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.

Infections with carbapenem-resistant (CR) Gram-negative (GN) pathogens have increased in many countries worldwide, leaving only few therapeutic options. Cefiderocol (CFDC) is approved in Europe for the treatment of aerobic GN infections in adults with limited treatment options. This study evaluated the in vitro activity of cefiderocol and comparators against multidrug-resistant (MDR) bacteria including meropenem-resistant (MR) or pandrug-resistant (PR) GN clinical isolates from France and Belgium. The minimum inhibitory concentrations (MICs) of CFDC were determined by broth microdilution, using iron-depleted cation-adjusted Mueller–Hinton broth, and were compared to those of 10 last-line antibiotics. The MICs were interpreted according to EUCAST and CLSI breakpoints, and in the absence of species-specific breakpoints, non-species-related pharmacokinetic/pharmacodynamic breakpoints were used. Among the 476 isolates tested, 322 were carbapenemase producers (CP), 58 non-CP-CRs, 52 intrinsically CR, 41 expanded-spectrum cephalosporin resistant and 5 were multi-susceptible. Susceptibility to CFDC was high using EUCAST breakpoints 81%, 99% and 84%, and was even higher using CLSI breakpoints to 93%, 100% and 88% for Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii, respectively. Susceptibility to cefiderocol using non-species-related breakpoints for Stenotrophomonas maltophilia, Achromobacter xylosoxydans and Burkholderia cepacia, was 100%, 100% and 92.3%, respectively. The susceptibility rates were lower with the NDM producers, with values of 48% and 30% using EUCAST breakpoints and 81% and 50% using CLSI breakpoints for Enterobacterales and Acinetobacter spp, respectively. CFDC demonstrated high in vitro susceptibility rates against a wide range of MDR GN pathogens, including MR and PR isolates.

Since the first description of OXA-48, more than forty variants have been recovered from Enterobacterales isolates. Whereas some OXA-48-related enzymes have been reported as conferring similar resistance patterns, namely, the hydrolysis of carbapenems and penicillins with very weak or almost no activity against expanded-spectrum cephalosporins, some have reduced carbapenem and temocillin hydrolysis, and others hydrolyze expanded-spectrum cephalosporins and carbapenems only marginally. With such drastic differences in the hydrolytic profile, especially of carbapenems, it becomes urgent to establish hydrolytic cutoffs in order to determine when an OXA-48-like enzyme may be considered as a carbapenemase or not. With this aim, the coefficient of activity for imipenem (k_cat/K_m) was determined for a total of 30 enzymes, including OXA-48, OXA-48-like natural variants, and OXA-48 synthetic mutants. In addition, six different methods for the detection of carbapenemase-producers were performed. The coefficients of activity for imipenem for all the different enzymes went from 550 mM⁻¹·s⁻¹ to 0.02 mM⁻¹·s⁻¹. In order to match the coefficient of activity results with the biochemical confirmatory tests, we suggest the value of 0.27 mM⁻¹·s⁻¹ as the cutoff above which an OXA-48 variant may be considered a carbapenem-hydrolyzing enzyme.

Carbapenem resistance (CR) is an emerging health issue. Epidemiological surveys on carbapenem-resistant Gram-negative bacilli (CR-GNB) in Lebanon remain scarce. In this study, we determined the prevalence of CR-GNB isolated between 2015 to 2019 in three hospitals in northern Lebanon: 311 CR-Enterobacterales (out of 11210; 2.8%), 155 CR-Pseudomonas (out of 1034; 15%) and 106 CR- Acinetobacter (out of 184; 57.6%) were identified. CR mechanisms were determined for 146 randomly chosen isolates: the Carba NP test revealed an enzymatic resistance to carbapenems in 109 isolates (out of 146, 74.7%). Produced carbapenemases were evaluated by the NG-Test Carba5, NG-Test OXA-23 immunochromatographic assays and PCR. Carbapenemase-producing (CP) Enterobacterales expressed bla_OXA-48-like, bla_NDM-like and bla_VIM-like genes and CP-Pseudomonas expressed bla_IMP-like and bla_VIM-like genes, whereas CP-Acinetobacter expressed bla_OXA-23-like genes. The NG-Test Carba5 results were confirmed by PCR sequencing and revealed several variants, such as NDM-19, VIM-62 and OXA-162, never described so far in Lebanon. Isolates with discordant results were sequenced by WGS and highlighted novel variants of the natural oxacillinases of Pseudomonas aeruginosa: bla_OXA-50-like genes. Their role in carbapenem resistance should be further studied. Overall, our findings highlight an alarming situation and encourage health care centers to establish performant registration systems that could help in limiting resistance spread.

Background: Avibactam, relebactam and vaborbactam are β-lactamase inhibitors that proved their efficiency against KPC-producing Enterobacterales. Regarding their inhibitor activity towards Ambler’s class A extended spectrum β-lactamases (ESBL) and Ambler’s class C cephalosporinase (AmpC), they should be active on most of the carbapenem-resistant non-carbapenemase-producing Enterobacterales (CR non-CPE). Objectives: Determine the in vitro activity of ceftazidime-avibactam, imipenem-relebactam and meropenem-vaborbactam and comparators against CR non-CPE. Methods: MICs to ceftazidime/avibactam, imipenem/relebactam, meropenem/vaborbactam, but also temocillin, ceftolozane/tazobactam, ertapenem, colistin, eravacycline and tigecycline were determined by broth microdilution (ThermoFisher) on a collection of 284 CR non-CPE (inhibition zone diameter < 22 mm to meropenem). Whole genome sequencing was performed on 90 isolates to assess the genetic diversity as well as resistome. Results: According to EUCAST breakpoints, susceptibility rates of ceftazidime, imipenem, meropenem and ertapenem used at standard dose were 0.7%, 45.1%, 14.8% and 2.5%, respectively. Increased exposure of ceftazidime, imipenem and meropenem led to reach 3.5%, 68.3% and 67.7% susceptibility, respectively. Using the EUCAST clinical breakpoints, susceptibility rates of ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam were 88.4%, 81.0% and 80.6%, respectively. Susceptibility rates of temocillin, ceftolozane/tazobactam, tigecycline, eravacycline, and colistin were 0%, 4.6%, 27.8%, 54.9% and 90.1%. MICs distributions with and without the presence of the inhibitor demonstrated a better ability of avibactam and relebactam compared to vaborbactam to restore susceptibility to the associated β-lactam. Conclusions: This study demonstrated the in vitro efficacy of ceftazidime/avibactam, imipenem/relebactam and to a lesser extent meropenem/vaborbactam against CR non-CPE. Moreover, to test all β-lactams/β-lactamases inhibitors combinations without a priori for CRE, non-CPE is crucial since resistance to one of the β-lactam/β-lactamase inhibitor combinations does not predict resistance to another molecule, depending on the resistance mechanisms involved.

Background: Despite the availability of new options (ceftazidime-avibactam, imipenem-relebactam, meropenem-vaborbactam and cefiderocol), it is still very difficult to treat infections caused by metallo-β-lactamase (MBLs)-producers resistant to aztreonam. The in vitro efficacy of aztreonam in association with avibactam, vaborbactam or relebactam was evaluated on a collection of MBL-producing Enterobacterales, MBL-producing P. aeruginosa and highly drug-resistant S. maltophilia. Methods: A total of fifty-two non-duplicate MBL-producing Enterobacterales, five MBL-producing P. aeruginosa and five multidrug-resistant S. maltophila isolates were used in this study. The minimum inhibitory concentrations (MICs) of aztreonam, meropenem-vaborbactam and imipenem-relebactam were determined by Etest^® (bioMérieux, La Balme-les-Grottes) according to EUCAST recommendations. For aztreonam-avibactam, aztreonam-vaborbactam and aztreonam-relebactam associations, the MICs were determined using Etest^® on Mueller-Hinton (MH) agar supplemented with 8 mg/L of avibactam, 8 mg/L of vaborbactam and 4 mg/L of relebactam. The MICs were interpreted according to EUCAST guidelines. Results: The susceptibility rates of aztreonam-avibactam, aztreonam-vaborbactam and aztreonam-relebactam with a standard exposure of aztreonam (1g × 3, IV) were 84.6% (44/52), 55.8% and 34.6% for Enterobacterales and 0% for all combinations for P. aeruginosa and S. maltophila. The susceptibility rates of aztreonam-avibactam, aztreonam-vaborbactam and aztreonam-relebactam with a high exposure of aztreonam (2g × 4, IV) were 92.3%, 78.9% and 57.7% for Enterobacterales, 75%, 60% and 60% for P. aeruginosa and 100%, 100% and 40% for S. maltophila. Conclusions: As previously demonstrated for an aztreonam/ceftazidime-avibactam combination, aztreonam plus imipenem-relebactam and aztreonam plus meropenem-vaborbactam might be useful options, but with potentially lower efficiency, to treat infections caused by aztreonam-non-susceptible MBL-producing Gram-negative strains.