Multiplex Lateral Flow Immunoassay for the Detection of Expanded-Spectrum Hydrolysis and CTX-M Enzymes

Abstract

Background: Early detection of expanded-spectrum cephalosporinase (ESC) hydrolyzing ß-lactamases is essential for antibiotic stewardship. Here we have developed a multiplex lateral flow immunoassay (LFIA) that detects cefotaxime-hydrolyzing activity as well as the most prevalent ESC-hydrolyzing ß-lactamases: the CTX-M-like.

Methods: The Rapid LFIA ESC test was evaluated retrospectively on 188 (139 Enterobacterales, 30 Pseudomonas spp. and 14 Acinetobacter spp.) agar-grown bacterial isolates with well-characterized ß-lactamase content. One single colony was resuspended in 150 µL extraction buffer containing cefotaxime, incubated at room temperature for 30 min prior to loading on the LFIA for reading within 10 min.

Results: Out of the 188 isolates, all 17 that did not express a β-lactamase hydrolyzing cefotaxime gave negative results, and all 171 isolates expressing a β-lactamase known to hydrolyze cefotaxime, gave a positive test result. In addition, all 86 isolates expressing a CTX-M-variant belonging to one of the five CTX-M-subgroups were correctly identified. The sensitivity and specificity was 100% for both tests.

Conclusions: The results showed that the multiplex LFIA was efficient, fast, low cost and easy to implement in routine laboratory work for the confirmation of ESC hydrolyzing activity and the presence of CTX-M enzymes.

Keywords: CTX-M enzymes; cephalosporin hydrolysis; lateral flow immunoassay; β-lactamase detection.

Figure 1

Structure of the strips. The strip consists of the sample pad with dried gold-labelled mAbs (anti-cefotaxime + anti-CTX-Ms); the nitrocellulose membrane with a Test Line 1 consisting of intact CTX coupled with BSA; a Test Line 2 consisting of mAbs recognizing CTX-Ms; a control line consisting of mAbs recognizing the colloidal gold-labelled mAbs; and the absorption pad which allows the sample to migrate along the strip.

Figure 2

Test procedure. One colony is resuspended in 150 µL of extraction buffer with 15 ng/mL of CTX. After a 30-min incubation at room temperature (RT), 100 µL are loaded on the cassette. The results are obtained after 10 min of migration. Data acquisition is by naked eye readings.

Figure 3

Test interpretation. Case 1: In the absence of enzymatic activity, all the anti-cefotaxime mAb’s paratopes are occupied by the CTX added to the sample before the test. On both tests lines, the results are negative: “N”. The control line is positive: “P”. Case 2: In the presence of enzymatic activity other than CTX-Ms, the hydrolysed CTX is not recognized by the anti-cefotaxime mAbs, which are able to react with the CTX immobilized on the Test Line 1. A signal appears only on the Test Line 1 and the control line “P”, when cephalosporinase expressing strains, other than CTX-Ms, are present. Case 3: In the presence of CTX-Ms, the hydrolysed CTX is not recognized by the anti-cefotaxime mAbs, which are able to react with the CTX immobilized on the Test Line 1. The labelled anti-CTX-Ms mAbs fix CTX-Ms and react with the anti-CTX-Ms mAbs on the Test Line 2. A signal appears on the Test Line 1, the Test Line 2 and the control line “P”. Case 4: In rare cases with some cephalosporinase-expressing strains such as CTX-M-93, the CTX is not hydrolysed. All the paratopes of anti-cefotaxime mAbs are then occupied by the CTX added to the sample before the test. The labelled anti-CTX-Ms mAbs fix CTX-Ms and react with the mAbs on the Test Line 2. A signal appears only on the Test Line 2 and the control line “P”. The associated number of “P” is relative to the intensities observed on the test lines.