Evaluation of a new rapid immunochromatographic assay for the detection of GES-producing Gram-negative bacteria.

BACKGROUND: As carbapenemase-producing Enterobacterales are increasingly reported worldwide, their rapid detection is crucial to reduce their spread and prevent infections and outbreaks. Lateral flow immunoassays (LFIAs) have become major tools for the detection of carbapenemases. However, as for most commercially available assays, only the five main carbapenemases are targeted. OBJECTIVES: Here, we have developed and evaluated an LFIA prototype for the rapid and reliable detection of the increasingly identified GES-type ß-lactamases. METHODS: The GES LFIA was validated on 103 well-characterized Gram-negative isolates expressing various ß-lactamases grown on Mueller-Hinton (MH) agar, chromogenic, and chromogenic/selective media. RESULTS: The limit of detection of the assay was 106 cfu per test with bacteria grown on MH agar plates. GES LFIA accurately detected GES-type ß-lactamases irrespective of the culture media and the bacterial host. The GES LFIA was not able to distinguish between GES-ESBLs and GES-carbapenemases. Because GES enzymes are still rare, their detection as an ESBL or a carbapenemase remains important, especially because extensive use of carbapenems to treat ESBL infections may select for GES variants capable of hydrolysing carbapenems. CONCLUSIONS: The GES LFIA is efficient, rapid and easy to implement in the routine workflow of a clinical microbiology laboratory for the confirmation of GES-type ß-lactamases. Combining it with immunochromatographic assays targeting the five main carbapenemases (KPC, NDM, VIM, IMP and OXA-48) would improve the overall sensitivity for the most frequently encountered carbapenemases and ESBLs, especially in non-fermenters.

A Lateral Flow Immunoassay for the Rapid Identification of CTX-M-Producing Enterobacterales from Culture Plates and Positive Blood Cultures.

We have developed a lateral flow immunoassay (LFIA), named NG-Test CTX-M MULTI (NG-Test), to detect group 1, 2, 8, 9, 25 CTX-M producers from agar plates and from positive blood cultures in less than 15 min. The NG-Test was validated retrospectively on 113 well-characterized enterobacterial isolates, prospectively on 102 consecutively isolated ESBL-producers from the Bicêtre hospital and on 100 consecutive blood cultures positive with a gram-negative bacilli (GNB). The NG-Test was able to detect all CTX-M producers grown on the different agar plates used in clinical microbiology laboratories. No false positive nor negative results were observed. Among the 102 consecutive ESBL isolates, three hyper mucous isolates showed an incorrect migration leading to invalid results (no control band). Using an adapted protocol, the results could be validated. The NG-Test detected 99/102 ESBLs as being CTX-Ms. Three SHV producers were not detected. Among the 100 positive blood cultures with GNB tested 10/11 ESBL-producers were detected (8 CTX-M-15, 2 CTX-M-27). One SHV-2-producing-E. cloacae was missed. The NG-Test CTX-M MULTI showed 100% sensitivity and specificity with isolates cultured on agar plates and was able to detect 98% of the ESBL-producers identified in our clinical setting either from colonies or from positive blood cultures.

[Formula: see text]H, [Formula: see text]C and [Formula: see text]N assignments of human Grb2 free of ligands.

Growth factor receptor-bound 2 (Grb2) is an important link in the receptor tyrosine kinase signaling cascades. It is involved in crucial processes, both physiological (mainly embryogenesis) and pathological (different types of cancer). Several binding partners of all three domains (SH3-SH2-SH3) of this adaptor protein are well described, such as ErbB family members for the SH2 domain and Sos for the SH3 domains. How the different domains interact with each other, both structurally and functionally, is still unclear. These interactions could be essential for regulation processes, and therefore are of great interest. Although a lot of structural data on Grb2 exist, they describe either individual domains, ligand-bound conformations, or frozen pictures of the protein captured by crystallography. Here we report the assignment of backbone and of [Formula: see text] chemical shifts of full-length, apo-Grb2 in solution. In addition to the assigned conformation corresponding to three well-folded domains, a set of peaks compatible with the presence of an unfolded conformation of the N-terminal SH3 domain is observed. This assignment paves the way for future studies of inter-domain interactions and dynamics that have to be taken into account when studying the regulation of Grb2 interactions and signaling pathways.

False-Positive Carbapenem-Hydrolyzing Confirmatory Tests Due to ACT-28, a Chromosomally Encoded AmpC with Weak Carbapenemase Activity from Enterobacter kobei.

In Enterobacter cloacae complex (ECC), the overproduction of the chromosome-encoded cephalosporinase (cAmpC) associated with decreased outer membrane permeability may result in carbapenem resistance. In this study, we have characterized ACT-28, a cAmpC with weak carbapenemase activity, from a single Enterobacter kobei lineage. ECC clinical isolates were characterized by whole-genome sequencing (WGS), susceptibility testing, and MIC, and carbapenemase activity was monitored using diverse carbapenem hydrolysis methods. ACT-28 steady-state kinetic parameters were determined. Among 1,039 non-carbapenemase-producing ECC isolates with decreased susceptibility to carbapenems received in 2016-2017 at the French National Reference Center for antibiotic resistance, only 8 had a positive carbapenemase detection test (Carba NP). These eight ECC isolates were resistant to broad-spectrum cephalosporins due to AmpC derepression, showed decreased susceptibility to carbapenems, and were categorized as carbapenemase-producing Enterobacteriaceae (CPE) according to several carbapenemase detection assays. WGS identified a single clone of E. kobei ST125 expressing only its cAmpC, ACT-28. The blaACT-28 gene was expressed in a wild-type and in a porin-deficient Escherichia coli background and compared to the blaACT-1 gene. Detection of carbapenemase activity was positive only for E. coli expressing the blaACT-28 gene. Kinetic parameters of purified ACT-28 revealed a slightly increased imipenem hydrolysis compared to that of ACT-1. In silico porin analysis revealed the presence of a peculiar OmpC-like protein specific to E. kobei ST125 that could impair carbapenem influx into the periplasm and thus enhance carbapenem-resistance caused by ACT-28. We described a widespread lineage of E. kobei ST125 producing ACT-28, with weak carbapenemase activity that can lead to false-positive detection by several biochemical and phenotypic diagnostic tests.

A multiplex lateral flow immunoassay for the rapid identification of NDM-, KPC-, IMP- and VIM-type and OXA-48-like carbapenemase-producing Enterobacteriaceae.

Objectives: The global spread of carbapenemase-producing Enterobacteriaceae represents a substantial challenge in clinical practice and rapid and reliable detection of these organisms is essential. The aim of this study was to develop and validate a lateral flow immunoassay (Carba5) for the detection of the five main carbapenemases (KPC-, NDM-, VIM- and IMP-type and OXA-48-like). Methods: Carba5 was retrospectively and prospectively evaluated using 296 enterobacterial isolates from agar culture. An isolated colony was suspended in extraction buffer and then loaded on the manufactured Carba5. Results: All 185 isolates expressing a carbapenemase related to one of the Carba5 targets were correctly and unambiguously detected in <15 min. All other isolates gave negative results except those producing OXA-163 and OXA-405, which are considered low-activity carbapenemases. No cross-reaction was observed with non-targeted carbapenemases, ESBLs, AmpCs or oxacillinases (OXA-1, -2, -9 and -10). Overall, this assay reached 100% sensitivity and 95.3% (retrospectively) to 100% (prospectively) specificity. Conclusions: Carba5 is efficient, rapid and easy to implement in the routine workflow of a clinical microbiology laboratory for confirmation of the five main carbapenemases encountered in Enterobacteriaceae.

Dissemination of carbapenemase-producing Enterobacteriaceae and Pseudomonas aeruginosa in Romania.

Fifteen carbapenemase-producing Enterobacteriaceae isolates and 12 carbapenemase-producing Pseudomonas aeruginosa isolates were recovered from patients hospitalized between August 2011 and March 2013 at the Hospital of Infectious Disease, Cluj-Napoca, Romania. One KPC-, nine NDM-1-, four OXA-48-, and one VIM-4-producing Enterobacteriaceae isolates along with 11 VIM-2-producing and one IMP-13-producing P. aeruginosa isolates were recovered from clinical samples. All carbapenemase genes were located on self-conjugative plasmids and were associated with other resistance determinants, including extended-spectrum ß-lactamases and RmtC methylases.