April M. Bobenchik

Comparison of the Vitek MS and Bruker Microflex LT MALDI-TOF MS platforms for routine identification of commonly isolated bacteria and yeast in the clinical microbiology laboratory

This study compared the diagnostic performance of Brukers Microflex LT and bioMérieuxs Vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry systems. A total of 477 isolates were tested on both instruments. Discrepant results were resolved by sequencing. Overall, there was no statistically significant difference between the proportion of isolates correctly identified, miscalled or not called by each instrument. Although both systems were good at identifying yeast (66/69 to species level), the confidence level was high only to genus level for 30% of the isolates on the Bruker. Both systems performed with high accuracy when evaluated solely on Food and Drug Administration-approved organisms for each database. A user-based assessment of the 2 instruments revealed an overall preference for the Vitek MS instrument.

Performance of Vitek 2 for Antimicrobial Susceptibility Testing of Staphylococcus spp. and Enterococcus spp.

ABSTRACT Vitek 2 (bioMérieux, Inc., Durham, NC) is a widely used commercial antimicrobial susceptibility testing system. We compared MIC results obtained by Vitek 2 to those obtained by the Clinical and Laboratory Standards Institute (CLSI) broth microdilution (BMD) reference method for 134 staphylococcal and 84 enterococcal clinical isolates. Nineteen agents were evaluated, including all those available on Vitek 2 for testing staphylococci and enterococci. The resistance phenotypes tested included methicillin-resistant Staphylococcus aureus (MRSA) (n = 58), S. aureus with inducible clindamycin resistance (ICR) (n = 30), trimethoprim-sulfamethoxazole-resistant MRSA (n = 10), vancomycin-resistant Enterococcus (n = 37), high-level gentamicin-resistant Enterococcus (n = 15), linezolid-resistant Enterococcus (n = 5), and daptomycin-nonsusceptible Enterococcus faecalis (n = 6). For the staphylococci, there was 98.9% categorical agreement (CA). There was one very major error (VME) for gentamicin in a Staphylococcus hominis isolate, six VMEs for inducible clindamycin in S. aureus isolates, and two major errors (ME) for daptomycin in an S. aureus and a Staphylococcus epidermidis isolate. For enterococci, there was 97.3% CA. Two VMEs were observed for daptomycin in isolates of E. faecalis and 2 ME, 1 for high-level gentamicin resistance and 1 for nitrofurantoin, in E. faecium isolates. Overall, there was 98.3% CA and 99% essential agreement for the testing of staphylococci and enterococci by the Vitek 2. With the exception of detecting ICR in S. aureus, Vitek 2 performed reliably for antimicrobial susceptibility testing of staphylococci and enterococci.

Modified Carbapenem Inactivation Method for Phenotypic Detection of Carbapenemase Production among Enterobacteriaceae

ABSTRACT The ability of clinical microbiology laboratories to reliably detect carbapenemase-producing carbapenem-resistant Enterobacteriaceae (CP-CRE) is an important element of the effort to prevent and contain the spread of these pathogens and an integral part of antimicrobial stewardship. All existing methods have limitations. A new, straightforward, inexpensive, and specific phenotypic method for the detection of carbapenemase production, the carbapenem inactivation method (CIM), was recently described. Here we describe a two-stage evaluation of a modified carbapenem inactivation method (mCIM), in which tryptic soy broth was substituted for water during the inactivation step and the length of this incubation was extended. A validation study was performed in a single clinical laboratory to determine the accuracy of the mCIM, followed by a nine-laboratory study to verify the reproducibility of these results and define the zone size cutoff that best discriminated between CP-CRE and members of the family Enterobacteriaceae that do not produce carbapenemases. Bacterial isolates previously characterized through whole-genome sequencing or targeted PCR as to the presence or absence of carbapenemase genes were tested for carbapenemase production using the mCIM; isolates with Ambler class A, B, and D carbapenemases, non-CP-CRE isolates, and carbapenem-susceptible isolates were included. The sensitivity of the mCIM observed in the validation study was 99% (95% confidence interval [95% CI], 93% to 100%), and the specificity was 100% (95% CI, 82% to 100%). In the second stage of the study, the range of sensitivities observed across nine laboratories was 93% to 100%, with a mean of 97%; the range of specificities was 97% to 100%, with a mean of 99%. The mCIM was easy to perform and interpret for Enterobacteriaceae, with results in less than 24 h and excellent reproducibility across laboratories.

Mechanisms of Linezolid Resistance among Coagulase-Negative Staphylococci Determined by Whole-Genome Sequencing

ABSTRACT Linezolid resistance is uncommon among staphylococci, but approximately 2% of clinical isolates of coagulase-negative staphylococci (CoNS) may exhibit resistance to linezolid (MIC, ≥8 µg/ml). We performed whole-genome sequencing (WGS) to characterize the resistance mechanisms and genetic backgrounds of 28 linezolid-resistant CoNS (21 Staphylococcus epidermidis isolates and 7 Staphylococcus haemolyticus isolates) obtained from blood cultures at a large teaching health system in California between 2007 and 2012. The following well-characterized mutations associated with linezolid resistance were identified in the 23S rRNA: G2576U, G2447U, and U2504A, along with the mutation C2534U. Mutations in the L3 and L4 riboproteins, at sites previously associated with linezolid resistance, were also identified in 20 isolates. The majority of isolates harbored more than one mutation in the 23S rRNA and L3 and L4 genes. In addition, the cfr methylase gene was found in almost half (48%) of S. epidermidis isolates. cfr had been only rarely identified in staphylococci in the United States prior to this study. Isolates of the same sequence type were identified with unique mutations associated with linezolid resistance, suggesting independent acquisition of linezolid resistance in each isolate. IMPORTANCE Linezolid is one of a limited number of antimicrobials available to treat drug-resistant Gram-positive bacteria, but resistance has begun to emerge. We evaluated the genomes of 28 linezolid-resistant staphylococci isolated from patients. Multiple mutations in the rRNA and associated proteins previously associated with linezolid resistance were found in the isolates investigated, underscoring the multifocal nature of resistance to linezolid in Staphylococcus. Importantly, almost half the S. epidermidis isolates studied harbored a plasmid-borne cfr RNA methylase gene, suggesting that the incidence of cfr may be higher in the United States than previously documented. This finding has important implications for infection control practices in the United States. Further, cfr is commonly detected in bacteria isolated from livestock, where the use of phenicols, lincosamides, and pleuromutilins in veterinary medicine may provide selective pressure and lead to maintenance of this gene in animal bacteria. Linezolid is one of a limited number of antimicrobials available to treat drug-resistant Gram-positive bacteria, but resistance has begun to emerge. We evaluated the genomes of 28 linezolid-resistant staphylococci isolated from patients. Multiple mutations in the rRNA and associated proteins previously associated with linezolid resistance were found in the isolates investigated, underscoring the multifocal nature of resistance to linezolid in Staphylococcus. Importantly, almost half the S. epidermidis isolates studied harbored a plasmid-borne cfr RNA methylase gene, suggesting that the incidence of cfr may be higher in the United States than previously documented. This finding has important implications for infection control practices in the United States. Further, cfr is commonly detected in bacteria isolated from livestock, where the use of phenicols, lincosamides, and pleuromutilins in veterinary medicine may provide selective pressure and lead to maintenance of this gene in animal bacteria.

Performance of Vitek 2 for Antimicrobial Susceptibility Testing of Enterobacteriaceae with Vitek 2 (2009 FDA) and 2014 CLSI Breakpoints

ABSTRACT Vitek 2 (bioMérieux Inc., Durham, NC) is a widely used commercial antimicrobial susceptibility test system. We compared the MIC results obtained using the Vitek 2 AST-GN69 and AST-XN06 cards to those obtained by CLSI broth microdilution (BMD) for 255 isolates of Enterobacteriaceae, including 25 isolates of carbapenem-resistant Enterobacteriaceae. In total, 25 antimicrobial agents were examined. For 10 agents, the MIC data were evaluated using two sets of breakpoints: (i) the Vitek 2 breakpoints, which utilized the 2009 FDA breakpoints at the time of the study and are equivalent to the 2009 CLSI M100-S19 breakpoints, and (ii) the 2014 CLSI M100-S24 breakpoints. There was an overall 98.7% essential agreement (EA). The categorical agreement was 95.5% (CA) using the Vitek 2 breakpoints and 95.7% using the CLSI breakpoints. There was 1 very major error (VME) (0.05%) observed using the Vitek 2 breakpoints (cefazolin) and 8 VMEs (0.5%) using the CLSI breakpoints (2 each for aztreonam, cefepime, and ceftriaxone, and 1 for cefazolin and ceftazidime). Fifteen major errors (MEs) (0.4%) were noted using the Vitek 2 breakpoints and 8 (0.5%) using the CLSI breakpoints. Overall, the Vitek 2 performance was comparable to that of BMD for testing a limited number of Enterobacteriaceae commonly isolated by clinical laboratories. Ongoing studies are warranted to assess performance in isolates with emerging resistance.

Ciprofloxacin-Resistant Aeromonas hydrophila Cellulitis following Leech Therapy

ABSTRACT We report a case of surgical site infection with ciprofloxacin-resistant Aeromonas hydrophila following leech therapy. Antimicrobial and genetic analyses of leech and patient isolates demonstrated that the resistant isolates originated from the leech gut microbiota. These data suggest that ciprofloxacin monotherapy as a prophylaxis regimen prior to leech therapy may not be effective in preventing infection.

Rapid Detection of Campylobacter Antigen by Enzyme Immunoassay Leads to Increased Positivity Rates

ABSTRACT Campylobacter antigen detection by enzyme immunoassay (EIA) provides rapid results compared to traditional culture. However, concern exists regarding specificity. Verification studies of an EIA compared to culture revealed a positive predictive value (PPV) of 91%, whereas PPV fell to 42% during routine diagnostic testing. We suggest all positive EIA results be confirmed via culture.

Performance of Vitek 2 for Antimicrobial Susceptibility Testing of Acinetobacter baumannii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia with Vitek 2 (2009 FDA) and CLSI M100S 26th Edition Breakpoints

ABSTRACT The performances of Vitek 2 AST-GN69 and AST-XN06 cards were compared to Clinical and Laboratory Standards Institute (CLSI) reference broth microdilution (BMD) for 99 isolates of Pseudomonas aeruginosa, 26 Acinetobacter baumannii isolates, and 11 Stenotrophomonas maltophilia isolates. In total, 15 antimicrobials were evaluated, with 11 for P. aeruginosa, 14 for A. baumannii, and 2 for S. maltophilia. Categorical agreement (CA) was assessed using both Vitek 2 breakpoints and 2016 CLSI M100S 26th edition breakpoints. The essential agreement values for P. aeruginosa, A. baumannii, and S. maltophilia were 99.5%, 99.2%, and 100%, respectively. The CA values for P. aeruginosa, A. baumannii, and S. maltophilia were 94.1%, 92.7%, and 95.5%, respectively, by the Vitek 2 breakpoints, and 93.4%, 92.3%, and 95.5%, respectively, by the CLSI breakpoints. Overall, the Vitek 2 performance was comparable to that of BMD using both Vitek 2 breakpoints and 2016 CLSI M100S 26th edition breakpoints. Improved performance was noted for the reformulated piperacillin-tazobactam and imipenem found on the AST-GN69 card, with no very major or major errors noted when using the CLSI breakpoints.

Use of rapid diagnostic tests for diagnosis of malaria in the United States.

In the September issue of this journal, performance of BinaxNOW for the diagnosis of malaria in a major U.S. academic medical center was reported ([1][1]). Herein, we report performance of this test in a second large academic medical center in the United States. BinaxNOW was introduced at our

Frequency of Susceptibility Testing for Patients with Persistent Methicillin-Resistant Staphylococcus aureus Bacteremia

ABSTRACT Currently, no standards exist for determining the optimal frequency of repeat antimicrobial susceptibility testing (AST) when an organism is recurrently isolated from the same specimen source. Although testing every 2 to 5 days is thought sufficient, we present three cases of methicillin-resistant Staphylococcus aureus (MRSA) bacteremia where current laboratory protocol for repeating AST every 5 days was inadequate to identify resistant organisms.