Abstract
Objectives:
To evaluate the biochemical Rapid ESBL NP® (Liofilchem, Italy) for the rapid detection of extended-spectrum β-lactamases (ESBLs) in Enterobacterales.
Methods:
A total of 100 clinical gram-negative strains (40 ESBL producers with or without cephalosporinase, 43 carbapenemase producers with or without additional ESBLs, 8 AmpC-type producers, 6 penicillinase producers, and 3 non-β-lactamase producers) were tested using this colorimetric technique.
Results:
The overall sensitivity and specificity of the test were found to be 93.9% and 98.5%, respectively. This test is rapid (turn-around-time of 40–45 minutes), easy to perform, and reliable for identification of ESBL producers.
Conclusion:
The Rapid ESBL NP test allows differentiation between ESBL producers on one hand, and non-ESBL producers or isolates expressing combined mechanisms of resistance on the other hand.
Multidrug resistance in Enterobacterales represents a serious threat to public health, since the accumulation of resistance determinants may be the source of difficult-to-treat infections in humans. One of the most important resistance traits is plasmid-mediated resistance to broad-spectrum cephalosporins through production of extended-spectrum β-lactamases (ESBLs). ESBLs hydrolyze oxyiminocephalosporins and aztreonam, while being inhibited by β-lactamase inhibitors such as clavulanic acid and tazobactam. 1 The most frequent ESBLs are CTX-M-type enzymes, followed by TEMs and SHVs, and to a lesser extent PER, VEB, and GES variants. 2
ESBL-producing Enterobacterales constitute a major source of hospital-acquired infections (bacteremia mainly caused by Klebsiella pneumoniae) and community infections (urinary tract infections mainly caused by Escherichia coli). 3 Therefore, the rapid identification of this resistance trait is important to optimize the treatment of infections caused by ESBL producers and to prevent the spread of ESBL producers in nosocomial settings.
Several techniques have been developed to identify ESBL producers, such as phenotypic techniques based on the inhibition of ESBL activity by clavulanic acid or tazobactam. 4 Those techniques require a preliminary growth step of 24–48 hours. 5 Molecular detection of ESBL-encoding genes is also interesting but remains costly, requires expertise, and detects only known ESBL-encoding genes. 5
Other techniques, such as those adapted from the matrix-assisted laser desorption ionization-time of flight mass spectrometry, are being developed, but they do require additional material and a significant degree of expertise. 5 The β-Lacta test (Bio-Rad, Cressier, Switzerland) has been developed and its principle is based on detection of hydrolysis of a chromogenic cephalosporin, namely HMRZ-86. 6 Indeed, the β-Lacta test turns positive when either ESBL, cephalosporinase, or carbapenemase enzymes are produced, considering that all those enzymes basically hydrolyze HMRZ-86. The recently introduced immunological detection of ESBLs is rapid, sensitive, and specific (NG-CTX-M [NG Biotech, France], e.g.), but detects only ESBLs of the CTX-M types. 7
Therefore, developing tests based on biochemical detection of any type of ESBL activity is quite interesting. For that purpose, the Rapid ESBL NDP had been developed based on the detection of hydrolysis of the cefotaxime β-lactam ring revealing the production of a broad-spectrum β-lactamase, coupled with a tube containing tazobactam that inhibits the ESBL activity, signaling ESBL production.8–10 This colorimetric test is based on the change of color of the pH indicator by acidification of the medium. The hydrolysis of cephalosporin, here cefotaxime, gives a carboxyl group, which decreases the pH. The red phenol present in the reagent of the test subsequently turns yellow upon acidification of the pH.
A commercial version of this test, the Rapid ESBL NP® (Liofilchem, Roseto degli Abruzzi, Italy), is now available, based on mostly on the same protocol as the home-made Rapid ESBL NDP test. It can identify any ESBL producer in 40–45 minutes, regardless of the ESBL type. The aim of our study was to evaluate this novel Rapid ESBL NP test using a collection of ESBL-producing isolates.
A well-characterized panel of 100 clinical enterobacterial strains (40 ESBL producers with or without additional AmpC-type β-lactamases, 43 carbapenemase producers with or without additional ESBL, 8 AmpC-type producers, 6 penicillinase producers, and 3 non-β-lactamase producers) from the collection of clinical strains of the Medical and Molecular Microbiology Unit, Faculty of Science and Medicine, University of Fribourg, Switzerland, was tested. All strains were characterized for their β-lactamase content at their molecular level (polymerase chain reaction and sequencing) before any testing.
The antimicrobial resistance profiles of the strains were determined by disk diffusion according to EUCAST guidelines (www.eucast.org/clinical_breakpoints/); when needed, precise determination of minimal inhibitory concentration (MIC) values was performed by using the MIC test strip technique (Liofilchem). 11
Rapid ESBL NP
The Rapid ESBL NP assay was performed according to the recommendations of the manufacturer from fresh overnight bacterial colonies grown on selective or nonselective agar plates at 37°C. URISelect™ 4 agar plates (Bio-Rad) were used in this study. One to two full calibrated loops (10-μL loop) of bacterial colonies were suspended in 400 μL of lysis buffer. After 15 minutes, 100 μL of suspension was dispensed in three wells (A, B, and C) of the gallery. Well A does not contain antibiotic. Well B contains cefotaxime and well C contains cefotaxime and tazobactam.
The results were interpreted according to the recommendations of the manufacturer (Fig. 1) as follows. When all wells A, B, and C remain red, no ESBL is produced (negativity). When wells A and C remain red and well B turns orange/yellow, an ESBL is produced (positivity). When well A remains red and wells B and C both turn orange/yellow, different possibilities are possible, namely the production of an AmpC-type β-lactamase, coproduction of ESBL+AmpC, or production of a carbapenemase with or without an additional ESBL and/or AmpC (undetermined result) (Fig. 1). Production of an AmpC β-lactamase may be detected by comparing susceptibility results with β-lactams, obtained after an additional 18-hour culture step on media containing or not cloxacillin, 4 although carbapenemase detection may be performed by using the Rapid Carba NP test. 12
Rapid ESBL NP test. Positive result (Klebsiella pneumoniae producing CTX-M-15, raw 1); negative result (Escherichia coli wild type, raw 2); undetermined (K. pneumoniae producing NDM-1, raw 3). ESBL, extended-spectrum β-lactamases. Color images are available online.
Reading of the results of the test was done after 20 minutes of incubation at 37°C. All strains have been tested in duplicate. Around 2 minutes per isolate was needed for sample preparation, with a total turn-around time of ∼40 minutes to obtain the result.
The Rapid ESBL NP test could detect 31 out of 33 ESBL producers (Table 1). Two E. coli strains producing SHV-12 and VEB-1 with low expression levels of their ESBL were not detected. MIC values of cefotaxime were 1 and 2 μg/mL, respectively, for the SHV-12 and the VEB-1 strains, indicating that those strains, despite being ESBL producers, were still susceptible to cefotaxime, which is the substrate for ESBL detection of this test. No carbapenemase producer was detected as an ESBL producer. All the AmpC producers that coproduced or not an ESBL did not give a positive result that would subsequently sign an ESBL production, except for a single DHA-1 producer that coproduced the SHV-11ESBL. That strain was susceptible to cefotaxime, with an MIC value of 0.25 mg/L.
Results of the Rapid ESBL NP with Enterobacterales
ESBL, extended-spectrum β-lactamases.
Altogether, the sensitivity and specificity of the Rapid ESBL NP test were 93.9% and 98.5%, respectively. The main advantage of this test is the ability to detect all types of ESBLs, being either known or unknown, and clearly differentiate isolates that only produce an ESBL from those exhibiting other multidrug resistance patterns. This test is, therefore, more specific than the β-Lacta test that detects all strains hydrolyzing the cephalosporin HMRZ-86, corresponding not only to ESBL producers, but also to AmpC or carbapenemase producers.
By testing 30 strains producing or not an ESBL using different culture media such as Drigalski, Columbia blood agar, or the ESBL screening medium, namely ChromID ESBL (bioMérieux), the ESBL detection was found to be optimal after culture on the Columbia blood and ChromID ESBL selective media. Prior culture on Drigalski medium before testing gave totally inconsistent results with the Rapid ESBL NP test (data not shown).
Use of the Rapid ESBL NP test will be of clinical value in particular for antibiotic stewardship in the context of bacteremia to identify patients deserving a carbapenem-containing therapy. 13
The Rapid ESBL NP test may be also interesting for immediate isolation of the patient's carriers of ESBL producers before waiting for the results of antibiogram (24-hour delay). The excellent positive predictive value of the Rapid ESBL NP test is also a crucial feature. Actually, strains that co-produced a CTX-M enzyme along with a carbapenemase (e.g., KPC) may be misidentified as a CTX-M-only producing isolate by using the NG-CTX-M test.
Similarly, the use of molecular techniques for identification of ESBL encoding genes is limited to screening of few ESBL genes, thus leading to false-negative results when some minor ESBL enzymes not included in the screening pool are produced. Therefore, use of molecular techniques for detection of ESBL producers shall gather not only ESBL encoding genes but also carbapenemase genes, to eventually prevent mistaken interpretations when an ESBL is actually produced together with an additional carbapenemase. In addition, the immunological and molecular tests cannot detect the never-ending variety of ESBL encoding genes that are emerging worldwide.
In conclusion, the Rapid ESBL NP test is a rapid, easy to perform, reliable, and low-cost technique for detection of any type of ESBL producer with good sensitivity and specificity.
Footnotes
Disclosure Statement
The Rapid ESBL NP kits used in this evaluation were kindly provided by Liofilchem, Italy.
Funding Information
The study was funded by the National Reference Center for Emerging Antibiotic Resistance for Switzerland.