Saoussen Oueslati

: Carbapenem resistance in Acinetobacter spp. is frequently mediated by acquired carbapenemases, whose rapid detection can inform antimicrobial therapy and infection-control decisions. NG-TEST ^® Acineto -5 ^® is a lateral flow immunoassay designed to detect major acquired carbapenemases in Acinetobacter spp. We evaluated its performance using 433 whole-genome-sequenced isolates, including single producers of OXA-23-like (n = 126), OXA-24-like (n = 14), OXA-58-like (n = 8), OXA-143/253/542 (n = 10), VIM-like (n = 9), and NDM-like enzymes (n = 25), as well as isolates co-producing two (n = 20) or three (n = 2) carbapenemases. The collection was predominantly composed of Acinetobacter baumannii (n = 365) and Acinetobacter pittii (n = 33), with 13 additional species represented by 1 to 7 isolates each. The assay detected all targeted carbapenemases, including all tested variants and combinations, yielding 100% sensitivity. OXA-24-like, OXA-143-like, and the distantly related OXA-542 were detected by the same antibody pair, demonstrating cross-reactivity within this group. No false-positive results occurred among isolates lacking targeted carbapenemases, resulting in 100% specificity. Weak positive signals were observed in four of five A. radioresistens isolates carrying intrinsic bla _OXA-23-like genes, consistent with low-level expression. Overall, NG-TEST® Acineto -5 ^® enables rapid detection of major acquired carbapenemases in Acinetobacter spp., supporting infection control, epidemiological surveillance and therapeutic decision-making, as carbapenemase-producing isolates are often multidrug resistant. Detection of VIM and NDM may be particularly valuable because these enzymes are not inhibited by durlobactam and have been associated with increased cefiderocol MICs, potentially limiting treatment options. IMPORTANCE: Carbapenem-resistant Acinetobacter spp. are important causes of healthcare-associated infections and are frequently multidrug resistant, leaving few therapeutic options. The diversity of acquired carbapenem-hydrolyzing class D β-lactamases (CHDLs) and metallo-β-lactamases (MBLs) complicates their rapid phenotypic detection. We evaluated the NG-TEST ^® Acineto -5 ^® lateral flow immunoassay using a large, whole-genome-sequenced collection representing diverse carbapenemase variants and clinically relevant Acinetobacter spp. The assay reliably detected all targeted carbapenemases, including OXA-23-, OXA-24-, OXA-58-, OXA-143/253/542-, VIM-, and NDM-type enzymes, as well as isolates producing multiple carbapenemases. These findings support the use of NG-TEST ^® Acineto -5 ^® as a rapid diagnostic tool for detecting Acinetobacter -specific carbapenemases, facilitating infection-control measures and antimicrobial therapy. This is particularly relevant with the use of novel β-lactamase inhibitors such as durlobactam in combination with sulbactam. Durlobactam inhibits class D OXA-type carbapenemases but not MBLs such as NDM and VIM; therefore, rapid identification of the underlying carbapenemase mechanism may help inform appropriate therapeutic decisions.

Les antibiotiques, et plus particulièrement les β-lactamines, furent pendant longtemps considérés comme l’arme absolue pour le traitement des infections bactériennes, du fait de leur efficacité, leur bonne tolérance et de leur faible coût. Cependant, les bactéries ont développé des mécanismes de résistance, y compris vis-à-vis des β-lactamines possédant le spectre d’activité le plus large, les carbapénèmes. Ces derniers sont considérés comme les antibiotiques de derniers recours pour le traitement des infections sévères à bacilles à Gram négatif en milieu hospitalier. Chez les entérobactéries, cette résistance émergente aux carbapénèmes est principalement due à l’expression d’enzymes appelées les carbapénèmases capables d’inactiver les carbapénèmes. L’émergence exponentielle à l’échelle mondiale de ces entérobactéries productrices de carbapénèmases (EPC) constitue un problème majeur de santé publique. Actuellement, les carbapénémases les plus répandues dans le monde sont les KPC (Klebsiella pneumoniae carbapenemase), NDM-1 (New Delhi metallo- β- lactamase) et OXA-48 (Oxacillinase). La première KPC a été identifiée en 1996 aux Etats- Unis, NDM-1 en 2009 en Suède chez une patiente en provenance d’Inde et OXA-48 en 2003 en Turquie. En France, les carbapénèmases de type OXA-48 sont largement majoritaires et représentent près de 70% des EPC. Ainsi, le retentissement de leur dissémination et le besoin de développer de nouveaux inhibiteurs capables d’inactiver les carbapénèmases se fait de plus en plus pressante.Cette thèse a pour objectif de mieux comprendre le mode d’ action de ces 3 carbapénèmases d’un point de vue moléculaire et biochimique, afin d’ identifier les éléments structuraux nécessaires à leur fonctionnement, mais aussi de poser les bases du développement de « pan-inhibiteurs » et de nouveaux outils de diagnostics. Pour cela, la caractérisation biochimique des carbapénèmases de type KPC, NDM et OXA-48, de leurs variants naturels et de mutants générés in vitro a été entreprise. Il a pu être mis en exergue l’implication de résidus spécifiques et d’éléments structuraux nécessaires à l’ hydrolyse des carbapénèmes. L ’ étude cristallographique et RMN ainsi que l’étude in silico (modélisation) de ces enzymes et de leurs mutants respectifs, nous a permis de mieux comprendre le mode d’ interaction enzyme- substrat. Nous avons ainsi pu comprendre les bases de la capacité impressionnante de ces carbapénèmases à s’adapter et à évoluer en fonction des pressions de sélections exercées.En collaborations avec différentes équipes de chimistes nous avons développés différentes séries de molécules « pan-inhibiteurs » capables d’inhiber les 3 classes de carbapénèmases. Ainsi nous avons montré un effet inhibiteur de certains composés de la famille des flavonoïdes dont la myrécétine, molécule la plus active. Nous avons également identifié une série de composés, les imidazolines, possédant un effet pan-inhibiteur avec des valeurs de CI50 sub-micromolaires et donc compatible avec une utilisation in vivo.Enfin, nous avons participé au développement (en collaboration avec le CEA) d’ un outil de diagnostic rapide basé sur l’immunochromatographie, permettant détection des EPC en moins de 15 minutes.

OXA-244, an R214G variant of OXA-48, is silently spreading worldwide likely because of difficulties in detection using classical screening media. Here, we characterized two clinical isolates of Escherichia coli and Citrobacter youngae that displayed reduced susceptibility to carbapenems but were lacking significant carbapenemase activity as revealed by negative Carba NP test results. However, positive test results were seen for OXA-48-like enzymes by lateral flow immunoassays. WGS revealed the presence of a blaOXA-181-like gene that codes for OXA-484, an R214G variant of OXA-181. BlaOXA-484 gene was located on a 58.4-kb IncP1-like plasmid (pN-OXA-484), that upon transfer into E. coli HB4 with impaired permeability, conferred carbapenem and temocillin resistance (MICs > 32 mg/L). E. coli TOP10 (pTOPO-OXA-484) revealed reduced MICs in most substrates as compared to E. coli TOP10 (pTOPO-OXA-181), especially for imipenem (0.25 mg/L versus 0.75 mg/L) and temocillin (16 mg/L versus 1028 mg/L). Catalytic efficiencies of OXA-484 were reduced as compared to OXA-181 for most ß-lactams including imipenem and temocillin with 27.5- and 21.7-fold reduction, respectively. Molecular modeling confirmed that the salt bridges between R214, D159, and the R1 substituent’s carboxylate group of temocillin were not possible with G214 in OXA-484, explaining the reduced affinity for temocillin. In addition, changes in active site’s water network may explain the decrease in hydrolysis rate of carbapenems. OXA-484 has weak imipenem and temocillin hydrolytic activities, which may lead to silent spread due to underdetection using selective screening media or biochemical imipenem hydrolysis confirmatory tests.

Abscesses represent the most prominent emerging problem in the red meat industry, leading to great economic constraints and public health hazards. Data on etiological agents present in these purulent lesions in Algeria are very scarce. The aim of this study was to identify the bacteria responsible for these abscesses and to determine their antibiotic susceptibility profiles. A total of 123 samples of abscesses from 100 slaughtered sheep and 23 slaughtered cattle were cultured in several media. A total of 114 bacterial isolates were cultured from 103 abscesses. Bacteria were identified using MALDI–TOF, and antibiotic susceptibility was determined by the disk diffusion method on Mueller–Hinton agar. A total of 73.6% (n = 84) corresponded to Enterobacterales, of which four were multidrug-resistant (MDR). These isolates, together with Staphylococcus aureus, coagulase negative Staphylococci, and seven randomly chosen susceptible Escherichia coli isolates, were further characterized using WGS. Resistome analysis of the four MDR Enterobacterales isolates revealed the presence of OXA-48 carbapenemase in two Klebsiella pneumoniae ST985 and one E. coli ST10 isolates and a CTX-M-15 ESBL in one E. coli isolate ST1706. Two coagulase-negative Staphylococci isolates were found to carry the mecA gene. WGS showed the presence of different resistance genes and virulence genes. Our study revealed 5% of MDR Enterobacterales (including ESBLs and carbapenemases) identified from abscesses, thus urging the need for abscess monitoring in slaughterhouses.

The development of novel antibiotics is mandatory to curb the growing antibiotic resistance problem resulting in difficult-to-treat bacterial infections. Here, we have determined the spectrum of activity of cystobactamids and chelocardins, two novel and promising classes of molecules with different modes of action. A panel of 297 clinically relevant Gram-negative and Gram-positive isolates with different antibiotic susceptibility profiles, going from wild type to multi- or even extremely drug resistant (MDR, XDR) and including carbapenem-resistant isolates, were tested using broth microdilution assays to determine the minimal inhibitory concentrations (MICs), MIC50s and MIC90s of two cystobactamids derivatives (CN-861-2 and CN-DM-861) and two chelocardin derivatives (CHD and CDCHD). Cystobactamids revealed potent activities on the majority of tested Enterobacterales (MIC50s ranging from 0.25 to 4 µg/mL), except for Klebsiella pneumoniae isolates (MIC50s is 128 µg/mL). Pseudomonas aeruginosa and Acinetobacter baumannii showed slightly higher MIC50s (4 µg/mL and 8 µg/mL, respectively) for cystobactamids. Chelocardins inhibited the growth of Enterobacterales and Stenotrophomas maltophilia at low to moderate MICs (0.25–16 µg/mL) and the chemically modified CDCHD was active at lower MICs. A. baumannii and P. aeruginosa were less susceptible to these molecules with MICs ranging from 0.5 to 32 µg/mL. These molecules show also interesting in vitro efficacies on clinically relevant Gram-positive bacteria with MICs of 0.125–8 µg/mL for cystobactamids and 0.5–8 µg/mL for chelocardins. Taken together, the cystobactamid CN-DM-861 and chelocardin CDCHD showed interesting antibiotic activities on MDR or XDR bacteria, without cross-resistance to clinically relevant antibiotics such as carbapenems, fluoroquinolones, and colistin.

Culture media is fundamental in clinical bacteriology for the detection and isolation of bacterial pathogens. However, in-house media preparation could be challenging in low-resource settings. InTray^® cassettes (Biomed Diagnostics) could be a valid alternative as they are compact, ready-to-use media preparations. In this study, we evaluate the use of two InTray media as a subculture alternative for the diagnosis of bloodstream infections: the InTray^® Müller-Hinton (MH) chocolate and the InTray^® Colorex™ Screen. The InTray MH chocolate was evaluated in 2 steps: firstly, using simulated positive blood cultures (reference evaluation study), and secondly, using positive blood cultures from a routine clinical laboratory (clinical evaluation study). The Colorex Screen was tested using simulated poly-microbial blood cultures. The sensitivity and specificity of the InTray MH chocolate were respectively 99.2% and 90% in the reference evaluation study and 97.1% and 88.2% in the clinical evaluation study. The time to detection (TTD) was ≤20 h in most positive blood cultures (99.8% and 97% in the two studies, respectively). The InTray^® MH Chocolate agar showed good performance when used directly from clinical blood cultures for single bacterial infections. However, mixed flora is more challenging to interpret on this media than on Colorex™ Screen, even for an experienced microbiologist.

This study evaluated the performances of immunoassays (LFIA and ELISA) designed for SARS-CoV-2 Antigen (Ag)-detection in nasopharyngeal (NP) and serum samples in comparison to RT-PCR. NP samples from patients with respiratory symptoms (183 RT-PCR-positive and 74 RT-PCR-negative samples) were collected from March to April and November to December 2020. Seroconversion and antigen dynamics were assessed by symptom onset and day of RT-PCR diagnosis. Serum samples from 87 COVID-19 patients were used to investigate the added value of Ag quantification, at diagnosis and during follow-up. The sensitivity of COVID-VIRO-LFIA on samples with Ct ≤ 33, considered as the contagious threshold, was 86% on NPs (CI 95%: 79–90.5) and 76% on serum samples (CI 95%: 59.4–88), with a specificity of 100%. Serum N-Ag was detected during active infection as early as day two from symptom onset, with a diagnostic sensitivity of 81.5%. Within one week of symptom onset, diagnostic sensitivity and specificity reached 90.9% (95% CI, 85.1%–94.6%) and 98.3% (95% CI, 91.1%–99.9%), respectively. Serum N-Ag concentration closely correlated with disease severity. Longitudinal analysis revealed the simultaneous increase of antibodies and decrease of N-Ag. Sensitivities of COVID-VIRO-LFIA and COV-QUANTO-ELISA tests on NP and serum samples were close to 80%. They are suitable COVID-19-laboratory diagnostic tests, particularly when blood samples are available, thus reducing the requirement for NP sampling, and subsequent PCR analysis. ELISA titers may help to identify patients at risk of poor outcomes.