Multicenter evaluation of rapid antimicrobial susceptibility testing by VITEK®2 directly from positive blood culture.

STUDY OBJECTIVE: To compare the antimicrobial susceptibility testing (AST) performance of positive blood cultures (PBC) VITEK®2 off-label use (D0) and traditional VITEK®2 workflow using isolated colonies after overnight (D1). METHODS: Patient samples with monomicrobial Gram-negative rod or Gram-positive cocci in clusters bacteremia were tested on D0 and compared to D1 AST results in 7 laboratories in France. RESULTS: Overall, categorical and essential agreement rates were 98.4% and 96.7%, respectively. Very major discrepancy and major discrepancy rates for Enterobacterales and Staphylococci satisfied the NF EN ISO 20776-2 (2007) criteria for sepsis-relevant drugs. Very major discrepancies were >3% for amoxicillin-clavulanate (4.9%, 6/122), piperacillin-tazobactam (7.5%, 4/53) and meropenem (33%,1/3) for Enterobacterales and gentamicin for Staphylococci (4.6%, 4/87). CONCLUSION: Direct AST from PBC broths by VITEK®2 for Enterobacterales and Staphylococci is reliable and fast and may positively influence antimicrobial stewardship.

Multicenter Clinical Evaluation of ETEST Eravacycline for Susceptibility Testing of Enterobacteriaceae and Enterococci.

Eravacycline (ERV) (brand name Xerava [Tetraphase]) is a new tetracycline-class antibacterial that has been approved by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for treatment of complicated intra-abdominal infections (cIAIs). ETEST is a gradient diffusion method that represents a simple alternative to the broth microdilution (BMD) method for performing antimicrobial susceptibility testing (AST). A multicenter evaluation of the performance of the new ETEST ERV (bioMérieux) in comparison with BMD was conducted following FDA and International Standards Organization (ISO) recommendations, using FDA- and EUCAST-defined breakpoints. Clinical isolates of Enterobacteriaceae (n = 542) and Enterococcus spp. (n = 137) were included. Based on the BMD reference method, 92 Enterobacteriaceae isolates and 9 enterococcal isolates were nonsusceptible to ERV according to the FDA breakpoints, while 7 Escherichia coli isolates and 3 Enterococcus sp. isolates were classified as ERV resistant according the EUCAST breakpoints. Referring to FDA performance criteria, the ETEST ERV demonstrated 99.4% and 100.0% essential agreement (EA), 98.0% and 94.9% categorical agreement (CA), very major error (VME) rates of 5.4% and 33.33%, and major error (ME) rates of 1.3% and 3.1% with clinical and challenge isolates, respectively, of Enterobacteriaceae and Enterococcus spp. According to EUCAST breakpoints, E. coli and Enterococcus sp. isolate results also met ISO acceptance criteria for EA and CA (EA of 99.0% and 100.0%, respectively, and CA of 100.0% for both), without any VMEs or MEs. In conclusion, we report that ETEST ERV represents an accurate tool for performing ERV AST of Enterobacteriaceae and Enterococcus sp. isolates.

Multicenter Clinical Evaluation of ETEST Plazomicin (PLZ) for Susceptibility Testing of Enterobacterales.

Plazomicin (PLZ), brand name ZEMDRI (Cipla Therapeutics), is a novel aminoglycoside antibiotic approved by the U.S. Food and Drug Administration (FDA) for treatment of complicated urinary tract infections including pyelonephritis. ETEST® is a gradient diffusion method that represents an alternative to the more laborious broth micro-dilution (BMD) method for performing antimicrobial susceptibility testing (AST). A multi-center evaluation of the performance of the new ETEST PLZ (bioMérieux) was conducted in comparison with BMD following FDA and International Standards Organization (ISO) recommendations using FDA-defined breakpoints. Clinical isolates of Enterobacterales (n = 598) were included. Fifty-three isolates were resistant to PLZ according to BMD. Overall, the ETEST PLZ demonstrated 99.0% essential agreement (EA), 92.8% category agreement (CA), 1.9% very major errors (VME), 0% major errors (ME), and 7.0% minor errors (mE) with both clinical and challenge isolates of Enterobacterales. The VME was found for a single Serratia marcescens strain. Individual species demonstrated EA rates ≥ 90%. In conclusion, we report that ETEST PLZ represents an accurate tool for performing PLZ AST of Enterobacterales.

A simple phenotypic test for detecting the contribution of outer membrane permeability to carbapenem resistance.

Introduction. The worldwide emergence of carbapenem resistance in Gram-negative bacteria makes the development of simple tests mandatory to identify antimicrobial resistance mechanisms. Enzymatic and membrane barriers are the prominent resistance mechanisms described in these bacteria. Several tests are currently used to detect carbapenemase activities.Aim. However, a simple test for the identification of membrane-associated mechanisms of resistance is not yet available and this mechanism is often inferred after the exclusion of a carbapenemase in carbapenem-resistant Gram-negative bacteria.Methodology. Different media (liquid and solid) containing a membrane permeabilizer were tested to identify the existence of a membrane barrier. Here, polymyxin B nonapeptide (PMBN) was selected to bypass the role of impermeability in clinical carbapenem-resistant Enterobacteriaceae, including Escherichia coli, Enterobacter cloacae , Klebsiella pneumoniae and Klebsiella aerogenes isolates. In parallel, the expression of porins (OmpC and OmpF types) was checked in the various bacterial strains in order to search for a correlation between the restoration of susceptibility and the expression of porin.Results. Using a large number of clinical isolates, PMBN associated with a carbapenem allowed us to detect porin-deficient isolates with a sensitivity ranging from 89 to 93 % and a specificity ranging from 86 to 100 %.Conclusion. This paves the way for a diagnostic assay allowing the detection of this membrane-associated mechanism of resistance in Enterobacteriaceae.

Multicenter Evaluation of the New Etest Gradient Diffusion Method for Piperacillin-Tazobactam Susceptibility Testing of Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii Complex.

Piperacillin-tazobactam (P/T) is a ß-lactam-ß-lactamase inhibitor combination frequently used in the hospital setting. Etest is a gradient diffusion method that represents an alternative to broth microdilution (BMD) for performing antimicrobial susceptibility testing. We conducted a multicenter evaluation of the performance of the new P/T Etest compared to that of BMD following U.S. Food and Drug Administration (FDA) and International Standards Organization (ISO) standard ISO 20776-2 criteria using Clinical and Laboratory Standards Institute (CLSI)-FDA and European Committee on Antimicrobial Susceptibility Testing (EUCAST) interpretive breakpoints, respectively. A total of 977 isolates (775 Enterobacterales isolates, 119 Pseudomonas aeruginosa isolates, and 83 Acinetobacter baumannii complex isolates) were tested. Overall essential agreement (EA) was 96.4% and 96.6% for Enterobacterales when FDA and ISO 20776-2 criteria, respectively, were followed. EA was 98.3% for P. aeruginosa and 91.6% for the A. baumannii complex when both the FDA and ISO criteria were followed. Applying CLSI-FDA breakpoints, categorical agreement (CA) reached 93.0%, 93.3%, and 89.2% for the Enterobacterales, P. aeruginosa, and the A. baumannii complex, respectively. Two very major errors (VMEs; 1.1%) were found among the Enterobacterales (for 2 Klebsiella pneumoniae isolates). No additional major errors (MEs) or VMEs were found. Applying EUCAST breakpoints, CA was 94.8% and 95.8% for Enterobacterales and P. aeruginosa, respectively (no breakpoints are currently available for the A. baumannii complex). No VMEs were observed among the Enterobacterales, but 2 (0.4%) MEs were found. Among the P. aeruginosa isolates, 2 (6.9%) VMEs and 3 (3.3%) MEs were observed. These errors resulted when P/T Etest MICs were 1 doubling dilution apart from the BMD MICs. In conclusion, the new P/T Etest represents an accurate tool for performing antimicrobial susceptibility testing of Enterobacterales, P. aeruginosa, and A. baumannii complex isolates with limited category errors.

Breakpoint to MIC quotient: A parameter to rapidly evaluate the in vitro bactericidal activity of ß-lactams on Enterobacteriaceae.

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) do not completely predict the bactericidal nature of the antimicrobial agent. Patients with pathogens having MICs near the clinical breakpoint experience a higher risk of clinical failure. This study defined an indicator, breakpoint to MIC quotient (BMQ), that incorporates MIC and the European Committee on Antimicrobial Susceptibility Testing breakpoint. The BMQ was inversely correlated with MBC in antibiotic combinations against Enterobacteriaceae strains (Spearman coefficient ≤ -0.96). This new parameter may provide timely additional insight for choosing an antibiotic for a severe bacterial infection.

Performance of CHROMID® Colistin R agar, a new chromogenic medium for screening of colistin-resistant Enterobacterales.

Recent emergence of transferable plasmid-borne colistin resistance (mcr genes) raised fear for pan-resistance. We evaluated the performance of a new chromogenic medium [CHROMID® Colistin R agar (COLR)] for the screening of colistin-resistant Enterobacterales. Specificity was evaluated using 89 rectal swabs and 89 stools prospectively collected. COLR sensitivity was evaluated by seeding 59 negative clinical samples artificially contaminated (105 CFU/mL) with 59 colistin-resistant Enterobacterales, including 20 mcr-1-positive strains. Twelve samples with an Enterobacterales with nonintrinsic resistance to colistin were recovered during the specificity study, including one mcr-1-positive Escherichia coli, representing a 6.7% prevalence of colistin resistance in fecal carriage. Overall, specificity was 100.0% [95% CI: 97.8-100.0] and sensitivity yielded 88.1% [95% CI: 77.5-94.1]. False negatives corresponded to 3 Enterobacter spp. (MIC>64 mg/L), 2 Salmonella spp. (MIC = 16 mg/L), 1 E. coli (MIC = 4 mg/L), and 1 K. pneumoniae (MIC = 8 mg/L). COLR appears to be a sensitive and specific chromogenic agar for screening colistin-resistant Enterobacterales, including those carrying mcr-1 gene.

Correlation between phenotypic antibiotic susceptibility and the resistome in Pseudomonas aeruginosa.

Genetic determinants of antibiotic resistance (AR) have been extensively investigated. High-throughput sequencing allows for the assessment of the relationship between genotype and phenotype. A panel of 672 Pseudomonas aeruginosa strains was analysed, including representatives of globally disseminated multidrug-resistant and extensively drug-resistant clones; genomes and multiple antibiograms were available. This panel was annotated for AR gene presence and polymorphism, defining a resistome in which integrons were included. Integrons were present in >70 distinct cassettes, with In5 being the most prevalent. Some cassettes closely associated with clonal complexes, whereas others spread across the phylogenetic diversity, highlighting the importance of horizontal transfer. A resistome-wide association study (RWAS) was performed for clinically relevant antibiotics by correlating the variability in minimum inhibitory concentration (MIC) values with resistome data. Resistome annotation identified 147 loci associated with AR. These loci consisted mainly of acquired genomic elements and intrinsic genes. The RWAS allowed for correct identification of resistance mechanisms for meropenem, amikacin, levofloxacin and cefepime, and added 46 novel mutations. Among these, 29 were variants of the oprD gene associated with variation in meropenem MIC. Using genomic and MIC data, phenotypic AR was successfully correlated with molecular determinants at the whole-genome sequence level.

Phylogenetic Distribution of CRISPR-Cas Systems in Antibiotic-Resistant Pseudomonas aeruginosa.

UNLABELLED: Pseudomonas aeruginosa is an antibiotic-refractory pathogen with a large genome and extensive genotypic diversity. Historically, P. aeruginosa has been a major model system for understanding the molecular mechanisms underlying type I clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (CRISPR-Cas)-based bacterial immune system function. However, little information on the phylogenetic distribution and potential role of these CRISPR-Cas systems in molding the P. aeruginosa accessory genome and antibiotic resistance elements is known. Computational approaches were used to identify and characterize CRISPR-Cas systems within 672 genomes, and in the process, we identified a previously unreported and putatively mobile type I-C P. aeruginosa CRISPR-Cas system. Furthermore, genomes harboring noninhibited type I-F and I-E CRISPR-Cas systems were on average ~300 kb smaller than those without a CRISPR-Cas system. In silico analysis demonstrated that the accessory genome (n = 22,036 genes) harbored the majority of identified CRISPR-Cas targets. We also assembled a global spacer library that aided the identification of difficult-to-characterize mobile genetic elements within next-generation sequencing (NGS) data and allowed CRISPR typing of a majority of P. aeruginosa strains. In summary, our analysis demonstrated that CRISPR-Cas systems play an important role in shaping the accessory genomes of globally distributed P. aeruginosa isolates. IMPORTANCE: P. aeruginosa is both an antibiotic-refractory pathogen and an important model system for type I CRISPR-Cas bacterial immune systems. By combining the genome sequences of 672 newly and previously sequenced genomes, we were able to provide a global view of the phylogenetic distribution, conservation, and potential targets of these systems. This analysis identified a new and putatively mobile P. aeruginosa CRISPR-Cas subtype, characterized the diverse distribution of known CRISPR-inhibiting genes, and provided a potential new use for CRISPR spacer libraries in accessory genome analysis. Our data demonstrated the importance of CRISPR-Cas systems in modulating the accessory genomes of globally distributed strains while also providing substantial data for subsequent genomic and experimental studies in multiple fields. Understanding why certain genotypes of P. aeruginosa are clinically prevalent and adept at horizontally acquiring virulence and antibiotic resistance elements is of major clinical and economic importance.

Rapid Bacterial Identification, Resistance, Virulence and Type Profiling using Selected Reaction Monitoring Mass Spectrometry.

Mass spectrometry (MS) in Selected Reaction Monitoring (SRM) mode is proposed for in-depth characterisation of microorganisms in a multiplexed analysis. Within 60-80 minutes, the SRM method performs microbial identification (I), antibiotic-resistance detection (R), virulence assessment (V) and it provides epidemiological typing information (T). This SRM application is illustrated by the analysis of the human pathogen Staphylococcus aureus, demonstrating its promise for rapid characterisation of bacteria from positive blood cultures of sepsis patients.

Role of the culture medium in porin expression and piperacillin-tazobactam susceptibility in Escherichia coli.

The continuing emergence of the multidrug resistance phenotype in Gram-negative bacteria makes the development of rapid susceptibility tests mandatory. To achieve this goal, proprietary specific media for bacterial growth can be used but may have some adverse effects. In this study, we dissected the role of media on porin, efflux pump and ß-lactamase expression. Depending on the medium used, we observed a change in piperacillin-tazobactam susceptibility for some isolates, such as increases in MIC values. No significant alteration in efflux activity or in ß-lactamase production was detected after changing the incubation medium. The ratio of piperacillinase:nitrocefinase showed no specific alteration, indicating that the various media did not affect significantly the relative enzymic affinity for the substrates. In contrast, osmotic variation was able to modulate both porin expression and OmpC : OmpF balance, thus modulating the antibiotic uptake. This study suggests that porin expression may be impacted by a susceptibility testing medium, which may modify the antibiotic diffusion into the bacteria, thus affecting MIC results.

Meropenem/colistin synergy testing for multidrug-resistant Acinetobacter baumannii strains by a two-dimensional gradient technique applicable in routine microbiology.

OBJECTIVES: Precise assessment of potential therapeutic synergy, antagonism or indifference between antimicrobial agents currently depends on time-consuming and hard-to-standardize in vitro chequerboard titration methods. We here present a method based on a novel two-dimensional antibiotic gradient technique named Xact™. METHODS: We used a test comprising a combination of perpendicular gradients of meropenem and colistin in a single quadrant. We compared test outcomes with those obtained with classical chequerboard microbroth dilution testing in a study involving 27 unique strains of multidrug-resistant Acinetobacter baumannii from diverse origins. RESULTS: We were able to demonstrate 92% concordance between the new technology and classical chequerboard titration using the A. baumannii collection. Two strains could not be analysed by Xact™ due to their out-of-range MIC of meropenem (>128 mg/L). CONCLUSIONS: The new test was shown to be diagnostically useful, easy to implement and less labour intensive than the classical method.

Population analysis of Escherichia coli isolates with discordant resistance levels by piperacillin-tazobactam broth microdilution and agar dilution testing.

Population analysis was performed for 42 Escherichia coli isolates to determine whether heterogeneity of resistance was a factor in piperacillin-tazobactam category differences between agar dilution and broth microdilution. Of 20 isolates discordant between methods, 80% were heterogeneous. Of 22 isolates in agreement, 59% were homogeneous. Heterogeneity and homogeneity rates for those in agreement were significantly different from those that were discordant (P value, 0.010). Heterogeneity of resistance expression appears to be an important factor in category differences observed between broth microdilution and agar dilution for piperacillin-tazobactam.

Comparative evaluation of a novel chromogenic medium (chromID OXA-48) for detection of OXA-48 producing Enterobacteriaceae.

Comparative evaluation of the recently developed chromogenic culture medium chromID OXA-48 (bioMérieux) with chromID CARBA (bioMérieux) and SUPERCARBA showed that chromID OXA-48 and SUPERCARBA media have the highest sensitivity for detection of OXA-48 producing Enterobacteriaceae (91% and 93%) comparatively to chromID CARBA (21 %). The chromID OXA-48 has the highest specificity, with 100%, as compared to 53% and 68% for the SUPERCARBA and chromID CARBA media for detecting those OXA-48 producers.

Evaluation of Etest® strips for detection of KPC and metallo-carbapenemases in Enterobacteriaceae.

The performance of Etest KPC and MBL strips (bioMérieux) was evaluated as compared to other phenotypic tests for detecting carbapenemases of the KPC-type and metallo-ß-lactamases, respectively, on 133 well-characterized enterobacterial isolates. KPC and meropenem-containing MP/MPI Etest had high sensitivity (>92 %) and specificity (>97 %).

Rapid clinical bacteriology and its future impact.

Clinical microbiology has always been a slowly evolving and conservative science. The sub-field of bacteriology has been and still is dominated for over a century by culture-based technologies. The integration of serological and molecular methodologies during the seventies and eighties of the previous century took place relatively slowly and in a cumbersome fashion. When nucleic acid amplification technologies became available in the early nineties, the predicted "revolution" was again slow but in the end a real paradigm shift did take place. Several of the culture-based technologies were successfully replaced by tests aimed at nucleic acid detection. More recently a second revolution occurred. Mass spectrometry was introduced and broadly accepted as a new diagnostic gold standard for microbial species identification. Apparently, the diagnostic landscape is changing, albeit slowly, and the combination of newly identified infectious etiologies and the availability of innovative technologies has now opened new avenues for modernizing clinical microbiology. However, the improvement of microbial antibiotic susceptibility testing is still lagging behind. In this review we aim to sketch the most recent developments in laboratory-based clinical bacteriology and to provide an overview of emerging novel diagnostic approaches.

Comparative evaluation of a prototype chromogenic medium (ChromID CARBA) for detecting carbapenemase-producing Enterobacteriaceae in surveillance rectal swabs.

Carbapenemase-producing Enterobacteriaceae (CPE) are an increasing problem worldwide, and rectal swab surveillance is recommended as a component of infection control programs. The performance of a prototype chromogenic medium (chromID CARBA) was evaluated and compared with media tested by four other screening methods: (i) overnight selective enrichment in 5 ml tryptic soy broth with a 10-µg ertapenem disk followed by plating onto MacConkey agar (CDC-TS), (ii) short selective enrichment in 9 ml brain heart infusion broth with a 10-µg ertapenem disk followed by plating onto chromID ESBL medium (ESBL-BH), (iii) direct plating onto chromID ESBL, and (iv) direct plating onto MacConkey agar supplemented with meropenem (1 µg/ml) (MCM). The screening methods were applied to detect CPE in 200 rectal swab specimens taken from different hospitalized patients. Identification and antimicrobial susceptibility were performed by the Vitek 2 system. Carbapenem MICs were checked by Etest. Carbapenemase production was confirmed using the modified Hodge test, combined-disk tests, and PCR assays. In total, 133 presumptive CPE strains were detected. Phenotypic and genotypic assays confirmed 92 strains to be CPE (56 KPC-positive Klebsiella pneumoniae, 29 VIM-positive K. pneumoniae, and 7 KPC-positive Enterobacter aerogenes strains) recovered from 73 patients, while the remaining 41 strains were confirmed to be CPE negative (19 ESBL producers and 22 nonfermenters). chromID CARBA, ESBL-BH, and chromID ESBL exhibited the highest sensitivity (92.4%), followed by CDC-TS and MCM (89.1%) (P = 0.631). The specificity was greater for chromID CARBA (96.9%) and ESBL-BH (93.2%) than for CDC-TS (86.4%), MCM (85.2%), and chromID ESBL (84.7%) (P = 0.014). In conclusion, chromID CARBA was found to be a rapid and accurate culture screening method for active CPE surveillance.

Methicillin-resistant Staphylococcus aureus expressing low-level methicillin resistance may not be detected by the VITEK2® system.

Low-level methicillin-resistant Staphylococcus aureus may be difficult to detect with the VITEK® 2 system (VK2). Here, we suggest that S. aureus exhibiting VK2-oxacillin MIC of 1 or 2 mg/L and a negative cefoxitin screen should be tested for the presence of mecA or its gene product.