key: cord-0023389-zid9owoz
authors: Boattini, Matteo; Bianco, Gabriele; Comini, Sara; Iannaccone, Marco; Casale, Roberto; Cavallo, Rossana; Nordmann, Patrice; Costa, Cristina
title: Direct detection of extended-spectrum-β-lactamase-producers in Enterobacterales from blood cultures: a comparative analysis
date: 2021-11-25
journal: Eur J Clin Microbiol Infect Dis
DOI: 10.1007/s10096-021-04385-1
sha: 5784f94c517705d1b5271dd383c2b4041882c686
doc_id: 23389
cord_uid: zid9owoz

Accurate detection of extended-spectrum-β-lactamase (ESBL)-producing Enterobacterales from bloodstream infection (BSI) is of paramount importance for both epidemiological and clinical purposes, especially for optimization of antibiotic stewardship interventions. Three phenotypic methods for the detection of ESBL phenotype in Klebsiella pneumoniae and Escherichia coli BSI were compared over a 4-month period (May–August 2021) in a main University Hospital from Northern Italy. The methods were the biochemical Rapid ESBL NP®, the immunological NG-Test CTX-M MULTI®, and the E-test technique based on ESBL E-test®. One hundred forty-two blood cultures (BCs) positive for K. pneumoniae or E. coli were included. ESBL and carbapenemase phenotype were detected in 26.1% (n = 37) and 16.9% (n = 24), respectively. The Rapid ESBL NP®, NG-Test CTX-M MULTI®, and direct ESBL E-test® positive and negative predictive values with 95% confidence intervals were 1 (0.87–1) and 0.97 (0.92–0.99), 1 (0.87–1) and 0.97 (0.92–0.99), and 1 (0.88–1) and 1 (0.96–1), respectively. The three phenotypic methods evaluated showed good performance in the detection of ESBL phenotype from K. pneumoniae– or E. coli–positive BCs. Rapid ESBL NP® and NG-test CTX-M® offer the important advantage of a turnaround time of 15 to 45 min, and the Rapid ESBL NP test in addition detects any type of ESBL producers.

Extended-spectrum-β-lactamase (ESBL)-producing Enterobacterales (EB) bloodstream infections (BSI) represent a worldwide clinical issue, especially given their association with multidrug resistance, severity of illness, poor outcomes, and growing number in the community [1] [2] [3] . The spread of plasmids carrying CTX-M-type genes in the community beginning mostly in the 2000s is the main driver of ESBL dissemination in EB and replaced other ESBL enzymes (i.e., mostly TEM, SHV derivatives) that were mostly identified in hospital-acquired EB infections [1, 4, 5] . However, given the heterogeneity of ESBL family, other-than-CTX-M-type enzymes have been also reported as a source of outbreaks of ESBL producers [6] . Therefore, accurate detection of ESBLs is of paramount importance for both epidemiological and clinical purposes, given the ability of ESBLs of hydrolyzing penicillins, monobactams, and cephalosporins. Several phenotypic tests are available for ESBL detection [7] [8] [9] . They are mostly performed on bacterial colonies and associated with non-negligible turnaround time (TAT) contributing Matteo Boattini and Gabriele Bianco contributed equally to this work.

* Matteo Boattini matteo.boattini@unito.it to delay obtention of results in critical scenarios as that of management of BSI patients. Various phenotypic tests that can detect ESBLs have been evaluated directly from EBpositive blood cultures (BCs) to provide result on the same day of positive BC processing with variable specificity and sensitivity [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] . Although the benefit in reducing TAT is recognized [12, 23] , evidence of the detection of ESBL producers directly from EB-positive BCs in clinical routine are limited. Therefore, this study was aimed at evaluating the performance of three methods for direct detection of ESBL producers from BSI, none of them being molecular based and all of them being very recently commercialized. 

The bacterial pellets of positive BCs with Gram-negative bacilli obtained using the MBT Sepsityper IVD Kit (Bruker DALTONIK GmbH, Bremen, Germany) from 1 mL of positive BC broth underwent MALDI-TOF MS analysis using the MALDI BioTyper system in accordance with the manufacturer's instructions (Bruker DALTONIK GmbH, Bremen, Germany). In case of identification of Klebsiella pneumoniae or Escherichia coli, three phenotypic methods were used in parallel for the detection of ESBL phenotype and results were compared with conventional culture-based result over a 4-month period (May-August 2021). The Rapid ESBL NP® (Liofilchem, Roseto degli Abbruzzi, Italy) is a colorimetric cefotaxime hydrolysis-based assay able to detect the presence/absence of any type of ESBLs or the presence of an enzyme or combination of enzymes that can hydrolyze cefotaxime, but which is not inhibited by the addition of tazobactam (i.e., cephalosporinase, ESBL + cephalosporinase, carbapenemase with or without an ESBL) [25, 26] . Rapid ESBL NP® test was performed using the bacterial pellet recovered with MBT Sepsityper IVD Kit from 1 mL of positive BC broth, as previously described [11, 12, 27] . Briefly, after keeping test panel at room temperature for 10 min, 400 μL of lysis buffer was added to the bacterial pellet and vortexed for 5 s. After 15 min, 100 μL of the solution obtained was dispensed into each well (A, B, and C) of the test cassette. Then, the panel was covered with the lid provided and incubated at 36 ± 2 °C for 20 min in ambient air. Rapid ESBL NP test results were read within 20 min. Positive result for ESBL phenotype was considered if wells A and C remained red and well B turned orange/yellow (Fig. 1a) .

The NG CTX-M MULTI® assay (NG Biotech, Guipry, France) is a lateral flow immunoassay exploiting monoclonal antibodies specific for the specific detection of CTX-M-type (groups 1, 2, 8, 9, and 25) ESBL enzymes only [10] [11] [12] . As opposed to the Rapid ESBL NP test, this technique is not aimed to identify all types of ESBL producers. NG CTX-M MULTI® assay was also performed using the bacterial pellet recovered with MBT Sepsityper IVD Kit from 1 mL of positive BC broth. Briefly, five drops of lysis buffer provided with the kit were added to bacterial pellet and vortexed for 5 s. One hundred microliters was added to the sample well of the test cassette. Assay results were read within 15 min. Positive result for ESBL phenotype was based on the presence of visible line specific for CTX-M ESBL enzyme (Fig. 1c) .

The E-test® (bioMérieux, Marcy l'Étoile, France) is an antimicrobial gradient method that combines the principle of dilution methods with that of diffusion methods to determine the MIC value. Both ESBL E-test® ceftazidime ± clavulanic acid and cefotaxime ± clavulanic acid (CTX/CTXL) are the antimicrobial agents required for ESBL confirmation. To confirm the presence of ESBLs in isolates with high-level expression of AmpC β-lactamases, it is recommended that an additional ESBL confirmation test is performed with cefepime as the indicator cephalosporin, as cefepime is usually not hydrolyzed by AmpC β-lactamases. Direct ESBL E-test® was performed modifying EUCAST inoculum recommendations for rapid AST from positive BCs (https:// www. eucast. org/ filea dmin/ src/ media/ PDFs/ EUCAST_ files/ RAST/ EUCAST_ RAST_ metho dology_ v1.1_ Final. pdf). In this modified procedure of E-test®, 200 μL of positive BC broth was mixed to 200 μL of normal saline and the total suspension was inoculated and spread gently on a cation-adjusted MHA plate. MHA surface was allowed to dry under the fume hood for 2 min. Then, a CTX/CTXL and cefepime ± clavulanic acid (PM/PML) strips were deposited on the agar surface and the plate was incubated at 37 °C in 5% CO 2 . Direct ESBL E-test® results were read after 5 h of incubation only if the growth was confluent and zone edges were clearly visible. Growth of microcolonies inside the entire inhibition zone was ignored. The test was considered positive for ESBL phenotype if ≥ eightfold reduction was observed in the MIC of CTX and/or PM combined with clavulanic acid compared with the MIC of the CTX and/or PM alone or if a phantom zone or deformed ellipse was present (Fig. 1e) .

All identified isolates were further analyzed with a multiplex real-time polymerase chain reaction assay specific for blaCTX-M-like genes (ESBL ELITe MGB Kits, ELITechGroup Molecular Diagnostics, Turin, Italy) as previously described [28] .

In case of discordant result between the direct phenotypic methods and conventional culture-based diagnostics, phenotypic tests were performed on bacterial colony.

Descriptive data are shown as absolute (n) and relative (%) frequencies for categorical data. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the three phenotypic methods for direct detection of ESBL phenotype from positive BCs with 95% confidence interval (95% CI) were computed using the online calculator at http:// vassa rstats. net/ clin1. html.

Over the study period, 242 positive BCs deemed representative of a single Gram-negative bacilli BSI event were processed. Of these, 58.7% (n = 142) were associated with K. pneumoniae (n = 62) or E. coli (n = 80) and were included in the study (Table 1 ). ESBL and carbapenemase phenotype were detected in 26.1% (n = 37) and 16.9% (n = 24), respectively. All the isolates displaying a carbapenemase phenotype were K. pneumoniae, being KPC (95.8%, n = 23) the most prevalent enzyme followed by VIM type (4.2%, n = 1). Among KPC producers, 21.7% (n = 5) were co-producers of CTX-M.

Among the three methods for direct detection of ESBL phenotype, the best performance was obtained by direct ESBL E-test® that showed no false-negative/positive results when combining the results of the CTX/CTXL and PM/PML strips ( (Table 2 ). In fact, for both the tests, sensitivity, specificity, and positive and negative predictive values with 95% confidence intervals were 0.92 (0.77-0.98), 1 (0.96-1), 1 (0.87-1), and 0.97 (0.92-0.99). The falsenegative results obtained with the Rapid ESBL NP® were as follows: two were positive for CTX-M according to the PCR results while one presented with both bacterial pellets and colony with hypermucoid phenotype and PM, CTX, and CAZ MICs of 4 mg/L, > 32 mg/L, and > 32 mg/L, respectively. By repeating the tests from overnight subcultures, one of the isolates carrying CTX-M tested positive and showed PM, CTX, and CAZ MICs of 2 mg/L, 32 mg/L, and 2 mg/L, respectively, while the other two isolates tested negative. Moreover, the Rapid ESBL NP® failed to provide indications on the presence of other combined mechanisms of cephalosporin resistance in four cases: one KPC-producing K. pneumoniae isolate; one CZA-resistant KPC-producing K. pneumoniae isolate with PM, CTX, and CAZ MICs of 8 mg/L, 4 mg/L, and > 32 mg/L, respectively; one VIM-producing K. pneumoniae; and one AmpC-producing E. coli with PM, CTX, and CAZ MICs of ≤ 0.5 mg/L, 4 mg/L, and 32 mg/L, respectively. However, by repeating the Rapid ESBL NP® tests from overnight subcultures, only the CZA-resistant KPC-producing K. pneumoniae isolate remained negative.

The three false-negative results obtained with the NG CTX-M MULTI® were all negative for CTX-M PCR. Of these, one was the same hypermucoid K. pneumoniae strain mentioned above.

Technical comparison of the three methods used for direct detection of ESBL phenotype from K. pneumoniae-or E. coli-positive BCs is summarized in Table 3 . ESBL phenotype could be detected within 40 to 45 min (Rapid ESBL NP®), 15 to 20 min (NG CTX-M MULTI®), or 5 h (direct ESBL E-test®).

The global CTX-M pandemic underlines the need for rapid ESBL detection to accelerate clinical decision-making and infection control measures and finally contribute to improve patient outcomes. Our data make obvious the high prevalence of ESBL producers in K. pneumoniae and E. coli BSI in our hospital located in the northern part of Italy, confirming the relevant burden of resistance rates to expanded-spectrum cephalosporins in those species in Italy compared to Europe [29] . Therefore, we compared the performance of two rapid tests, the biochemical-based Rapid ESBL NP® test very recently commercialized and lateral flow immunoassay NG CTX-M MULTI® for the detection of ESBL producers from BSI and compared their results with the direct ESBL E-test® performed also from BC samples. Our results demonstrated that with the [15] . Similarly, we excluded the direct β-lactam inactivation method despite its claimed sensitivity and specificity, both being 100% since it remains a homemade technique with a long TAT [18, 19] . We did not also evaluate MALDI-TOF MS-based approach for the detection of ESBL producers (sensitivity 91.1-100% and specificity 91.5-100%) since it required equipment and careful adaptation for its implemetation in routine microbiology [20] [21] [22] [23] .

The direct ESBL E-test® allows a reliable detection of ESBL phenotype but with a TAT unsuitable for antibiotic management of BSI patients. The Rapid ESBL NP® and NG CTX-M MULTI® also allowed a reliable phenotypic detection of ESBL producers with a TAT ranging from 15 to 45 min compatible with antibiotic stewardship. In addition, the Rapid ESBL NP® test offers the advantage to detect any type of ESBL (and not only CTX-M) and to identify combined mechanisms such as carbapenemase production. This is an important point to consider for implementing a carbapenem-containing therapy.

Based on these results, the following strategy for ESBL phenotype detection from K. pneumoniae-and E. coli-positive BC is proposed. Klebsiella spp.-or E. coli-positive BCs should be screened with the NG CTX-M MULTI® assay or the Rapid ESBL NP®. In countries where the prevalence of other-than-CTX-M-type ESBL enzymes is important such as those from Asia, South America, or Middle East, the Rapid ESBL NP® offers the advantage to detect any kind of ESBL producers. In addition, one shall be aware that false detection of ESBL has been noted by using the NG CTX-M MULTI® assay for several K. oxytoca strains that possess specific naturally occurring ESBLs of OXY type [30] . All the tests evaluated are low cost and require no specialized equipment or personnel; further studies on cost-benefit analysis are needed however.

In conclusion, the three phenotypic methods evaluated showed good performance for the detection of ESBL phenotype directly from K. pneumoniae-and E. coli-positive BCs. The rapid ESBL tests directly from BC samples might be implemented worldwide in particular for patients hospitalized in acute care facilities for promptly optimizing their antibiotic therapy.

Emergence of Enterobacteriaceae producing extended-spectrum betalactamases (ESBLs) in the community

Epidemiology and clinical features of community-onset bacteremia caused by extended-spectrum β-lactamase-producing Klebsiella pneumoniae

Clinical significance of extended-spectrum beta-lactamases

Global epidemiology of CTX-M β-lactamases: temporal and geographical shifts in genotype

Longitudinal analysis of ESBL and carbapenemase carriage among Enterobacterales and Pseudomonas aeruginosa isolates collected in Europe as part of the International Network for Optimal Resistance Monitoring (INFORM) global surveillance programme, 2013-17

Epidemiology of β-lactamaseproducing pathogens

Recent advances in biochemical and molecular diagnostics for the rapid detection of antibiotic-resistant Enterobacteriaceae: a focus on ß-lactam resistance

Detection of multidrugresistant Enterobacterales-from ESBLs to carbapenemases

Extended-spectrum β-lactamases: an update on their characteristics, epidemiology and detection

A lateral flow immunoassay for the rapid identification of CTX-M-producing Enterobacterales from culture plates and positive blood cultures

Evaluation of the NG-Test CTX-M MULTI immunochromatographic assay for the rapid detection of CTX-M extendedspectrum-β-lactamase producers from positive blood cultures

Fast-track identification of CTX-Mextended-spectrum-β-lactamase-and carbapenemase-producing Enterobacterales in bloodstream infections: implications on the likelihood of deduction of antibiotic susceptibility in emergency and internal medicine departments

Evaluation of the β-Lacta test for detection of extended-spectrum-β-lactamase (ESBL)-producing organisms directly from positive blood cultures by use of smudge plates

Early detection of extended-spectrum β-lactamase from blood culture positive for an Enterobacteriaceae using βLACTA test

Comparison of three biochemical tests for rapid detection of extended-spectrumβ-lactamase-producing Enterobacteriaceae

Rapid detection of extended-spectrum-β-lactamase-producing Enterobacteriaceae

Rapid detection of ESBLproducing Enterobacteriaceae in blood cultures

Direct β-lactam inactivation method: a new lowcost assay for rapid detection of carbapenemase-or extendedspectrum-β-lactamase-producing Enterobacterales directly from positive blood culture bottles

Direct Ethylenediaminetetraaceticacid-modified β-lactam inactivation method: an improved method to identify serine-carbapenemase-, metallo-β-lactamase-, and extendedspectrum-β-lactamase-producing Enterobacterales directly from positive blood culture

Rapid detection of Enterobacteriaceae producing extended-spectrum-β-lactamases directly from positive blood cultures by matrix-assisted laser desorption ionization-time of flight mass spectrometry

Evaluation of matrix-assisted laser desorption ionizationtime of flight mass spectrometry for rapid detection of β-lactam resistance in Enterobacteriaceae derived from blood cultures

Performance of a MALDI-TOF mass spectrometry-based method for rapid detection of third-generation oxymino-cephalosporin-resistant Escherichia coli and Klebsiella spp. from blood cultures

Rapid identification and detection of β-lactamase-producing Enterobacteriaceae from positive blood cultures by MALDI-TOF/MS

Rapid ESBL NP test for rapid detection of expandedspectrum β-lactamase producers in Enterobacterales

Evaluation of the performance of rapid tests for screening carriers of acquired ESBL-producing Enterobacterales and their impact on turnaround time

RESIST-5 O.O.K.N.V. and NG-Test Carba 5 assays for the rapid detection of carbapenemase-producing Enterobacterales from positive blood cultures: a comparative study

Evaluation of the CRE and ESBL ELITe MGB® kits for the accurate detection of carbapenemase-or CTX-M-producing bacteria

Trends of major antimicrobial resistance phenotypes in Enterobacterales and gram-negative non-fermenters from ATLAS and EARS-net surveillance systems: Italian vs. European and global data

False immunological detection of CTX-M enzymes in Klebsiella oxytoca