Over a period of 26 months, we have evaluated in a prospective fashion the use of 16S rRNA gene sequencing as a means of identifying clinically relevant isolates of nonfermenting gram-negative bacilli (non-Pseudomonas aeruginosa) in the microbiology laboratory. The study was designed to compare phenotypic with molecular identification. Results of molecular analyses were compared with two commercially available identification systems (API 20 NE, VITEK 2 fluorescent card; bioMérieux, Marcy l'Etoile, France). By 16S rRNA gene sequence analyses, 92% of the isolates were assigned to species level and 8% to genus level. Using API 20 NE, 54% of the isolates were assigned to species and 7% to genus level, and 39% of the isolates could not be discriminated at any taxonomic level. The respective numbers for VITEK 2 were 53%, 1%, and 46%, respectively. Fifteen percent and 43% of the isolates corresponded to species not included in the API 20 NE and VITEK 2 databases, respectively. We conclude that 16S rRNA gene sequencing is an effective means for the identification of clinically relevant nonfermenting gram-negative bacilli. Based on our experience, we propose an algorithm for proper identification of nonfermenting gram-negative bacilli in the diagnostic laboratory.

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This study compared three sample preparation methods (direct transfer, the direct transfer-formic acid method with on-target formic acid treatment, and ethanol-formic acid extraction) for the identification of Gram-positive cocci with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). A total of 156 Gram-positive cocci representing the clinically most important genera, Aerococcus, Enterococcus, Staphylococcus, and Streptococcus, as well as more rare genera, such as Gemella and Granulicatella, were analyzed using a Bruker MALDI Biotyper. The rate of correct genus-level identifications was approximately 99% for all three sample preparation methods. The species identification rate was significantly higher for the direct transfer-formic acid method and ethanol-formic acid extraction (both 77.6%) than for direct transfer (64.1%). Using direct transfer-formic acid compared to direct transfer, the total time to result was increased by 22.6%, 16.4%, and 8.5% analyzing 12, 48, and 96 samples per run, respectively. In a subsequent prospective study, 1,619 clinical isolates of Gram-positive cocci were analyzed under routine conditions by MALDI-TOF MS, using the direct transfer-formic acid preparation, and by conventional biochemical methods. For 95.6% of the isolates, a congruence between conventional and MALDI-TOF MS identification was observed. Two major limitations were found using MALDI-TOF MS: the differentiation of members of the Streptococcus mitis group and the identification of Streptococcus dysgalactiae. The Bruker MALDI Biotyper system using the direct transfer-formic acid sample preparation method was shown to be a highly reliable tool for the identification of Gram-positive cocci. We here suggest a practical algorithm for the clinical laboratory combining MALDI-TOF MS with phenotypic and molecular methods.

Two mechanisms account for AmpC activity in Escherichia coli, namely, mutations in the ampC promoter and attenuator regions resulting in ampC overexpression and acquisition of plasmid-carried ampC genes. In this study, we analyzed 51 clinical E. coli isolates with reduced susceptibility to amoxicillin-clavulanic acid, piperacillin-tazobactam, or extended-spectrum cephalosporins for the presence of AmpC production. Three phenotypic AmpC confirmation assays (cefoxitin-cloxacillin disk diffusion test, cefoxitin-EDTA disk diffusion test, and AmpC Etest) were compared for the detection of AmpC activity. All 51 isolates were characterized genetically by mutational analysis of the chromosomal ampC promoter/attenuator region and by PCR detection of plasmid-carried ampC genes. Altogether, 21/51 (41%) E. coli isolates were considered true AmpC producers. AmpC activity due to chromosomal ampC promoter/attenuator mutations was found in 12/21 strains, and plasmid-carried ampC genes were detected in 8/21 isolates. One strain contained both ampC promoter mutations and a plasmid-carried ampC gene. All three phenotypic tests were able to detect the majority (>90%) of AmpC-positive strains correctly. Cefoxitin resistance was found to be a discriminative parameter, detecting 20/21 AmpC-producing strains. Susceptibility to extended-spectrum cephalosporins, e.g., ceftriaxone, ceftazidime, and cefotaxime, was found in 9 of the 21 AmpC-positive strains. Considering the elevated zone diameter breakpoints of the 2010 CLSI guidelines, 2/21 AmpC-positive strains were categorized as susceptible to extended-spectrum cephalosporins.

We analyzed surgically resected endocardial specimens from 49 patients by broad-range PCR. PCR results were compared with (1) results of previous blood cultures, (2) results of culture and Gram staining of resected specimens, and (3) clinical data (Duke criteria). Molecular analyses of resected specimens and previous blood cultures showed good overall agreement. However, in 18% of patients with sterile blood cultures, bacterial DNA was found in the resected materials. When data from patients with definite or rejected cases of infective endocarditis (IE) were included, the sensitivity, specificity, and positive and negative predictive values of broad-range PCR were 82.6%, 100%, 100%, and 76.5%, respectively, overall, and 94.1%, 100%, 100%, and 90%, for cases of native valve endocarditis. The sensitivity, specificity, and positive and negative predictive values of culture of resected specimens from patients with native valve endocarditis were 17.6%, 88.9%, 75%, and 36.4%. We recommend broad-range PCR of surgically resected endocardial material in cases of possible IE, in cases of suspected IE in which blood cultures are sterile, and in cases in which organisms grow in blood cultures but only Duke minor criteria are met. We propose to add molecular techniques to the pathologic criteria of the Duke classification scheme.