine species of bacteria commonly associated with PLT contamination were prepared in one single-donor apheresis PLT unit per organism. Four mL of dilutions containing less than 1 to greater than 10(3) colony-forming units (CFUs) per mL was inoculated into blood culture bottles (Standard 10 Aerobic/F, Becton-Dickinson Diagnostic Systems) and incubated in a BACTEC 9240 continuously monitored blood culture system. Positive bottles were removed from the system and subcultured to insure the identity of bacterial growth. With the exception of Streptococcus mitis, the BACTEC system provided a detection sensitivity of less than 10 CFUs per mL for Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Serratia marcescens, Klebsiella pneumoniae, Bacillus cereus, Enterobacter cloacae, and Pseudomonas aeruginosa. The limit of detection for the S. mitis test strain was 61 CFUs per mL. Detection of positive bottles ranged from 6.5 to 17.6 hours depending on the species tested and the cell density of the inoculum. Ongoing use of this system for bacterial detection yielded two true-positive samples from 3879 apheresis PLT products collected at our hospital-based donor center over 9 months. This study validates the use of the BACTEC 9240 continuously monitored blood culture system for the detection of low-level bacterial contamination in single donor apheresis PLTs in less than 24 hours.