Evaluation of the Vitek 2 System for Rapid Identification of Clinical Isolates of Gram-Negative Bacilli and Members of the Family Streptococcaceae

Accuracy of the Vitek 2 automated system (bioMérieux Vitek, USA) for rapid identification of bacteria was evaluated using a collection of 858 epidemiologically unrelated gram-negative and 99 gram-positive clinical isolates. Isolates were tested after subculturing to ensure purity. Conventional agar-based biochemical tests (Steers replicator) were used as a reference method of identification. Gram-negative bacteria were identified to the species level with 95.3% accuracy by the system (Enterobacteriaceae, 95.9%; and non-Enterobacteriaceae, 92.5%), and gram-positive isolates with 72% accuracy. Although Vitek 2 identified routine clinical isolates of gram-negative bacilli and Enterococcus faecalis and Enterococcus faecium reliably, rapidly, and reproducibly, improvement is required in the identification of less common species of enterococci and viridans group streptococci. Electronic Publication     

Direct Antimicrobial Susceptibility Testing of Gram-Negative Bacilli in Blood Cultures by an Electrochemical Method

Nonfastidious aerobic gram-negative bacilli (GNB) are commonly isolated from blood cultures. The feasibility of using an electrochemical method for direct antimicrobial susceptibility testing of GNB in positive blood cultures was evaluated. An aliquot (10 μl) of 1:10-diluted positive blood cultures containing GNB was inoculated into the Bactometer module well (bioMérieux Vitek, Hazelwood, Mo.) containing 1 ml of Mueller-Hinton broth supplemented with an antibiotic. Susceptibility tests were performed in a breakpoint broth dilution format, with the results being categorized as resistant, intermediate, or susceptible. Seven antibiotics (ampicillin, cephalothin, gentamicin, amikacin, cefamandole, cefotaxime, and ciprofloxacin) were used in this study, with each agent being tested at the two interpretive breakpoint concentrations. The inoculated modules were incubated at 35°C, and the change in impedance in each well was continuously monitored for 24 h by the Bactometer. The MICs of the seven antibiotics for each blood isolate were also determined by the standardized broth microdilution method. Of 146 positive blood cultures (1,022 microorganism-antibiotic combinations) containing GNB tested by the direct method, the rates of very major, major, and minor errors were 0, 1.1, and 2.5%, respectively. The impedance method was simple; no centrifugation, preincubation, or standardization of the inocula was required, and the susceptibility results were normally available within 3 to 6 h after inoculation. The rapid method may allow proper antimicrobial treatment almost 30 to 40 h before the results of the standard methods are available.     

Colistin Susceptibility Testing of Gram-Negative Bacilli: Better Performance of Vitek2 System than E-Test Compared to Broth Microdilution Method as the Gold Standard Test

Introduction: Unavailability of optimal susceptibility testing (ST) challenges the clinical use of colistin. Broth microdilution (BMD), which is the reference for colistin ST, is inconvenient for diagnostics. Vitek2 and E-test although technically easier, are no longer recommended. Materials and Methods: For the evaluation of Vitek2 and E-test in reference with BMD, a total of 138 Gram-negative bacilli (GNB) especially carbapenem-resistant isolates from Tata Medical Center, Kolkata, India, were included during 2017–2018. The evaluation was performed only for Enterobacteriaceae (n = 102), but not for non-fermentative GNB (n = 36) due to lack of colistin-resistant (COL^R) isolates. Results and Conclusion: Of 138 isolates, meropenem, colistin and dual resistance were detected in 110 (79.7%), 31 (22.5%) and 21 (15.2%) of isolates, respectively. Using the European Committee on Antimicrobial Susceptibility Testing guidelines (susceptible, ≤2 μg/ml), Vitek2 performed better than E-test (essential agreement, 92.2% vs. 63.7%; categorical agreement, 94.1% vs. 93.1%; very major error [VME], 10% vs. 23.3%). However, Vitek2 overcalled resistance than E-test (major error, 4.2% vs. 0%). Considering Chew et al. proposed breakpoints (susceptible, ≤1 μg/ml), VMEs declined for both test (6.7% vs. 10%), but still remained unacceptable. Of eight colistin-heteroresistant isolates, two VME were categorised by Vitek2, one VME was by E-test, and two were uninterpretable. Both Vitek2 and E-test are unreliable. Further studies correlating minimum inhibitory concentrations with clinical outcome are needed to determine the accurate breakpoints for better patient management.     

Clinical Impact of Pretransplant Multidrug-Resistant Gram-Negative Colonization in Autologous and Allogeneic Hematopoietic Stem Cell Transplantation

Multidrug-resistant Gram-negative bacteria (MDR-GNB) are an emerging cause of morbidity and mortality after hematopoietic stem cell transplantation (HSCT). Three-hundred forty-eight consecutive patients transplanted at our hospital from July 2012 to January 2016 were screened for a pretransplant MDR-GNB colonization and evaluated for clinical outcomes. A pretransplant MDR-GNB colonization was found in 16.9% of allo-HSCT and in 9.6% of auto-HSCT recipients. Both in auto- and in allo-HSCT, carriers of a MDR-GNB showed no significant differences in overall survival (OS), transplant-related mortality (TRM), or infection-related mortality (IRM) compared with noncarriers. OS at 2 years for carriers compared with noncarriers was 85% versus 81% (P?=?.262) in auto-HSCT and 50% versus 43% (P?=?.091) in allo-HSCT. TRM at 2 years was 14% versus 5% (P?=?.405) in auto-HSCT and 31% versus 25% (P?=?.301) in allo-HSCT. IRM at 2 years was 14% versus 2% (P?=?.142) in auto-HSCT and 23% versus 14% (P?=?.304) in allo-HSCT. In multivariate analysis, only grade III to IV acute graft-versus-host disease was an independent factor for reduced OS (P?<?.001) and increased TRM (P?<?.001) and IRM (P?<?.001). During the first year after transplant, we collected 73 GNB bloodstream infectious (BSI) episodes in 54 patients, 42.4% of which sustained by a MDR-GNB. Rectal swabs positivity associated with the pathogen causing subsequent MDR-GNB BSI episodes in 13 of 31 (41.9%). Overall, OS at 4 months from MDR-GNB BSI episode onset was of 67.9%, with a 14-day attributed mortality of 12.9%, not being significantly different between carriers and noncarriers (P?=?.207). We conclude that in this extended single-center experience, a pretransplant MDR-GNB colonization did not significantly influence OS, TRM, and IRM both in auto- and allo-HSCT settings and that MDR-GNB attributed mortality can be controlled in carriers when an early pre-emptive antimicrobial therapy is started in case of neutropenic fever.     

Major Variation in MICs of Tigecycline in Gram-Negative Bacilli as a Function of Testing Method

Tigecycline is one of the few remaining therapeutic options for extensively drug-resistant (XDR) Gram-negative bacilli (GNB). MICs of tigecycline to Acinetobacter baumannii have been reported to be elevated when determined by the Etest compared to determinations by the broth microdilution (BMD) method. The study aim was to compare the susceptibility of GNB to tigecycline by four different testing methods. GNB were collected from six health care systems (25 hospitals) in southeast Michigan from January 2010 to September 2011. Tigecycline MICs among A. baumannii, carbapenem-resistant Enterobacteriaceae (CRE), extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae, and susceptible Enterobacteriaceae isolates were determined by Etest, BMD, Vitek-2, and MicroScan. Nonsusceptibility was categorized as a tigecycline MIC of ≥4 μg/ml for both A. baumannii and Enterobacteriaceae. The study included 4,427 isolates: 2,065 ESBL-producing Enterobacteriaceae, 1,105 A. baumannii, 888 susceptible Enterobacteriaceae, and 369 CRE isolates. Tigecycline nonsusceptibility among A. baumannii isolates was significantly more common as determined by Etest compared to that determined by BMD (odds ratio [OR], 10.3; P < 0.001), MicroScan (OR, 12.4; P < 0.001), or Vitek-2 (OR, 9.4; P < 0.001). These differences were not evident with the other pathogens. Tigecycline MICs varied greatly according to the in vitro testing methods among A. baumannii isolates. Etest should probably not be used by laboratories for tigecycline MIC testing of A. baumannii isolates, since MICs are significantly elevated with Etest compared to those determined by the three other methods.     

A Universal Culture Medium for Screening Polymyxin-Resistant Gram-Negative Isolates

The colistin-containing SuperPolymyxin medium was developed for screening polymyxin-resistant Gram-negative bacteria. It was evaluated with 88 polymyxin-susceptible or polymyxin-resistant cultured Gram-negative isolates. Its sensitivity and specificity of detection were ca. 100%. The SuperPolymyxin medium is the first screening medium that is able to detect intrinsic and acquired polymyxin-resistant bacteria.     

Sensitivity of Surveillance Testing for Multidrug-Resistant Gram-Negative Bacteria in the Intensive Care Unit

We tested intensive care unit patients for colonization with multidrug-resistant Gram-negative bacilli (MDR GNB) and compared the results with those of concurrent clinical cultures. The sensitivity of the surveillance test for detecting MDR GNB was 58.8% (95% confidence interval, 48.6 to 68.5%). Among 133 patients with positive surveillance tests, 61% had no prior clinical culture with MDR GNB.     

Detection of Colonization by Carbapenemase-Producing Gram-Negative Bacilli in Patients by Use of the Xpert MDRO Assay

Detecting colonization of patients with carbapenemase-producing bacteria can be difficult. This study compared the sensitivity and specificity of a PCR-based method (Xpert MDRO) for detecting bla_KPC, bla_NDM, and bla_VIM carbapenem resistance genes using GeneXpert cartridges to the results of culture with and without a broth enrichment step on 328 rectal, perirectal, and stool samples. The culture method included direct inoculation of a MacConkey agar plate on which a 10-μg meropenem disk was placed and plating on MacConkey agar after overnight enrichment of the sample in MacConkey broth containing 1 μg/ml of meropenem. Forty-three (13.1%) samples were positive by PCR for bla_KPC and 11 (3.4%) were positive for bla_VIM; none were positive for bla_NDM. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the PCR assay for bla_KPC were 100%, 99.0%, 93.0%, and 100%, respectively, compared to broth enrichment culture and sequencing of target genes. The sensitivity, specificity, PPV, and NPV of the assay for bla_VIM were 100%, 99.4%, 81.8%, and 100%, respectively. Since none of the clinical samples contained organisms with bla_NDM, 66 contrived stool samples were prepared at various dilutions using three Klebsiella pneumoniae isolates containing bla_NDM. The PCR assay showed 100% positivity at dilutions from 300 to 1,800 CFU/ml and 93.3% at 150 CFU/ml. The Xpert MDRO PCR assay required 2 min of hands-on time and 47 min to complete. Rapid identification of patients colonized with carbapenemase-producing organisms using multiplex PCR may help hospitals to improve infection control activities.     

Performance of Three Commercial Assays for Colistin Susceptibility in Clinical Isolates and Mcr-1 Carrying Reference Strain

Objective: Commercially available antibiotic susceptibility tests (cAST) for colistin are reported to shows variable performance. The current controversy on the colistin susceptibility testing and scarce data from India has left the clinical laboratories in a dilemma on the appropriate and practical approach to tackle the colistin antimicrobial susceptibility testing (AST) issue. This study was aimed to evaluate the performance of commonly used cAST for colistin against broth microdilution (BMD) as the reference method in the clinical isolates. Materials and Methods: Colistin AST was performed on 225 nonduplicate isolates of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii by BMD as the reference method and compared with Vitek-2, Micronaut-S and E-test. The accuracy of the various cASTs was analysed by assessing categorical and essential agreement (EA). Results: We observed an overall categorical agreement of 98.2%, 99.6% and 96.4% and EA of 92%, 92.4% and 72% for Vitek-2, Micronaut-S and E-test, respectively. Unacceptable rates of major error (10.5%) and very major error (21%) were observed for P. aeruginosa with Vitek-2 and E-test, respectively. All the categorical errors (CEs) (7.7%) with Vitek-2 were seen for minimum inhibitory concentrations ranging within two-fold dilution breakpoint of 2 mg/L. Conclusion: Micronaut-S was found to be an acceptable method for colistin AST. In contrast, E-test was unreliable in terms of EA. Vitek-2 was found to be reliable for colistin AST, although it was more prone to CE near the colistin breakpoints.     

Comparison of Phenotypic and Genotypic Techniques for Identification of Unusual Aerobic Pathogenic Gram-Negative Bacilli

Rapid and accurate identification of bacterial pathogens is a fundamental goal of clinical microbiology, but one that is difficult or impossible for many slow-growing and fastidious organisms. We used identification systems based on cellular fatty acid profiles (Sherlock; MIDI, Inc., Newark, Del.), carbon source utilization (Microlog; Biolog, Inc., Hayward, Calif.), and 16S rRNA gene sequence (MicroSeq; Perkin-Elmer Applied Biosystems Division, Foster City, Calif.) to evaluate 72 unusual aerobic gram-negative bacilli isolated from clinical specimens at the Mayo Clinic. Compared to lengthy conventional methods, Sherlock, Microlog, and MicroSeq were able to identify 56 of 72 (77.8%), 63 of 72 (87.5%), and 70 of 72 (97.2%) isolates to the genus level (P = 0.002) and 44 to 65 (67.7%), 55 of 65 (84.6%), and 58 of 65 (89.2%) isolates to the species level (P = 0.005), respectively. Four Acinetobacter and three Bordetella isolates which could not be identified to the species level by conventional methods were identified by MicroSeq. In comparison to the full 16S rDNA sequences, the first 527 bp provided identical genus information for all 72 isolates and identical species information for 67 (93.1%) isolates. These data show that MicroSeq provides rapid, unambiguous identification of clinical bacterial isolates. The improved turnaround time provided by genotypic identification systems may translate into improved clinical outcomes.     

Rapid, Low-Technology MIC Determination with Clinical Mycobacterium tuberculosis Isolates by Using the Microplate Alamar Blue Assay

A colorimetric, microplate-based Alamar Blue assay (MABA) method was used to determine the MICs of isoniazid (INH), rifampin, streptomycin (SM), and ethambutol (EMB) for 34 Peruvian Mycobacterium tuberculosis isolates (including both pansensitive and multidrug-resistant strains) and the H₃₇Rv strain by using bacterial suspensions prepared directly from solid media. Results for all isolates were available within 8 days. Discordant results were observed on initial tests for 3 of 16 INH-susceptible isolates, 5 of 31 EMB-susceptible isolates, and 2 of 4 SM-resistant isolates (by the BACTEC 460 system). The overall agreements between the MICs obtained by MABA and the results obtained with the BACTEC 460 system were 87.9% for initial results and 93.6% after retesting 12 of 17 samples with discrepant results. Interpretation of MABA endpoints improved with technical experience. The MABA is a simple, rapid, low-cost, appropriate technology which does not require expensive instrumentation and which makes use of a nontoxic, temperature-stable reagent.     

Characterization of Clinical Multidrug-Resistant Escherichia coli and Klebsiella pneumoniae Isolates, 2007-2009, China

Various resistance mechanisms facilitate the emergence and spread of multidrug-resistance (MDR) phenotypes of Escherichia coli and Klebsiella pneumoniae. To elucidate the MDR mechanisms of E. coli and K. pneumoniae in China, we analyzed the antimicrobial susceptibilities of strains isolated from clinical samples in a large tertiary care hospital in Beijing, China, during 2007-2009 and characterized the isolates with a cefotaxime-ciprofloxacin-amikacin (CTX-CIP-AK) resistance pattern. In total, 98 and 52 clinical isolates of E. coli and K. pneumoniae, respectively, with a CTX-CIP-AK resistance pattern were subjected to antimicrobial susceptibility testing and screening of common β-lactamase genes, plasmid-mediated quinolone resistance (PMQR) genes, quinolone resistance-determining region (QRDR) substitutions, and 16S rRNA methylase genes by polymerase chain reaction amplification and DNA sequencing. Pulsed-field gel electrophoresis (PFGE) was used to determine the genetic relatedness of the isolates. Approximately 6.86% and 8.05% of the clinical E. coli and K. pneumoniae isolates, respectively, exhibited MDR phenotypes. The MDR K. pneumoniae isolates exhibited significantly higher ceftazidime resistance than the MDR E. coli isolates (90.4% vs. 76.5%, p=0.0339); a similar result was noted for piperacillin-tazobactam resistance (28.8% vs. 2%, p=0.0001). The common resistance determinants among the MDR E. coli and K. pneumoniae isolates were as follows: CTX-M (88.8% vs. 82.7%), PMQR genes (70.4% vs. 90.4%), gyrA mutations (100% vs. 90.4%), and 16S rRNA methylase genes (93.9% vs. 94.2%). Half (50%) of the MDR E. coli isolates belonged to phylogenetic group D, followed by group A (39.8%). For the E. coli isolates, 94 PFGE patterns and 23 clusters were identified, whereas 51 PFGE patterns and 11 clusters were identified for the K. pneumoniae isolates. Clinical E. coli and K. pneumoniae isolates seem to have a low prevalence of MDR phenotypes in China. The great genetic variation indicates a considerable transmission of common resistance determinants, including a high prevalence of QRDR substitutions in E. coli and K. pneumoniae.