The global spread of antimicrobial resistant (AMR) bacteria represents a considerable public health concern, yet their detection and identification of their resistance mechanisms remain challenging. Optimal diagnostic tests should provide rapid results at low cost to enable implementation in any microbiology laboratory. Lateral flow assays (LFA) meet these requirements and have become essential tools to combat AMR. This review presents the versatility of LFA developed for the AMR detection field, with particular attention to those directly triggering β-lactamases, their performances, and specific limitations. It considers how LFA can be modified by detecting not only the enzyme, but also its β-lactamase activity for a broader clinical sensitivity. Moreover, although LFA allow a short time-to-result, they are generally only implemented after fastidious and time-consuming techniques. We present a sample processing device that shortens and simplifies the handling of clinical samples before the use of LFA. Finally, the capacity of LFA to detect amplified genetic determinants of AMR by isothermal PCR will be discussed. LFA are inexpensive, rapid, and efficient tools that are easy to implement in the routine workflow of laboratories as new first-line tests against AMR with bacterial colonies, and in the near future directly with biological media.

Summary: Early detection of expanded‐spectrum cephalosporin (ESC) resistance is essential not only for an effective therapy but also for the prompt implementation of infection control measures to prevent dissemination in the hospital. We have developed and validated a lateral flow immunoassay (LFIA), called LFIA‐CTX test, for the detection of ESC (cefotaxime) hydrolytic activity based on structural discrimination between the intact antibiotic and its hydrolysed product. A single bacterial colony was suspended in an extraction buffer containing cefotaxime. After a 30‐min incubation, the solution is loaded on the LFIA for reading within 10 min. A total of 348 well‐characterized Gram‐negative isolates were tested. Among them, the 38 isolates that did not express any cefotaxime‐hydrolysing β‐lactamase gave negative results. Of the 310 isolates expressing at least one cefotaxime‐hydrolysing β‐lactamase, all were tested positive, except three OXA‐48‐like producers, which were repeatedly detected negative. Therefore, the sensitivity was 99.1% and the specificity was 100%. The LFIA‐CTX test is efficient, fast, low‐cost and easy to implement in the workflow of a routine microbiology laboratory.

Rapid detection of expanded-spectrum cephalosporins (ESC) hydrolysing enzymes is crucial to implement infection control measures and antibiotic stewardship. Here, we have evaluated three biochemical ESC hydrolysis assays (ESBL NDP test, β-LACTA™ test, LFIA-CTX assay) and the NG-Test^® CTX-M MULTI that detects CTX-M enzymes, on 93 well-characterized Gram-negative isolates, including 60 Enterobacterales, 21 Pseudomonas spp. and 12 Acinetobacter spp. The performances were good for all three hydrolysis assays, with the LFIA-CTX being slightly more sensitive and specific on the tested panel of isolates especially with Enterobacterales, without ambiguous results. This study showed that LFIA-CTX may be used for the detection of ESC hydrolysis as a competitive alternative to already available assays (β-LACTA™ test and ESBL NDP test) without any specific equipment and reduced hands-on-time. The lateral flow immunoassay NG-Test^® CTX-M MULTI has proven to be a useful, easy, rapid, and reliable confirmatory test in Enterobacterales for detection of CTX-M-type ESBLs, which account for most of the resistance mechanisms leading to ESC resistance in Enterobacterales, but it misses rare ESC hydrolysing β-lactamases (AmpC, minor ESBLs, and carbapenemases). Combining it with the LFIA-CTX assay would yield an assay detecting the most frequently-encountered ESBLs (CTX-M-like β-lactamases) together with ESC hydrolysis.

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.

La lutte contre les bactéries multi-résistantes aux antibiotiques (BMR) représente un des plus grands défis auxquels doivent faire face nos sociétés actuelles. Le nombre d'infections causées par des BMR étant en nette augmentation dans le monde entier, celles-ci représentent un enjeu crucial de santé publique. L'un des principaux mécanismes de résistance mis en place par les bactéries est la production de béta-lactamases, enzymes hydrolysant les antibiotiques de type béta-lactamine. Pour lutter efficacement contre la dissémination de bactéries produisant ces enzymes et fournir un traitement approprié aux patients il est essentiel de pouvoir détecter rapidement les béta-lactamases.Dans ce contexte, le projet de cette thèse est de développer deux nouveaux tests bandelettes permettant de détecter l'activité enzymatique de ces enzymes. Ils détecteront l'hydrolyse de deux classes de béta-lactamines : les céphalosporines et les carbapénèmes.Ces tests seront basés sur l'utilisation d'anticorps monoclonaux spécifiques de ces deux classes d'antibiotiques. Ces travaux de thèse impliqueront la production, la sélection et la caractérisation de ces anticorps. Les tests développés seront transférés à un industriel et validés avec différentes souches bactériennes produisant différentes béta-lactamases dans différents milieux biologiques (hémocultures, urines, écouvillons rectaux).