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Previous studies relating hormone and cytokine concentrations in follicular fluid to oocyte fertilizability were flawed by the uncertainty about the actual oocyte maturity status at the time of recovery and by the possible contribution of the male factor to failures of conventional in-vitro fertilization. This is the first study in which oocyte maturity was assessed immediately after recovery and only mature oocytes were selected for treatment by intracytoplasmic sperm injection. Fertilization outcomes were related to follicular fluid concentrations of 17β-oestradiol, progesterone, follicle stimulating hormone, luteinizing hormone (LH), growth hormone (GH), prolactin (PRL), interleukin-1 (IL-1) and tumour necrosis factor-α (TNFα). Those oocytes that subsequently showed normal fertilization were harvested from follicles with higher concentrations of progesterone, GH, PRL, IL-1 and TNFα as compared with those of oocytes that failed to fertilize. Among the normally fertilized oocytes, low GH concentrations were associated with the failure of cleavage and with poor morphology of cleaving embryos, whereas rapidly cleaving embryos developed from oocytes recovered from follicles with high concentrations of LH and IL-1. These data suggest important roles for GH, IL-1 and TNFα, and of residual LH after pituitary suppression, as positive regulators of the final phase of oocyte intrafollicular development. [ABSTRACT FROM PUBLISHER]

We examined the emergence and characteristics of oxacillinase-484-producing Enterobacterales in France during 2012-2023. Genomic analysis identified 2 predominant sequence types in Escherichia coli: ST410 and ST1722. Plasmid analysis revealed that bla OXA-484 genes were carried mostly on an IncX3-type plasmid associated with genetic elements including insertion sequences IS3000 and ISKpn19.

There is growing concern about effectively controlling cat populations due to their impact on biodiversity, especially on islands. To plan this management, it is essential to know the cat population size, sterilization rates, and space they use. Small inhabited islands can have very high cat densities; thus, this study aimed to evaluate cat density and home range on a small tourist island in the Spanish Mediterranean. Surveys in the urban area identified individual cats using a photographic catalog, and camera trapping was conducted in the scrubland area. GPS devices were fitted on three urban cats. The overall cat density was estimated to be 308 cats/km 2 , varying between the urban area (1084 cats/km 2 ) and the uninhabited scrubland (27 cats/km 2 ). Urban cats had smaller average home ranges (0.38 ha or 1.25 ha, depending on the estimation method) compared to scrubland cats (9.53 ha). Penetration of scrubland cats into the urban area was not detected. These results indicate that the urban area acts as a source of cats for the scrubland. Although the total sterilization rate was high (90.3%), the large cat population implies that the density would take over a decade to decrease to acceptable levels. Therefore, complementary measures for managing this cat population are recommended.

BackgroundSince 2021, an emergence of New Delhi metallo-β-lactamase (NDM)-14-producing Klebsiella pneumoniae has been identified in France. This variant with increased carbapenemase activity was not previously detected in Enterobacterales.AimWe investigated the rapid dissemination of NDM-14 producers among patients in hospitals in France.MethodsAll NDM-14-producing non-duplicate clinical isolates identified in France until June 2022 (n = 37) were analysed by whole genome sequencing. The phylogeny of NDM-14-producers among all K. pneumoniae sequence type (ST) 147 reported in France since 2014 (n = 431) was performed. Antimicrobial susceptibility testing, conjugation experiments, clonal relationship and molecular clock analysis were performed.ResultsThe 37 NDM-14 producers recovered in France until 2022 belonged to K. pneumoniae ST147. The dissemination of NDM-14-producing K. pneumoniae was linked to a single clone, likely imported from Morocco and responsible for several outbreaks in France. The gene bla NDM-14 was harboured on a 54 kilobase non-conjugative IncFIB plasmid that shared high homology with a known bla NDM-1 -carrying plasmid. Using Bayesian analysis, we estimated that the NDM-14-producing K. pneumoniae ST147 clone appeared in 2020. The evolutionary rate of this clone was estimated to 5.61 single nucleotide polymorphisms per genome per year. The NDM-14 producers were highly resistant to all antimicrobials tested except to colistin, cefiderocol (minimum inhibitory concentration 2 mg/L) and the combination of aztreonam/avibactam.ConclusionHighly resistant NDM-14 producing K. pneumoniae can rapidly spread in healthcare settings. Surveillance and thorough investigations of hospital outbreaks are critical to evaluate and limit the dissemination of this clone.

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.

Objectives: Ceftolozane-tazobactam (C/T) proved its efficacy for the treatment of infections caused by non-carbapenemase producing Pseudomonas aeruginosa and Enterobacterales. Here, we aimed to provide susceptibility data on a large series of Enterobacterales since the revision of EUCAST categorization breakpoints in 2020., Methods: First, C/T susceptibility was determined on characterized Enterobacterales resistant to third generation cephalosporins (3GCs) (extended spectrum β-lactamase [ESBL] production or different levels of AmpC overexpression) (n = 213) and carbapenem-resistant Enterobacterales (CRE) (n = 259), including 170 carbapenemase producers (CPE). Then, 1632 consecutive clinical Enterobacterales responsible for infection were prospectively collected in 23 French hospitals. C/T susceptibility was determined by E-test® (biomérieux) and broth microdilution (BMD) (Sensititre™, Thermo Scientific) to perform method comparison., Results: Within the collection isolates, 88% of 3GC resistant strains were susceptible to C/T, with important variation depending on the resistance mechanism: 93% vs. 13% susceptibility for CTX-M and SHV-ESBL producers, respectively. Only 20% of the CRE were susceptible to C/T. Among CPE, 80% of OXA-48-like producers were susceptible to C/T, whereas all metallo-β-lactamase producers were resistant. The prospective study revealed that 95.6% of clinical isolates were susceptible to C/T. Method comparison performed on these 1632 clinical isolates demonstrated 99% of categorization agreement between MIC to C/T determined by E-test® in comparison with the BMD (reference) and only 74% of essential agreement., Conclusion: Overall, C/T showed good activity against wild-type Enterobacterales, AmpC producers, and ESBL-producing Escherichia coli but is less active against ESBL-producing Klebsiella pneumoniae, and CRE. E-test® led to an underestimation of the MICs in comparison to the BMD reference., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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.

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.

Relebactam and vaborbactam are among the newest β-lactamase inhibitors marketed. They were originally designed to inhibit the Ambler class A carbapenemase KPC. In this study, susceptibility to imipenem/relebactam and meropenem/vaborbactam was determined against a collection of OXA-48-like-producing Enterobacterales (n = 407). The clonality and resistomes of the isolates were determined by whole-genome sequencing. Comparison was performed with other relevant antibiotics such as carbapenems alone, ceftazidime/avibactam and ceftolozane/tazobactam. Addition of relebactam and vaborbactam did not significantly modify the MIC 50 and MIC 90 values obtained for imipenem and meropenem alone. In contrast, addition of avibactam strongly restored ceftazidime susceptibility. According to European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints, MIC 50 /MIC 90 values were at 2/4, 2/4, 2/8, 2/8, 32/>32 and 0.5/2 mg/L for imipenem, imipenem/relebactam, meropenem, meropenem/vaborbactam, ceftazidime and ceftazidime/avibactam, respectively. No differences were observed depending on the species. This study highlights the lack of benefit in vitro for carbapenem/inhibitor combination compared with carbapenem alone against OXA-48-producing isolates as well as the difficulties in comparing molecules since carbapenem/inhibitor combinations were not developed with the same dosage of carbapenem., (Copyright © 2022. Published by Elsevier Ltd.)

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.

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) has become a staple in clinical microbiology laboratories. Protein-profiling of bacteria using this technique has accelerated the identification of pathogens in diagnostic workflows. Recently, lipid profiling has emerged as a way to complement bacterial identification where protein-based methods fail to provide accurate results. This study aimed to address the challenge of rapid discrimination between Escherichia coli and Shigella spp. using MALDI-TOF MS in the negative ion mode for lipid profiling coupled with machine learning. Both E. coli and Shigella species are closely related; they share high sequence homology, reported for 16S rRNA gene sequence similarities between E. coli and Shigella spp. exceeding 99%, and a similar protein expression pattern but are epidemiologically distinct. A bacterial collection of 45 E. coli, 48 Shigella flexneri, and 62 Shigella sonnei clinical isolates were submitted to lipid profiling in negative ion mode using the MALDI Biotyper Sirius® system after treatment with mild-acid hydrolysis (acetic acid 1% v/v for 15 min at 98°C). Spectra were then analyzed using our in-house machine learning algorithm and top-ranked features used for the discrimination of the bacterial species. Here, as a proof-of-concept, we showed that lipid profiling might have the potential to differentiate E. coli from Shigella species using the analysis of the top five ranked features obtained by MALDI-TOF MS in the negative ion mode of the MALDI Biotyper Sirius® system. Based on this new approach, MALDI-TOF MS analysis of lipids might help pave the way toward these goals., (© 2022 The Authors. Microbiology Open published by John Wiley & Sons Ltd.)

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 ·s 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.

Vancomycin-resistant enterococci (VREs) have become one of the most important nosocomial pathogens worldwide, associated with increased treatment costs, prolonged hospital stays and high mortality. Rapid detection is crucial to reduce their spread and prevent infections and outbreaks. The lateral flow immunoassay NG-Test VanB (NG Biotech) was evaluated for the rapid detection of VanB-producing vancomycin-resistant enterococci (VanB-VREs) using 104 well-characterized enterococcal isolates. The sensitivity and specificity were both 100% when bacterial cells were grown in the presence of vancomycin used as a VanB inducer. The NG-Test VanB is an efficient, rapid and easy to implement assay in clinical microbiology laboratories for the confirmation of VanB-VREs from colonies. Together with the NG-Test VanA, they could replace the already existing tests available for the confirmation of acquired vancomycin resistance in enterococci, especially from selective media or from antibiograms, with 100% sensitivity and specificity. Rapid detection in less than 15 min will result in more efficient management of carriers and infected patients. In addition, these tests may be used for positive blood cultures, given a 3.5 h sub-culturing step on Chocolate agar PolyViteX in the presence of a 5-µg vancomycin disk, which is routinely performed in many clinical microbiology laboratories for every positive blood culture for subsequent MALDI-TOF identification of the growing bacteria.

Serological rapid diagnostic tests (RDTs) are widely used across pathologies, often providing users a simple, binary result (positive or negative) in as little as 5 to 20 min. Since the beginning of the COVID-19 pandemic, new RDTs for identifying SARS-CoV-2 have rapidly proliferated. However, these seemingly easy-to-read tests can be highly subjective, and interpretations of the visible "bands" of color that appear (or not) in a test window may vary between users, test models, and brands. We developed and evaluated the accuracy/performance of a smartphone application (xRCovid) that uses machine learning to classify SARS-CoV-2 serological RDT results and reduce reading ambiguities. Across 11 COVID-19 RDT models, the app yielded 99.3% precision compared to reading by eye. Using the app replaces the uncertainty from visual RDT interpretation with a smaller uncertainty of the image classifier, thereby increasing confidence of clinicians and laboratory staff when using RDTs, and creating opportunities for patient self-testing., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Objectives: This study evaluated the performance of the Novodiag ® CarbaR+ an automated qualitative nucleic acid-based diagnostic assay detecting the bla VIM , bla NDM , bla IMP , bla OXA-48 , bla KPC , bla OXA-23 , bla OXA-58 , bla OXA-24 , and IS Aba 1 associated bla OXA-51 carbapenemase genes and colistin resistance mcr-1 gene from clinical isolates or directly from rectal swabs. Materials and Methods: CarbaR+ was evaluated on 201 clinical isolates and on 100 rectal swabs (80 selected swabs from patients that were evaluated by culture method and/or Xpert Carba-R assay and 20 spiked samples). PCR-sequencing on colonies was considered as the gold standard. Results: The CarbaR+ detected all the variants of the targeted resistance genes (39 bla VIM- , 30 bla NDM- , 20 bla IMP- , 19 bla OXA-48- , 15 bla KPC- , 19 bla OXA-23- , 13 bla OXA-58- , 4 bla OXA-24- , 8 IS Aba 1- bla OXA-51- , and 3 mcr-1 -like genes) with sensitivity and specificity of 98.2% and 99.7%, respectively. On the 80 rectal swabs, 71 CarbaR+ results were fully concordant with the results on selective culture media (66 positive samples with 1 to 3 carbapenemases and 5 negative samples). In eight rectal swabs, CarbaR+ identified additional carbapenemase genes. One false negative result with an Escherichia coli producing-OXA-181 was observed and one CarbaR+ result for OXA-48 was in agreement with Xpert Carba-R assay, without growth on culture media. A concordance of 100% was observed on spiked samples. Conclusions: Novodiag CarbaR+ is a random-access fully automated system that achieves excellent performances for the detection of carbapenemase and/or colistin resistance determinants either from cultured clinical isolates or directly from rectal swabs in 80 minutes.

Numerous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapid serological tests have been developed, but their accuracy has usually been assessed using very few samples, and rigorous comparisons between these tests are scarce. In this study, we evaluated and compared 10 commercially available SARS-CoV-2 rapid serological tests using the STARD (Standards for Reporting of Diagnostic Accuracy Studies) methodology. Two hundred fifty serum samples from 159 PCR-confirmed SARS-CoV-2 patients (collected 0 to 32 days after the onset of symptoms) were tested with rapid serological tests. Control serum samples ( n  = 254) were retrieved from pre-coronavirus disease (COVID) periods from patients with other coronavirus infections ( n   = 11), positivity for rheumatoid factors ( n   = 3), IgG/IgM hyperglobulinemia ( n   = 9), malaria ( n  = 5), or no documented viral infection ( n   = 226). All samples were tested using rapid lateral flow immunoassays (LFIAs) from 10 manufacturers. Only four tests achieved ≥98% specificity, with the specificities ranging from 75.7% to 99.2%. The sensitivities varied by the day of sample collection after the onset of symptoms, from 31.7% to 55.4% (days 0 to 9), 65.9% to 92.9% (days 10 to 14), and 81.0% to 95.2% (>14 days). Only three of the tests evaluated met French health authorities' thresholds for SARS-CoV-2 serological tests (≥90% sensitivity and ≥98% specificity). Overall, the performances varied greatly between tests, with only one-third meeting acceptable specificity and sensitivity thresholds. Knowledge of the analytical performances of these tests will allow clinicians and, most importantly, laboratorians to use them with more confidence; could help determine the general population's immunological status; and may help diagnose some patients with false-negative real-time reverse transcription-PCR (RT-PCR) results., (Copyright © 2021 American Society for Microbiology.)

Background: VRE are nosocomial pathogens with an increasing incidence in recent decades. Rapid detection is crucial to reduce their spread and prevent infections and outbreaks., Objectives: To evaluate a lateral flow immunoassay (LFIA) (called NG-Test VanA) for the rapid and reliable detection of VanA-producing VRE (VanA-VRE) from colonies and broth., Methods: NG-Test VanA was validated on 135 well-characterized enterococcal isolates grown on Mueller-Hinton (MH) agar (including 40 VanA-VRE). Different agar plates and culture broths widely used in routine laboratories for culture of enterococci were tested., Results: All 40 VanA-VRE clinical isolates were correctly detected in less than 15 min irrespective of the species expressing the VanA ligase and the medium used for bacterial growth. No cross-reaction was observed with any other clinically relevant ligases (VanB, C1, C2, D, E, G, L, M and N). Overall, the sensitivity and specificity of the assay were 100% for VanA-VRE grown on MH agar plates. NG-Test VanA accurately detects VanA-VRE irrespective of the culture medium (agar and broth). Band intensity was increased when using bacteria grown on vancomycin-containing culture media or on MH close to the vancomycin disc as a consequence of VanA induction. The limit of detection of the assay was 6.3 × 106 cfu per test with bacteria grown on MH plates and 4.9 × 105 cfu per test with bacteria grown on ChromID® VRE plates., Conclusions: NG-Test VanA is efficient, rapid and easy to implement in the routine workflow of a clinical microbiology laboratory for the confirmation of VanA-VRE., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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.

Introduction: Will-to-live, a central concept in well-being theories, represents a positive attitudinal component towards one's own life. It has been identified as a mediator between the self-perception of aging and longevity and health. The objective of this study is to characterise elderly people with high levels of will-to-live in the main dimensions of positive psychology., Method: The study included the voluntary participation of 165 adults, aged between 54-89 years, users of senior centres in the Community of Madrid. Will-to-live and other dimensions of well-being and health were evaluated. The correlations between the different evaluated dimensions were analysed, and comparisons made in terms of different levels of will-to-live, as well as an analysis of the dimensions that contribute most to the will-to-live., Results: Correlations among the majority of variables were statistically significant, with a decrease in the coefficients being observed when controlling the effect of the will-to-live. When groups with different levels of will-to-live are compared with well-being and health, there are statistically significant differences in practically all of the dimensions. Gratitude, positive affect, and depression are the dimensions that best predict will-to-live., Conclusion: Older adults that make up the group with high will-to-live are characterised by higher levels of optimism, gratitude, positive affect, sense of life, psychological prosperity, resilience, happiness, and satisfaction with life, as well as lower levels of depression and negative self-perception of aging. The implications of these results point towards the relevance of will-to-live in successful aging., (Copyright © 2019 SEGG. Publicado por Elsevier España, S.L.U. All rights reserved.)

A novel real-time multiplex PCR assay, BD MAX Check-Points CPO, was evaluated to detect carbapenemase-producing organisms in clinical settings on the BD MAX system. A total of 175 well-characterized isolates (including 123 carbapenemase producers) and 128 rectal swab specimens (including 83 positives) of patients considered at high risk for carriage of carbapenemase producers were included. Bacterial suspensions were used to spike true-negative rectal swabs to mimic a clinical sample. Sample (50 μL), containing either the spiked or the patient's sample, was processed. The BD MAX Check-Points CPO assay detected carbapenemases KPC, VIM/IMP, NDM, and OXA-48-like producers with a high sensitivity and specificity of 97.1% and 98.8%, respectively. Rare variants of the IMP type (IMP-11, IMP-13, and IMP-14) and one rare and distantly related OXA-48 variant (OXA-535) remained undetected. With patients' rectal swabs, sensitivity and specificity were 92.8% and 97.8%, respectively. Failure of detection was due to weak inoculum. The time to result was short: approximately. 2.5 hours for 12 samples (including extraction and PCR). The automated sample-in results-out platform is an efficient, quick, and easy-to-use tool for the detection of the main five carbapenemases. The lack of distinction between producers of VIM and IMP may be limiting in countries where these enzymes are widespread, as in Asia, but not in France, where IMP producers are extremely rare., (Copyright © 2020 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Background: With the dissemination of carbapenemase producers, a revival of colistin was observed for the treatment of infections caused by MDR Gram-negatives. Unfortunately, the increasing usage of colistin led to the emergence of resistance. In Klebsiella pneumoniae, colistin resistance arises through addition of 4-amino-l-arabinose (l-Ara4N) or phosphoethanolamine (pEtN) to the native lipid A. The underlying mechanisms involve numerous chromosome-encoded genes or the plasmid-encoded pEtN transferase MCR. Currently, detection of colistin resistance is time-consuming since it still relies on MIC determination by broth microdilution. Recently, a rapid diagnostic test based on MALDI-TOF MS detection of modified lipid A was developed (the MALDIxin test) and tested on Escherichia coli and Acinetobacter baumannii., Objectives: Optimize the MALDIxin test for the rapid detection of colistin resistance in K. pneumoniae., Methods: This optimization consists of an additional mild-acid hydrolysis of 15 min in 1% acetic acid. The optimized method was tested on a collection of 81 clinical K. pneumoniae isolates, including 49 colistin-resistant isolates (45 with chromosome-encoded resistance, 3 with MCR-related resistance and 1 with both mechanisms)., Results: The optimized method allowed the rapid (<30 min) identification of l-Ara4N- and pEtN-modified lipid A of K. pneumoniae, which are known to be the real triggers of polymyxin resistance. At the same time, it discriminates between chromosome-encoded and MCR-related polymyxin resistance., Conclusions: The MALDIxin test has the potential to become an accurate tool for the rapid determination of colistin resistance in clinically relevant Gram-negative bacteria., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

Background: KPC-like carbapenemases have spread worldwide with more than 30 variants identified that differ by single or double amino-acid substitutions., Objectives: To describe the steady-state kinetic parameters of KPC-28, which differs from KPC-2 by a H274Y substitution and the deletion of two amino acids (Δ242-GT-243)., Methods: The blaKPC-2, blaKPC-3, blaKPC-14 and blaKPC-28 genes were cloned into a pTOPO vector for susceptibility testing or into pET41b for overexpression, purification and subsequent kinetic parameter (Km, kcat) determination. Molecular docking experiments were performed to explore the role of the amino-acid changes in the carbapenemase activity., Results: Susceptibility testing revealed that Escherichia coli producing KPC-28 displayed MICs that were lower for carbapenems and higher for ceftazidime and ceftazidime/avibactam as compared with KPC-2. The catalytic efficiencies of KPC-28 and KPC-14 for imipenem were 700-fold and 200-fold lower, respectively, than those of KPC-2, suggesting that Δ242-GT-243 in KPC-28 and KPC-14 is responsible for reduced carbapenem hydrolysis. Similarly, the H274Y substitution resulted in KPC-28 in a 50-fold increase in ceftazidime hydrolysis that was strongly reversed by clavulanate., Conclusions: We have shown that KPC-28 lacks carbapenemase activity, has increased ceftazidime hydrolytic activity and is strongly inhibited by clavulanate. KPC-28-producing E. coli isolates display an avibactam-resistant ESBL profile, which may be wrongly identified by molecular and immunochromatographic assays as the presence of a carbapenemase. Accordingly, confirmation of carbapenem hydrolysis will be mandatory with assays based solely on blaKPC gene or gene product detection., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Colistin has become a last-resort antibiotic for the treatment of infections caused by highly drug-resistant Gram-negative bacteria. Moreover, it has been widely used in the livestock sector. As a consequence, colistin resistance is emerging worldwide. Among the colistin resistance mechanisms, the spread of the plasmid-encoded colistin resistance gene mcr-1 (mostly in Escherichia coli ) is of particular concern due to its increased transferability compared to that of chromosome-encoded resistance. The early detection of MCR-1-producing bacteria is essential to prevent further spread and provide appropriate antimicrobial therapy. Lateral flow immunoassays (LFIAs) were manufactured with selected monoclonal antibodies. A collection of 177 human and 121 animal enterobacterial isolates was tested in a multicentric study. One bacterial colony grown on agar plates was suspended in extraction buffer and dispensed on the cassette. Migration was allowed for 15 min, and the results were monitored by the appearance of a specific band. The positive results showed a pink line resulting in an unambiguous interpretation. All MCR-1-producing isolates were found to be positive by the LFIA, and no false-negative results were observed. Three out of four MCR-2-producing isolates were also found to be positive. Our test does not detect MCR-3-, MCR-4-, or MCR-5-producing isolates. LFIA allows the detection of MCR-1 with 100% sensitivity and 98% specificity. This test is fast, sensitive, specific, easy to use, and cost-effective and can therefore be implemented in any microbiology laboratory worldwide. LFIA is a major tool for the rapid detection and monitoring of MCR-1 producers in humans and animals., (Copyright © 2019 Volland et al.)

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 bla ACT-28 gene was expressed in a wild-type and in a porin-deficient Escherichia coli background and compared to the bla ACT-1 gene. Detection of carbapenemase activity was positive only for E. coli expressing the bla ACT-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., (Copyright © 2019 American Society for Microbiology.)